Guinea Pig Complement Research Articles

Serum Resistance of Mycoplasma agalactiae Strains and Mutants Bearing Different Lipoprotein Profiles.

In order to spread systemically, resistance against complement and other factors present in serum is an important trait in pathogenic bacteria. The variable proteins of Mycoplasma agalactiae (Vpmas) have been shown to affect differential adhesion, invasion and immune evasion, and undergo high-frequency phase-variation in expression. However, nothing is known about their involvement in M. agalactiae’s serum susceptibility. To evaluate this, the PG2 strain, the GM139 strain and the six Vpma phase-locked mutants (PLMs, PLMU to PLMZ) were tested for their ability to survive in the presence of non-sensitized and sensitized sheep serum, as well as guinea pig complement. Additionally, the reactivity of the sensitized sheep serum was analysed on the strains via western blotting. Overall data demonstrate PG2 strain to be more susceptible to sheep serum compared to the GM139 strain bearing a different Vpma profile. Significant differences were also observed between the different PLMs, with PLMU and PLMX showing the highest serum susceptibility in serum, while the other PLMs expressing longer Vpma proteins were more resistant. The results are in good correlation with previous studies where shorter lipoprotein variants contributed to a higher susceptibility to complement. Since none of the tested strains and PLMs were susceptible to non-sensitized sheep serum, antibodies seem to play an important role in serum killing.

Characterization of pyruvate dehydrogenase complex E1 alpha and beta subunits of Mycoplasma synoviae

Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.

Intradermal Immunization with Heat-Killed Klebsiella pneumoniae Leading to the Production of Protective Immunoglobulin G in BALB/c Mice.

Introduction:Klebsiella pneumoniae superbug is emerging as a serious health concern as resistance to last-resort antibiotics spreads. To bypass the therapeutic molecules used today, the development of an immunoprophylactic safe approach is of great clinical relevance. This study was conducted to determine the protective efficacy of antibodies elicited by killed vaccine against multidrug-resistant (MDR) K. pneumoniae.Materials and Methods:In this study, heat-killed MDR K. pneumoniae isolated from different clinical samples were employed for the intradermal immunization of 10 BALB/c mice. Two weeks after the third dose of immunization, the mice were intraperitoneally challenged with live K. pneumoniae and observed for 14 days. Tail blood was collected 7 days after each booster followed by cardiac puncture 14 days postchallenge. Bactericidal activity and antigen-binding capacity of the serum antibody produced by the vaccine were evaluated by serum bactericidal antibody (SBA) assay and ELISA, respectively.Results:In this study, 80% survival rates were observed at 14 days postchallenge among the immunized mice. Regarding SBA assay, 100% bactericidal activity of the immunized mouse sera was observed using 50% guinea pig complement at 1:10 serum dilution after 3 h of incubation, and all the pre- and postchallenge immunized serum immunoglobulin G antibody had significantly higher optical density values comparing the control mice in ELISA.Conclusion:In our study, intradermal immunization with heat-killed MDR K. pneumoniae produced protective antibodies in BALB/c mice. These findings suggest that the use of a first-generation vaccine provides the supply of a larger number of candidate antigens for eliciting required immune response.

