RSSMassive monoclonal expansion of CD4 T-cells specific for a M. tuberculosis ESAT-6 peptide.
Source
Saarland University, 66421 Homburg.
Abstract
T-cell responses towards tuberculin (PPD) or the M. tuberculosis-specific antigens ESAT-6 CFP-10 are indicative of prior contact with mycobacterial antigens. In this study, we investigated the exceptional case of a 75-year-old patient who devoted more than one third of his CD4 T-cells against PPD and ESAT-6.Antigen-specific T-cells were characterised using flow-cytometric intracellular cytokine-staining, ELISPOT-assay, proliferation-assays, and T-cell receptor spectratyping.T-cell frequencies were far above those found in age-matched controls (median 0.33%, 0.05-6.32%) and remained at high levels for more than two years. The patient initially presented with hemoptysis, but active tuberculosis was ruled out by repeated analysis of sputum and BAL-fluid. Skin-testing was negative and hemoptyses did not have a M. tuberculosis-related etiology. Phenotypical and functional properties of specific T-cells were consistent with a terminally differentiated effector-memory phenotype with capacity to produce IFN-γ, IL-2, and TNF-α. Epitope mapping showed that the CD4 T-cells were directed against a single peptide from ESAT-6 (amino-acid 5-20) that was presented in context of HLA-DR. T-cell receptor Vβ-spectratyping and sequencing of specific CD4 T-cells revealed a prominent peak-fraction of monoclonal origin.In conclusion, similar to monoclonal gammopathies of undetermined significance (MGUS), this may represent the first T-cell counterpart with known specificity against M. tuberculosis.
Novel myeloma-associated antigens revealed in the context of syngeneic hematopoietic stem cell transplantation.
Source
Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, MA, United States;
Abstract
Targets of curative donor-derived graft-versus-myeloma (GvM) responses following allogeneic hematopoietic stem cell transplantation (HSCT) remain poorly defined, partly because immunity against minor histocompatibility antigens(mHAgs) complicates the elucidation of multiple myeloma (MM)-specific targets. We hypothesized that syngeneic HSCT would facilitate the identification of GvM-associated antigens since donor immune responses in this setting should exclusively target unique tumor antigens in the absence of donor-host genetic disparities. We therefore studied the development of tumor immunity in an HLA-A0201+ MM patient who achieved durable remission after myeloablative syngeneic HSCT. Using high-density protein microarrays to screen post-HSCT plasma, we identified six antigens that elicited high-titer (1:5,000-1:10,000) antibodies that correlated with clinical tumor regression. Two antigens (DAPK2, PIM1) had enriched expression in primary MM tissues. Both elicited antibody responses in other MM patients following chemotherapy or HSCT (11 and 6 of 32 patients for DAPK2 and PIM1, respectively). The index patient also developed specific CD8+ T cell responses to HLA-A2 restricted peptides derived from DAPK2 and PIM1. Peptide-specific T cells recognized HLA-A2+ MM-derived cell lines and primary MM tumor cells. Coordinated T and B cell immunity develops against MM-associated antigens following syngeneic HSCT. DAPK1 and PIM1 are promising target antigens for MM-directed immunotherapy.
Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells.
Source
The Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People's Hospital), Jinan University, 518020 Shenzhen, PR China.
Abstract
Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic β cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas that expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic β cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.
Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.
A novel method for detecting antigen-specific human regulatory T cells.
Abstract
Antigenic epitopes recognized by FoxP3(+) regulatory T cells (Treg) are poorly defined, largely due to a lack of assays for determining Treg specificity. We have developed a novel approach for detecting human Treg specific to peptideantigen, utilizing down-regulation of surface CD3 as a read-out of antigen recognition. Culture conditions and re-stimulation time have been optimized, allowing the detection of even very rare Treg, such as those specific to tumorantigens.
Copyright © 2012 Elsevier B.V. All rights reserved.
Cytometry by Time-of-Flight Shows Combinatorial Cytokine Expression and Virus-Specific Cell Niches within a Continuum of CD8(+) T Cell Phenotypes.
Source
Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA.
Abstract
Cytotoxic CD8(+) T lymphocytes directly kill infected or aberrant cells and secrete proinflammatory cytokines. By using metal-labeled probes and mass spectrometric analysis (cytometry by time-of-flight, or CyTOF) of human CD8(+) T cells, we analyzed the expression of many more proteins than previously possible with fluorescent labels, including surface markers, cytokines, and antigen specificity with modified peptide-MHC tetramers. With 3-dimensional principal component analysis (3D-PCA) to display phenotypic diversity, we observed a relatively uniform pattern of variation in all subjects tested, highlighting the interrelatedness of previously described subsets and the continuous nature of CD8(+) T cell differentiation. These data also showed much greater complexity in the CD8(+) T cell compartment than previously appreciated, including a nearly combinatorial pattern of cytokine expression, with distinct niches occupied by virus-specific cells. This large degree of functional diversity even between cells with the same specificity gives CD8(+) T cells a remarkable degree of flexibility in responding to pathogens.
