371 research outputs found

    A population of Langerin-positive dendritic cells in murine Peyer\u27s patches involved in sampling beta-glucan microparticles

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    Glucan particles (GPs) are 2-4 mum hollow, porous shells composed of 1,3-beta-D-glucan that have been effectively used for oral targeted-delivery of a wide range of payloads, including small molecules, siRNA, DNA, and protein antigens. While it has been demonstrated that the transepithelial transport of GPs is mediated by Peyer\u27s patch M cells, the fate of the GPs once within gut-associated lymphoid tissue (GALT) is not known. Here we report that fluorescently labeled GPs administered to mice by gavage accumulate in CD11c+ DCs situated in Peyer\u27s patch sub-epithelial dome (SED) regions. GPs appeared in DCs within minutes after gavage and remained within the SED for days afterwards. The co-administration or sequential administration of GPs with differentially labeled GPs or poly(lactic-co-glycolic acid) nanoparticles demonstrated that the SED DC subpopulation in question was capable of internalizing particles of different sizes and material compositions. Phenotypic analysis identified the GP-containing DCs as being CD8alpha- and CD11blo/-, suggesting they are the so-called myeloid and/or double negative (DN) subset(s) of PP DCs. A survey of C-type lectin receptors (CLRs) known to be expressed by leukocytes within the intestinal mucosa revealed that GP-containing SED DCs were positive for Langerin (CD207), a CLR with specificity for beta-D-glucan and that has been shown to mediate the internalization of a wide range of microbial pathogens, including bacteria, viruses and fungi. The presence of Langerin+ DCs in the SED as determined by immunofluorescence was confirmed using Langerin E-GFP transgenic mice. In summary, our results demonstrate that following M cell-mediated transepithelial transport, GPs (and other micro/nanoparticles) are sampled by a population of SED DCs distinguished from other Peyer\u27s patch DC subsets by their expression of Langerin. Future studies will be aimed at defining the role of Langerin in antigen sampling and antigen presentation within the context of the GALT

    Inhibition of HIV-1 infectivity and epithelial cell transfer by human monoclonal IgG and IgA antibodies carrying the b12 V region

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    Both IgG and secretory IgA Abs in mucosal secretions have been implicated in blocking the earliest events in HIV-1 transit across epithelial barriers, although the mechanisms by which this occurs remain largely unknown. In this study, we report the production and characterization of a human rIgA(2) mAb that carries the V regions of IgG1 b12, a potent and broadly neutralizing anti-gp120 Ab which has been shown to protect macaques against vaginal simian/HIV challenge. Monomeric, dimeric, polymeric, and secretory IgA(2) derivatives of b12 reacted with gp120 and neutralized CCR5- and CXCR4-tropic strains of HIV-1 in vitro. With respect to the protective effects of these Abs at mucosal surfaces, we demonstrated that IgG1 b12 and IgA(2) b12 inhibited the transfer of cell-free HIV-1 from ME-180 cells, a human cervical epithelial cell line, as well as Caco-2 cells, a human colonic epithelial cell line, to human PBMCs. Inhibition of viral transfer was due to the ability of b12 to block both viral attachment to and uptake by epithelial cells. These data demonstrate that IgG and IgA MAbs directed against a highly conserved epitope on gp120 can interfere with the earliest steps in HIV-1 transmission across mucosal surfaces, and reveal a possible mechanism by which b12 protects the vaginal mucosal against viral challenge in vivo

    High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

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    We previously produced a heavy-chain-only antibody (Ab) VH domain (VHH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538–36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific VHHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the VHH-displayed phage library to additional “pannings” on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique VHHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 VHHs grouped into more than 20 different competition bins. The RTA-specific VHHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific VHHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development