1231. VIR-2482: A potent and broadly neutralizing antibody for the prophylaxis of influenza A illness

BackgroundInfluenza A viruses are responsible for seasonal epidemics and represent a constant pandemic threat. Influenza vaccines induce predominantly antibodies against the head region of hemagglutinin (HA) and are strain specific. Vaccine effectiveness is often suboptimal due to mismatch with drifting viruses and an inadequate immune response. Broadly neutralizing monoclonal antibodies (mAbs) targeting the conserved stem-region of HA may provide protection through multiple seasons and cover strains with pandemic potential. We report pre-clinical data on VIR-2482, a fully human anti-HA stem mAb with half-life extending Fc mutations.MethodsBinding of VIR-2482 to a panel of influenza HAs and neutralization of H1N1 and H3N2 viruses were measured by ELISA and microneutralization. Epitope conservation was evaluated using 49,462 HA sequences retrieved from GiSAID. Engagement of human FcγRs by VIR-2482 was assessed by biolayer interferometry. Antibody-dependendent cell-mediated cytoxicity (ADCC) was measured via in vitro killing of A549 cells expressing H1-HA glycoprotein by human NK cells. Complement-dependent cytotoxicity (CDC) was evaluated by incubating VIR-2482 with H1N1 infected cells in the presence of guinea pig complement. Protection studies were performed in Balb/c mice given VIR-2482 24h before intranasal infection with a lethal dose of H1N1 PR8 and H3N2 HK/68.ResultsVIR-2482 binds to the HA proteins representing all 18 influenza A HA subtypes and neutralizes a broad panel of H1N1 and H3N2 viruses spanning almost 100 years of evolution. Bioinformatic analysis revealed >98.8% conservation for the majority of key contact residues examined from sequences retrieved for H1N1 and H3N2 between 2009-2019. The half-life extending mutations in the Fc portion do not affect the ability of the antibody to engage FcγRIIIa, FcγRIIa, and C1q as evidenced by their lack of impact on ADCC and CDC in vitro. Prophylactic administration of VIR-2482 protects Balb/c mice from infection with lethal challenge doses of H1N1 and H3N2 viruses.ConclusionThe attributes of potency, broad recognition of a highly conserved epitope, retention of high-level effector functions in addition to half-life extension support the development of VIR-2482 as a universal prophylactic for influenza A illness.DisclosuresMatteo Samuele Pizzuto, PhD, VIR Biotechnology (Employee) Fabrizia Zatta, n/a, Vir Biotechnology (Employee) Andrea Minola, MS, Vir Biotechnology (Employee) Alessia Peter, n/a, Vir Biotechnology (Employee) Katja Culap, n/a, Vir Biotechnology (Employee) Leah Soriaga, PhD, Vir Biotechnology (Employee) Anna De Marco, n/a, Vir Biotechnology (Employee) Barbara Guarino, PhD, Vir Biotechnology (Employee) Nadia Passini, n/a, Vir Biotechnology (Employee) David K. Hong, MD, Vir Biotechnology (Employee) Fabio Benigni, PhD, Vir Biotechnology (Employee) Christy Hebner, PhD, Vir Biotechnology (Employee) Aurelio Bonavia, PhD, Vir Biotechnology (Employee) Davide Corti, PhD, Vir Biotechnology (Employee)

Development of an Improved Epstein-Barr Virus (EBV) Neutralizing Antibody Assay to Facilitate Development of a Prophylactic gp350-Subunit EBV Vaccine.

No licensed vaccine is available for prevention of EBV-associated diseases, and robust, high-throughput bioanalytical assays are needed to evaluate immunogenicity of gp350 subunit-based candidate EBV vaccines. Here we have developed an improved EBV-GFP based neutralization assay for such a vaccine’s pre-clinical and clinical validation to measure EBV specific neutralizing antibodies in human donors. The supplementation of guinea pig complement of our previously published high-throughput EBV-GFP fluorescent focus (FFA)-based neutralization assay allowed the detection of complement-dependent neutralizing antibodies using a panel of heat-inactivated healthy human sera. Anti-gp350 antibody titers, which were evaluated using a previously optimized anti-gp350 IgG ELISA assay, were moderately correlated to the FFA-based neutralization titers. Overall, this improved high-throughput neutralization assay is capable of characterizing the serologic neutralizing antibody response to natural EBV infection, with applications in evaluating EBV antibody status in epidemiologic studies and immunogenicity of candidate gp350-subunit EBV vaccines in clinical studies.

Development and characterization of novel anti-C5 monoclonal antibodies capable of inhibiting complement in multiple species.

SummaryOver the last decade there has been an explosion in complement therapies; one‐third of the drugs in the clinic or in development target C5 protein. Eculizumab, a monoclonal antibody (mAb) that binds C5 and blocks its cleavage by the convertase, is the current reference standard treatment for atypical haemolytic uraemic syndrome (aHUS) and paroxysmal nocturnal haemoglobinuria (PNH) and in clinical trials for many other diseases. Here we describe a panel of novel anti‐C5 mAb, including mAb that, like Eculizumab, are efficient inhibitors of complement but, unlike Eculizumab, inhibit across species, including human, rat, rabbit and guinea pig. Several inhibitory anti‐C5 mAb were identified and characterized for C5 binding and lytic inhibitory capacity in comparison to current therapeutic anti‐C5 mAb; three clones, 4G2, 7D4 and 10B6, were selected and further characterized for ligand specificity and affinity and cross‐species inhibitory activity. The mAb 10B6 was human‐specific whereas mAb 4G2 and 7D4 efficiently inhibited lysis by human, rabbit and rat serum, and weakly inhibited guinea pig complement; 7D4 also weakly inhibited mouse complement in vitro The rat C5‐cross‐reactive mAb 4G2, when administered intraperitoneally in a rat model of myasthenia gravis, effectively blocked the disease and protected muscle endplates from destruction. To our knowledge this is the first report of an anti‐C5 function blocking mAb that permits preclinical studies in rats.

