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Antiphospholipid Antibodies Inhibit Trophoblast Toll-like Receptor and Inflammasome Negative Regulators.

Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications associated with poor placentation and placental inflammation. While these antibodies are heterogeneous, some anti-β2 GPI antibodies can activate human first trimester trophoblast TLR4 and NLRP3. The objective of this study was to determine the role of negative regulators of TLR and inflammasome function in aPL-induced trophoblast inflammation.

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First serologic evidence of human hantavirus infection in Alagoas State in Northeastern Brazil.

Hantavirus cardiopulmonary syndrome (HCPS) is rare in Northeastern Brazil.

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The First Serological Study of Q Fever in Humans in Lebanon.

The aim of this study was to estimate, for the first time, the human seroprevalence of Q fever in Lebanon, by assessing the presence of antibodies against the causative agent, Coxiella burnetii. A total number of 421 serum samples (226 females and 196 males) were collected in February 2015 from hospitals and laboratories dispersed in five Lebanese provinces: Akkar, Bekaa, Mount Lebanon, Nabatieh, and South Lebanon.

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Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G.

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal's immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')2 and polyclonal anti-IgG F(ab')2 are useful tools in biomedical and biochemical researches and diagnostic kits.

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Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination.

Antibody subclasses exhibit extensive polymorphisms (allotypes) that could potentially impact the quality of HIV-vaccine induced B cell responses. Allotypes of immunoglobulin (Ig) G1, the most abundant serum antibody, have been shown to display altered functional properties in regard to serum half-life, Fc-receptor binding and FcRn-mediated mucosal transcytosis. To investigate the potential link between allotypic IgG1-variants and vaccine-generated humoral responses in a cohort of 14 HIV vaccine recipients, we developed a novel protocol for rapid IgG1-allotyping. We combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1) of trial participants, using human plasma and RNA isolated from PBMC. The IgG1-allotype distribution of our participants mirrored previously reported results for caucasoid populations. We observed elevated levels of HIV gp140-specific IgG1 and decreased IgG2 levels associated with the G1m1-allele, in contrast to G1m3 carriers. These data suggest that vaccinees homozygous for G1m1 are predisposed to develop elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. This elevated IgG1:IgG2 ratio was further associated with higher FcγR-dimer engagement, a surrogate for potential antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) function. Although preliminary, these results suggest that IgG1 allotype may have a significant impact on IgG subclass distribution in response to vaccination and associated Fc-mediated effector functions. These results have important implications for ongoing HIV vaccine efficacy studies predicated on engagement of FcγR-mediated cellular functions including ADCC and ADCP, and warrant further investigation. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.

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Serum IgG antibodies from healthy subjects up to 100 years old react to JC polyomavirus.

JC polyomavirus (JCPyV) was identified in 1971 in the brain tissue of a patient (J.C.) affected by the progressive multifocal leukoencephalopathy (PML). JCPyV encodes for the oncoproteins large T antigen (Tag) and small t-antigen (tag). These oncoproteins are responsible of the cell transformation and tumorigenesis in experimental animals. JCPyV is ubiquitous in human populations. After the primary infection, which is usually asymptomatic, JCPyV remains lifelong in the host in a latent phase. Its reactivation may occur in heathy subjects and immunocompromised patients. Upon reactivation, JCPyV could reach (i) the CNS inducing the PML, (ii) the kidney of transplant patients causing the organ rejection. Association between JCPyV, which is a small DNA tumor virus, and gliomas and colorectal carcinomas has been published. In the present investigation, we report on a new indirect ELISA with two specific synthetic peptides mimicking JCPyV VP1 immunogenic epitopes to detect specific serum IgG antibodies against JCPyV. Serum samples of healthy subjects (n = 355) ranging 2-100 years old, were analyzed by this new indirect ELISA. The linear peptides VP1 K and VP1 N resemble the natural JCPyV VP1 capsidic epitopes constituting a docking site for serum antibodies. Data from this innovative immunologic assay indicate that the overall prevalence of JCPyV-VP1 antibodies in healthy subjects is at 39%. The innovative indirect ELISA with JCPyV VP1 mimotopes seems to be a useful method to detect specific IgG antibodies against this virus, without cross-reactivity with the closely related SV40 and BKPyV polyomaviruses. This article is protected by copyright. All rights reserved.

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ELISA Serology for Antibodies Against Chlamydia trachomatis in Crohn's Disease.

Recently we reported IgA anti-Chlamydia antibodies in patients with Crohn's disease (CD), in particular in four patients from a single family of six with CD.