Copyright © 2012 Elsevier Inc. All rights reserved.
- PMID:
- 22265676
- [PubMed - as supplied by publisher]
The potential utility of methoxypoly(ethylene Glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine.
Source
Canadian Blood Services, Vancouver, BC, Canada.
Abstract
Red blood cell (RBC) transfusions are an important clinical intervention. However, RBC express hundreds of non-ABOantigens making alloimmunization a significant risk. RhD expression is the most immunologically important non-ABOantigen. Availability of RhD(-) blood, often problematic in North America and Europe, is a significant issue in Asia and Africa where RhD(-) blood is uncommon (<0.5% of supply). The immunocamouflage of RhD is readily accomplished by the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to the RBC membrane. To determine if RhD immunocamouflage would inhibit its immunologic recognition, an in vitro RhD-sensitized antigen presentation assay using PBMC and dendritic cells (DC) from RhD-sensitized women was used. The immunological effects of polymer grafting to an immunodominant RhD peptide, purified RhD protein and intact RhD(+) RBC were examined via T cell proliferation and cytokine release assays. At Day 11, PEGylation significantly attenuated T cell proliferation arising from RhD peptide (∼80 → 5%), protein (36 → 0.2%) and intact RBC (33 → 1.4%). Cytokine secretion was similarly blunted following PEGylation of the purified protein or intact RBC. These data support the immunomodulatory effects of PEGylation and the potential utility of this technology in transfusion medicine - especially in situations where RhD(-) blood is rare or in short supply.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization.
Abstract
ABSTRACT:
BACKGROUND:
To characterize the human humoral immune response against enterovirus 71 (EV71) infection and map human epitopes on the viral capsid proteins.
METHODS:
A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3) of BJ08 strain (genomic C4) were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA) was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 mug/mul). Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel.
RESULTS:
A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3) were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15) was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71) were identified and mapped to VP1.
CONCLUSION:
These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents.
Biomineralization and Size Control of Stable Calcium Phosphate Core Protein Shell Nanoparticles: Potential for Vaccine Applications.
Abstract
Nanoscale calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of E. coli Thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50-70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high affinity ACP binding since elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers three weeks post-injection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.
How the TCR balances sensitivity and specificity for the recognition of self and pathogens.
Source
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
Abstract
The T cell repertoire is generated during thymic development in preparation for the response to antigens from pathogens. The T cell repertoire is shaped by positive selection, which requires recognition by the T cell antigen receptor (TCR) of complexes of self peptide and major histocompatibility complex proteins (self-pMHC) with low affinity, and negative selection, which eliminates T cells with TCRs that recognize self-pMHC with high affinity. This generates a repertoire with low affinity for self-pMHC but high affinity for foreign antigens. The TCR must successfully engage both of these ligands for development, homeostasis and immune responses. This review discusses mechanisms underlying the interaction of the TCR with peptide-major histocompatibility complex ligands of varying affinity and highlights signaling mechanisms that enable the TCR to generate different responses to very distinct ligands.
Physical Characterization of the 'Immunosignaturing Effect'
Source
ASU, United States;
Abstract
Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimers disease display a characteristic and reproducible immunosignature on a microarray of 10,000 random sequencepeptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.
The Antimicrobial Peptide hLF1-11 Drives Monocyte-Dendritic Cell Differentiation toward Dendritic Cells That Promote Antifungal Responses and Enhance Th17 Polarization.
Source
Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
Abstract
The hLF1-11 peptide comprising the first 11 N-terminal residues of human lactoferrin exerts antimicrobial activity in vivo, enhances the inflammatory response of monocytes and directs monocyte-macrophage differentiation toward cells with enhanced antimicrobial properties. In this study, we investigated the effects of hLF1-11 on human monocyte-dendritic cell (DC) differentiation and subsequent T cell activation. Results revealed that - compared to control (peptide-incubated) DCs - hLF1-11-differentiated DCs displayed enhanced expression of HLA class II antigens and dectin-1, and increased phagocytosis of Candida albicans. In addition, hLF1-11-differentiated DCs produced enhanced amounts of reactive oxygen species, IL-6 and IL-10, but not IL-12p40 and TNF-α, upon stimulation with C. albicans. Moreover, 6-day-cultured hLF1-11-differentiated DCs and control (peptide-incubated) DCs that had been stimulated with a Th17-inducing mix ofantigens (including C. albicans) for 24 h were cocultured with autologous CD4+ T cells for 72 h and then the levels of IL-10, IL-17 and IFN-γ production and the percentage of cytokine-producing T cells were assessed. The results revealed that the hLF1-11-differentiated DCs induced an enhanced IL-17, but reduced IFN-γ, production by T cells as compared to control (peptide-incubated) DCs. Collectively, the hLF1-11 peptide drives monocyte-DC differentiation toward DCs that promote antifungal responses and enhance Th17 polarization.