    Studying Cat (Felis catus) Diabetes: Beware of the Acromegalic Imposter

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    Naturally occurring diabetes mellitus (DM) is common in domestic cats (Felis catus). It has been proposed as a model for human Type 2 DM given many shared features. Small case studies demonstrate feline DM also occurs as a result of insulin resistance due to a somatotrophinoma. The current study estimates the prevalence of hypersomatotropism or acromegaly in the largest cohort of diabetic cats to date, evaluates clinical presentation and ease of recognition. Diabetic cats were screened for hypersomatotropism using serum total insulin-like growth factor-1 (IGF-1; radioimmunoassay), followed by further evaluation of a subset of cases with suggestive IGF-1 (>1000 ng/ml) through pituitary imaging and/ or histopathology. Clinicians indicated pre-test suspicion for hypersomatotropism. In total 1221 diabetic cats were screened; 319 (26.1%) demonstrated a serum IGF-1>1000 ng/ml (95% confidence interval: 23.6-28.6%). Of these cats a subset of 63 (20%) underwent pituitary imaging and 56/63 (89%) had a pituitary tumour on computed tomography; an additional three on magnetic resonance imaging and one on necropsy. These data suggest a positive predictive value of serum IGF-1 for hypersomatotropism of 95% (95% confidence interval: 90-100%), thus suggesting the overall hypersomatotropism prevalence among UK diabetic cats to be 24.8% (95% confidence interval: 21.2-28.6%). Only 24% of clinicians indicated a strong pre-test suspicion; most hypersomatotropism cats did not display typical phenotypical acromegaly signs. The current data suggest hypersomatotropism screening should be considered when studying diabetic cats and opportunities exist for comparative acromegaly research, especially in light of the many detected communalities with the human disease

    Structural Elucidation of a Protective B Cell Epitope on Outer Surface Protein C (OspC) of the Lyme Disease Spirochete, Borreliella burgdorferi

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    Outer surface protein C (OspC) plays a pivotal role in mediating tick-to-host transmission and infectivity of the Lyme disease spirochete, Borreliella burgdorferi. OspC is a helical-rich homodimer that interacts with tick salivary proteins, as well as components of the mammalian immune system. Several decades ago, it was shown that the OspC-specific monoclonal antibody, B5, was able to passively protect mice from experimental tick-transmitted infection by B. burgdorferi strain B31. However, B5’s epitope has never been elucidated, despite widespread interest in OspC as a possible Lyme disease vaccine antigen. Here, we report the crystal structure of B5 antigen-binding fragments (Fabs) in complex with recombinant OspC type A (OspCA). Each OspC monomer within the homodimer was bound by a single B5 Fab in a side-on orientation, with contact points along OspC’s α-helix 1 and α-helix 6, as well as interactions with the loop between α-helices 5 and 6. In addition, B5’s complementarity-determining region (CDR) H3 bridged the OspC-OspCâ€Č homodimer interface, revealing the quaternary nature of the protective epitope. To provide insight into the molecular basis of B5 serotype specificity, we solved the crystal structures of recombinant OspC types B and K and compared them to OspCA. This study represents the first structure of a protective B cell epitope on OspC and will aid in the rational design of OspC-based vaccines and therapeutics for Lyme disease

    High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

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    RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the “back side” (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed ÎČ-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species

    Loss of tolerance to gut immunity protein; glycoprotein 2 (GP2) is associated with progressive disease course in primary sclerosing cholangitis

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    Abstract Glycoprotein 2[GP2] is a specific target of pancreatic autoantibodies[PAbs] in Crohn’s disease(CD) and is involved in gut innate immunity processes. Our aim was to evaluate the prevalence and prognostic potential of PAbs in primary sclerosing cholangitis(PSC). Sixty-five PSC patients were tested for PAbs by indirect immunofluorescence and compared with healthy (n = 100) and chronic liver disease controls(CLD, n = 488). Additionally, a panel of anti-microbial antibodies and secretory (s)IgA levels were measured, as markers of bacterial translocation and immune dysregulation. PAbs were more frequent in PSC(46.2%) compared to controls(healthy:0% and CLD:4.5%), [P < 0.001, for each]. Occurrence of anti-GP2 antibody was 30.8% (20/65) and was exclusively of IgA isotype. Anti-GP2 IgA positive patients had higher sIgA levels (P = 0.021). With flow-cytometry, 68.4% (13/19) of anti-GP2 IgA antibodies were bound with secretory component, suggesting an active retro-transportation of anti-GP2 from the gut lumen to the mucosa. Anti-GP2 IgA was associated with shorter transplant-free survival [PLogRank < 0.01] during the prospective follow-up (median, IQR: 87 [9–99] months) and remained an independent predictor after adjusting for Mayo risk score(HR: 4.69 [1.05–21.04], P = 0.043). These results highlight the significance of gut-liver interactions in PSC. Anti-GP2 IgA might be a valuable tool for risk stratification in PSC and considered as a potential therapeutic target
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