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Immunogenicity and safety of a novel tetanus toxoid-conjugated anti-gastrin vaccine in BALB/c mice.

The objective of this study is to determine the immunogenicity and safety of our novel anti-gastrin vaccine that is composed of the common amino-terminal portions of human carboxy-amidated gastrin-17 (G17) and glycine-extended gastrin-17 (gly-G17) as well as the common carboxy-terminal portion of the gastrin precursor progastrin (in a 50:50 mixture) all covalently linked to tetanus toxoid (TT) via peptide spacers. The vaccine, or immunogen, was injected intramuscularly into the legs of BALB/c mice, which produced high serum titres of specific IgG antibodies and IFN-γ in their spleen cells, identifiable by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT), respectively. TT as the protein carrier effectively enhanced the antigenic epitopes' humoural and cellular immune responses, unlike the antigenic epitopes alone or the immunogen's adjuvant emulsion system (AES), all of which failed to provoke any obvious immune response. Notably, the animals' body weights increased significantly after immunization (P < .01), while their haematology and serum biochemistry were all generally normal, and the gross anatomy of their main organs (e.g., heart, liver, spleen, lung, kidney) showed no obvious histopathological changes.

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Development and evaluation of a broad bead-based multiplex immunoassay to measure IgG seroreactivity against human polyomaviruses.

Introduction The family of polyomaviruses, which cause severe disease in immunocompromised hosts, has expanded substantially in recent years. To accommodate measurement of IgG seroresponses against all currently known human polyomavirus (HPyV), including the Lyon IARC polyomavirus (LIPyV), we extended our custom multiplex bead-based HPyV immunoassay and evaluated the performance of this pan-HPyV immunoassay.Methods VP1 protein of fifteen HPyVs belonging to 13 polyomavirus species were expressed as recombinant glutathione S-transferase (GST) fusion proteins and coupled to fluorescent Luminex beads. Sera from healthy blood donors and immunocompromised kidney transplant recipients were used to analyse seroreactivity against the different HPyVs. For BK polyomavirus (BKPyV) the GST-VP1 fusion protein-directed seroresponses were compared to those obtained against BKPyV VP1 virus-like particles (VLP).Results Seroreactivity against most HPyVs was common and generally high in both test populations. Low seroreactivity was observed against HPyV9, HPyV12, New Jersey PyV and LIPyV. The assay was reproducible (Pearson's r2> 0.84, P<0.001) and specific. Weak but consistent cross-reactivity was observed between related HPyV6 and HPyV7. Seroresponses measured by the GST-VP1-based immunoassay and the VP1 VLP-based ELISA were highly correlated (Spearman's ρ=0.823, P<0.001).Conclusions The bead-based pan-HPyV multiplex immunoassay is a reliable tool to determine HPyV-specific seroresponses with high reproducibility and specificity and is suitable for seroepidemiological studies.

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A Serological Point-of-Care Test for the Detection of IgG Antibodies against Ebola Virus in Human Survivors.

Ebola virus disease causes widespread and highly fatal epidemics in human populations. Today, there is still great need for point-of-care tests for diagnosis, patient management and surveillance, both during and post outbreaks. We present a point-of-care test comprising an immunochromatographic strip and a smartphone reader, which detects and semiquantifies Ebola-specific antibodies in human survivors. We developed a Sudan virus glycoprotein monoplex platform and validated it using sera from 90 human survivors and 31 local noninfected controls. The performance of the glycoprotein monoplex was 100% sensitivity and 98% specificity compared to standard whole antigen enzyme-linked immunosorbent assay (ELISA), and it was validated with freshly collected patient samples in Uganda. Moreover, we constructed a multiplex test for simultaneous detection of antibodies against three recombinant Sudan virus proteins. A pilot study comprising 15 survivors and 5 noninfected controls demonstrated sensitivity and specificity of 100% compared to standard ELISA. Finally, we developed a second multiplex subtype assay for the identification of exposure to three related EVD species: Sudan virus, Bundibugyo virus and Ebola virus (formerly Zaire) using recombinant viral glycoprotein. This multiplex test could distinguish between the host's immunity to specific viral species and identify cross-reactive immunity. These developed serological platforms consisted of capture ligands with high specificity and sensitivity, in-house developed strips and a compatible smartphone application. These platforms enabled rapid and portable testing, data storage and sharing as well as geographical tagging of the tested individuals in Uganda. This platform holds great potential as a field tool for diagnosis, vaccine development, and therapeutic evaluation.

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