Copyright © 2012 S. Karger AG, Basel.
Akt2 inhibits the activation of NFAT in lymphocytes by modulating calcium release from intracellular stores.
Source
Department of Medicinal Chemistry and Molecular Pharmacology and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA.
Abstract
The engagement of antigen receptors on lymphocytes leads to the activation of phospholipase C-γ, the mobilization of intracellular calcium and the activation of the NFAT transcription factor. The coupling of antigen receptors to the activation of NFAT is modulated by numerous cellular effectors including phosphoinositide 3-kinase (PI3K), which is activated following receptor cross-linking. The activation of PI3K has both positive and negative effects on the receptor-mediated activation of NFAT. An increase in the level and activity of Akt2, a target of activated PI3K, potently inhibits the subsequent activation of NFAT. In contrast, an elevation in Akt1 has no effect on signaling. Signaling pathways operating both upstream and downstream of inositol 1,4,5-trisphosphate (IP3)-stimulated calcium release from intracellular stores are unaffected by Akt2. An increase in the level of Akt2 has no significant effect on the initial amplitude, but substantially reduces the duration of calcium mobilization. The ability of Akt2 to inhibit prolonged calcium mobilization is abrogated by the administration of a cell permeable peptide that blocks the interaction between Bcl-2 and the IP3 receptor. Thus, Akt2 is a negative regulator of NFAT activation through its ability to inhibit calcium mobilization from the ER.
Copyright © 2012. Published by Elsevier Inc.
Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays.
Source
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University , Nanjing, Jiangsu 210029 , China.
Abstract
Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8(153-161) had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8(153-161) is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = - 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection ofpeptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.
The Pathway of Cross-Presentation is Influenced by the Particle Size of Phagocytosed Antigen.
Source
From Cancer Sciences Division, University of Southampton, Southampton, SO16 6YD, UK.
Abstract
Cross-presentation is the presentation by MHC class I of antigenic peptides from exogenous proteins that have been internalised and processed by professional antigen presenting cells, eg. dendritic cells (DC). We have investigated the influence of particle size and antigen load on cross-presentation following antigen delivery on microspheres (MS). Cross-presentation from small particles (0.8 μm) is sensitive to proteasome inhibition and the blockade of ER-resident MHC class I complex export, whereas cross-presentation from larger particles (aggregated clumps of 0.8 μm MS) is resistant to these antagonists. This observation may have been overlooked previously, due to the heterogeneity of particle size and MS uptake in unsorted DC populations. Whilst larger particles carry more antigen, we show that antigen load does not influence the cross-presentation pathway utilised. Whereas early endosome autoantigen 1 (EEA1) could be observed in all phagosomes, we observed endoplasmic reticulum SNARE of 24 kDa (ERS24) and cathepsin S in association with 3.0 μm and aggregated 0.8 μm MS, but not individual 0.8 μm MS. A potential mechanism underlying our observations may be the activation of β-catenin by disruption of E-cadherin-mediated adhesion. Activated β-catenin was detected in the cytoplasm of cells after phagocytosis of MS (highest levels for the largest particles). We propose that particle size can direct the use of different pathways for the cross-presentation of an identical antigen. Furthermore, these pathways have differing yields of MHC class I-peptide complexes, which is an important variable in designing vaccination strategies for maximal antigen expression and CD8(+) T cell priming.
Journal compilation © 2012 Blackwell Publishing Ltd.
Region Specific and Worldwide Distribution of Collagen-Binding M Proteins with PARF Motifs among Human Pathogenic Streptococcal Isolates.