Evaluation of the Stability of Performance of the Analytical Test Method Used for Determination of Anticomplementary Activity of Human Immunoglobulin Preparations

The evaluation of anticomplementary activity, being an obligatory component of the quality control ofhuman immunoglobulin preparations, requires the use of a number of reagents of biological origin that are difficult to standardise. In order to standardise the quality control method used for determination of antiсomplementary activity it is advisable to use a reference standard which demonstrates whether obtained results comply with the acceptance criteria, and helps to assess the stability of analytical performance. The aim of the study was to assess the stability of analytical performance of the test procedure used for determination of antiсomplementary activity of human immunoglobulin preparations using human immunoglobulin reference standards of various grades. Materials and methods: anticomplementary activity was determined by the complement fixation test in accordance with the general monograph of the State Pharmacopoeia of the Russian Federation (14th ed.) OFS.1.8.2.0007.15 using different batches of guinea pig complement and sheep red blood cells. The test procedure was standardised using the OSO 42-28-430-2018 human immunoglobulin reference standard and the human immunoglobulin BRP, batch 1 (cat. No. Y0001504). The obtained results of anticomplementary activity of the reference standards were used to construct Shewhart control charts, using the certified values as control limits. Results: the analysis of the constructed Shewhart charts helped to assess trends, and estimate the influence of different batches of guinea pig complement and sheep red blood cells on the stability of analytical performance of the test procedure used for determination of antiсomplementary activity of human immunoglobulin preparations. Conclusions: the use of the OSO 42-28-430-2018 reference standard in combination with Shewhart control charts for the quality control of human immunoglobulin preparations in terms of Anticomplementary activity makes it possible to control the testing process, and assess any of its changes associated with the replacement of the batch of the reagent. At the same time, the reference standard of the European Pharmacopoeia, which has a wide range of permissible values, can only be used to confirm the acceptance of the results. Further studies to determine the mean value of the anticomplementary activity of control samples, as well as the standard deviation, may increase the possibilities of using this reference standard for assessment of analytical performance stability.

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines.

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.

The innate immunity of guinea pigs against highly pathogenic avian influenza virus infection.

H5N1 avian influenza viruses are a major pandemic concern. In contrast to the highly virulent phenotype of H5N1 in humans and many animal models, guinea pigs do not typically display signs of severe disease in response to H5N1 virus infection. Here, proteomic and transcriptional profiling were applied to identify host factors that account for the observed attenuation of A/Tiger/Harbin/01/2002 (H5N1) virulence in guinea pigs. RIG-I and numerous interferon stimulated genes were among host proteins with altered expression in guinea pig lungs during H5N1 infection. Overexpression of RIG-I or the RIG-I adaptor protein MAVS in guinea pig cell lines inhibited H5N1 replication. Endogenous GBP-1 expression was required for RIG-I mediated inhibition of viral replication upstream of the activity of MAVS. Furthermore, we show that guinea pig complement is involved in viral clearance, the regulation of inflammation, and cellular apoptosis during influenza virus infection of guinea pigs. This work uncovers features of the guinea pig innate immune response to influenza that may render guinea pigs resistant to highly pathogenic influenza viruses.

Experimental immunological demyelination enhances regeneration in autograft-repaired long peripheral nerve gaps.

Peripheral nerve long gap defects are a clinical challenge in the regeneration field. Despite the wide variety of surgical techniques and therapies, autografting is the “gold standard” for peripheral nerve gap reconstruction. The pathological process of Wallerian degeneration from the time of acute injury to efficient regeneration requires several weeks. Regeneration time is critical for nerve reconstruction. Immunological demyelination induced by anti-galactocerebroside antibodies plus guinea pig complement was used to shorten the treatment time. Based on an antigen-antibody complex reaction, the demyelinating agent induced an acute and severe demyelination, leading to the pathological process of Wallerian degeneration during the demyelinating period. This method was used to treat a 12 mm-long sciatic nerve defect in rats. The control groups were injected with one of the demyelinating agent components. The results indicated that anti-galactocerebroside antibodies plus guinea pig complement can significantly shorten treatment time and promote nerve regeneration and functional recovery. In addition, the demyelinating agent can increase the mRNA levels of nerve growth factors and can regulate inflammation. In conclusion, treatment with anti-galactocerebroside antibodies plus guinea pig complement can promote axonal regeneration. This therapy provides a novel method to improve functional recovery in the treatment of long nerve defects.

Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses.

We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency.

The immunosuppression mechanism of hypodermin A on complement

Hypodermin A (HA), a serine protease secreted by first-instar larvae of Hypoderma lineatum (Diptera: Oestridae) is associated with inflammatory and the specific immune responses in cattle hosts. In the present study, the cDNA sequence of HA was synthesized, and found to have fifteen amino acids which differed from the sequence available in GenBank. We then examined the association between recombinant HA and guinea-pig complement component 3 (C3) through a co-immunoprecipitation assay. Cos7 cells stably expressing HA were generated, and were found to be more resistant to lysis by guinea-pig C3 than the controls. HA was also able to degrade the C6 and C5b-9 of guinea-pig C3. The presumed DNA binding site of HA with guinea-pig C3 was detected by an electrophoretic mobility shift assay (EMSA). In contrast, after stable transfection, mHA was unable to reduce the amount of C3 or to inhibit its cytotoxicity, while HA could degrade guinea-pig C3 and inhibit the complement pathway. The findings suggest that recombinant HA could serve as an immunosuppressive agent against organ rejection after xenotransplantation.

Development of a simple, rapid, sensitive, high-throughput luciferase reporter based microneutralization test for measurement of virus neutralizing antibodies following Respiratory Syncytial Virus vaccination and infection

We have established a new reporter gene-based RSV neutralization test using Renilla luciferase. The RSV-Luciferase Neutralization Test (RSV-Luc-NeuT) is a simple, rapid, high throughput, and less labor intensive functional serological assay than the traditional RSV-PRNT, capable of measuring a broad range of anti-RSV neutralizing antibodies targeting both RSV-F and RSV-G proteins. Specificity and sensitivity of the RSV-Luc-NeuT are comparable to the RSV-PRNT. Panels of pre-vaccination and post-vaccination animal sera, monoclonal antibodies and animal polyclonal anti-RSV sera confirmed assay specificity. A panel of 60 human sera demonstrated high assay sensitivity for measurement of RSV neutralizing antibodies that strongly correlated with the RSV-PRNT titers (R2=0.864). Neutralization in the presence of guinea pig complement (GPC) increased PRNT titers more than the RSV-Luc-NeuT neutralizing antibody titers for these human sera. This newly developed simple, high throughput, RSV-Luc-NeuT could be easily automated and applied in measurement of RSV neutralization titers in large vaccine trials.

383 IDENTIFICATION OF PLURIPOTENCY MARKERS IN SWINE EMBRYOS

Embryonic stem cells (ESC) are a useful tool for studying embryonic development, cell differentiation, and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral, and religious considerations by scientists and laymen alike. Therefore, an animal model such as the pig (Sus scrofa) is valuable in overcoming such hurdles because this species holds physiologic parameters similar to humans. In spite of the great biomedical potential of ESC, many difficulties have been faced in maintaining these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro- and in vivo-produced swine blastocysts; (2) to compare 2 in vitro culture conditions to maintain isolated inner cell masses (ICM), murine embryonic fibroblasts (MEF), or Matrigel; and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2, and FoxD3 at ESC and in vitro- and in vivo-produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vivo-produced blastocysts were obtained by superovulation followed by AI of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing 5 days after ovulation. In vitro- and in vivo-produced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution, and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. Resultant ICM (14 and 66 ICM from in vitro- and in vivo-produced blastocysts, respectively) were cultured in stem cells media (GMEM added by 15% FCS, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, and 4 ng mL-1 of basic fibroblast growth factor) over monolayer of irradiated mouse embryonic fibroblasts (MEF) or Matrigel for 3 weeks. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro- and in vivo-produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis was performed only with whole in vitro- and in vivo-produced blastocysts. Results showed that Nanog and Sox2 were less expressed in in vitro-produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Because no ESC lineage was obtained in swine until now, we believe this species has different requirements compared with murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC. Acknowledgments are given to FAPESP (processes 06/58507-0 and 07/51732-0).

A liposome immune lysis assay for enrofloxacin in carp and chicken muscle

A homogenous complement-mediated liposome immune lysis assay (LILA) was developed for the determination of enrofloxacin (ENRO) in carp and chicken muscle. ENRO was covalently coupled to DPPE, and then immobilized onto the surface of liposomes by reverse-phase evaporation method. The performed liposome would be specifically lysed by the sequential additions of anti-ENRO monoclonal antibody (MAb) and guinea pig complement. Through a competitive assay format, the performed liposome can be used to detect ENRO in a range of 5.0–20 ng mL −1 in assay buffer. The limit of detection of ENRO in carp and chicken muscle was 1 ng g −1 and the limit of quantification was 2 ng g −1. Recoveries ranged from 58.3% to 65.2% for carp and 55.6–63.8% for chicken muscle at spiked levels of 2–8 ng g −1, with intra-assay and inter-assay variations 5.6–12.3% and 7.1–19.2%, respectively.

THE USE OF MTT [3-(4, 5-DIMETHYL-2-THIAZOLYL) -2, 5-DIPHENYL - 2H- TETRAZOLIUM BROMIDE]-REDUCTION AS AN INDICATOR OF THE EFFECTS OF STRAIN-SPECIFIC, POLYCLONAL RABBIT ANTISERA ON CANDIDA ALBICANS AND C. KRUSEI

There is only scanty data on the effects of specific antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro, although previously published work has shown that specific antibody mediates anti-Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing.The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-specific, polyclonal anti-Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell-mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without specific antibody, also, had no such effect on these two Candida species.

Preparation of a respiratory syncytial virus human reference serum for use in the quantitation of neutralization antibody

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, young children and the elderly. Yet, the development of a vaccine to protect against RSV infection still remains an unmet need. At present, immune responses to experimental vaccines under investigation are usually evaluated by ELISA and/or by neutralization assays against RSV. However, both types of assays are generally performed somewhat differently at different laboratories. An important step towards standardization of serology is the use of a standard human reference serum enabling normalization of results generated within and between laboratories. To fill this need, we prepared and characterized a human reference serum against the A2 strain of respiratory syncytial virus. The serum represents a pool of more than 400 individual human sera obtained from commercial sources. The sera were screened and selected on the basis of individual RSV neutralization titers. A final neutralization titer of 973 (95% C.I., 884–1072) was assigned to the final reference serum pool after it was tested three times in the presence of 10% guinea pig complement and a titer of 286 (95% C.I., 243–337) was assigned to the serum when it was tested in the absence of an exogenous complement source. Sterilely reconstituted lyophilized aliquots of the serum exhibited a stable neutralization titer for at least 1 month at room temperature and at 4 °C, as well as after 5 weekly freeze-and-thaw cycles at −20 °C. In the lyophilized state, the neutralization titer of the lyophilized reagent was stable for at least 6 months, the last time point tested. Two additional smaller pools of serum with high and medium neutralization titers of 2692 and 575, respectively, were also produced in parallel for use as positive controls and were designated as control sera. The reference serum can be used to normalize neutralization and/or other RSV-specific assay results from different laboratories and the control sera can be used for quality control purposes or as part of a panel to test operator proficiency. Individual lyophilized aliquots of the reference and control sera may be obtained from the US National Institute of Allergy and Infectious Diseases (NIAID) Reference Reagent Repository.

The use of mtt [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2h- tetrazolium bromide]-reduction as an indicator of the effects of strain-specific, polyclonal rabbit antisera on <i> Candida albicans</i> and <i> C. krusei</i>

There is only scanty data on the effects of specific antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro, although previously published work has shown that specific antibody mediates anti- Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing. The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-specific, polyclonal anti- Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell-mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without specific antibody, also, had no such effect on these two Candida species.

Derivation of a Xeno‐Free Human Embryonic Stem Cell Line

Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [Stem Cells 2006; 24:221-229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611.

Bacteriological study of autologous cryoprecipitate‐derived fibrin glue as the operative sealant

Fibrin glue (FG) is frequently used to seal and cover the anastomoses in many operations such as cardiovascular surgery or orthopaedic surgery. However, in case of gastrointestinal surgery, anastomoses are potentially contaminated, and FG may promote bacterial growth, increasing the risk of leakage. The purpose of this study was to examine the effect of cryoprecipitate-derived FG (CryoFG) on bacterial growth. Bacterial growth on the CryoFG and on the commercial FG (Beriplast P) was evaluated and compared with that on control medium. In addition, the complement activities were evaluated by heat inactivation or addition of guinea-pig complement to the experimental settings. The CryoFG inhibited the growth of Escherichia coli, whereas the commercial FG had no effect. Heat inactivation of the CryoFG inhibited the bactericidal effect of CryoFG. Addition of guinea-pig complement to the heat-inactivated CryoFG could almost restore the bactericidal activity, suggesting the important role of complement. This study showed that the CryoFG preserved the complement activity, which inhibited the in vitro growth of E. coli. Therefore, we concluded that the application of the CryoFG for gastrointestinal surgical anastomoses not only would be safe but also has the advantage of reducing bacterial infection.