Source
Department of Microbial Pathogenesis, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Abstract
Some of the variety of Streptococcus pyogenes and Streptococcus dysgalactiae ssp. equisimilis (SDSE) M proteins act as collagen-binding adhesins that facilitate acute infection. Moreover, their potential to trigger collagen autoimmunity has been implicated in the pathogenesis of acute rheumatic fever and attributed to a collagen-binding motif called PARF (peptide associated with rheumatic fever). For the first time we determine the rate of clinical isolates with collagen-binding M proteins that use a PARF motif (A/T/E)XYLXX(L/F)N in a defined geographic region, Vellore in South India. In this region both, incidence of streptococcal infections and prevalence of acute rheumatic fever are high. M proteins with PARF motif conferred collagen-binding activity to 3.9% of 153 S. pyogenes and 10.6% of 255 SDSE clinical isolates from Vellore. The PARF motif occurred in three S. pyogenes and 22 SDSE M protein types. In one of the S. pyogenes and five of the SDSE M proteins that contained the motif, collagen-binding was impaired, due to influences of other parts of the M protein molecule. The accumulated data on the collagen binding activity of certain M protein types allowed a reanalysis of published worldwide emm-typing data with the aim to estimate the rates of isolates that bind collagen via PARF. The results indicate that M proteins, which bind collagen via a PARF motif, are epidemiologically relevant in human infections, not only in Vellore. It is imperative to include the most relevant collagen-binding M types in vaccines. But when designing M protein based vaccines it should be considered that collagen binding motifs within the vaccineantigen remain potential risk factors.
- PMID:
- 22253902
- [PubMed - as supplied by publisher]
- PMCID: PMC3256231
Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection.
Source
Divisions of Bacteriology & Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United States of America.
Abstract
The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4-6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnosticpeptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.
Selective binding of CD4 and CD8 T-cells to antigen presenting cells for enrichment of CMV and HIV specific T-lymphocytes.
Source
Department of Pediatric Hematology and Oncology, Bambino Gesù Children Hospital, Rome, Italy.
Abstract
Adherent antigen presenting cells (APC) pulsed with protein or peptide antigens were used to capture specific CD4 or CD8 T-cells derived from established T-cell lines or from PBMC of immune subjects based on physiological interaction between TCR and MHC-peptide complex. This method could be applied independently of epitope specificity, HLA restriction alleles, activation markers and secreted cytokines, parameters required by other methods for selection of specific T cells. Non specific T-cells were removed by applying a 1g force that did not affect binding of specific T-lymphocytes. Lymphocyte selection was specific and the average recovery was 36% for CD4 T-cells. CD8 T-cells proved trickier to purify, since solid phase APC were recognized as targets for cytotoxicity. Specificity was comparable to CD4 cells, but the average recovery for CD8 cells was 26%. No residual alloreactivity was detected in expanded T-cells. Frequency and recovery of specific T-cells were comparable to other current technologies, such as generation of T-cell lines and cytokine capture method. Since antigen and IL2 are the only reagents added to the cultures, this physiological procedure can be proposed for selection and expansion of pathogen specific T-cells not only for research purposes, but also for adoptive reconstitution of immunocompromised subjects if performed under GMP conditions.
Copyright © 2012. Published by Elsevier B.V.
Allele-specific amplification of the complete HLA-C gene from genomic DNA - a novel Cw4 allele (C*04:71) with a Cw1 motif in the peptide-binding site.
Source
Laboratorio de Inmunogenética-HLA, Servicio de Inmunología, Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain Departamento de Histocompatibilidad, Centro de Transfusión de la Comunidad de Madrid, Madrid, Spain.
Abstract
To determine the complete sequence of a newly identified human leukocyte antigen (HLA)-C allele, we designed a method where the full genomic sequence of HLA-C*04 was amplified in isolation from the patient second HLA-C allele in a single polymerase chain reaction (PCR), using primers spanning its 5'- and 3'-untranslated regions. The new allele, officially designated HLA-C*04:71, differs from HLA-C*04:01:01:01 by two single-nucleotide polymorphisms: one determines substitution of phenylalanine for serine 9 at the B pocket of the peptide-binding site; the second substitution is a new polymorphism in intron 5. Phe-9 is characteristic of Cw1 alleles and its presence in C*04:71 most likely affects its peptide-binding repertoire. The principle we have used for C*04:71 isolation could be adapted for unambiguous sequence-based HLA-C typing of selected samples in a clinical setting.
© 2012 John Wiley & Sons A/S.
B Lymphocytes Treated In Vitro with Antigen Coupled to Cholera Toxin B Subunit Induce Antigen-Specific Foxp3+ Regulatory T Cells and Protect against Experimental Autoimmune Encephalomyelitis.
Source
University of Gothenburg Vaccine Research Institute, Sahlgrenska Academy at the University of Gothenburg, SE-405 30, Göteborg, Sweden.
Abstract
The ability of activated B cells to protect against various experimental autoimmune or allergic diseases makes them attractive for use in cell-based therapies. We describe an efficient way to generate B cells with strong suppressive functions by incubating naive B cells with a relevant Ag conjugated to cholera toxin B subunit (CTB). This allows most B cells, irrespective of BCR, to take up and present Ag and induces their expression of latency-associated polypeptide (LAP)/TGF-β and after adoptive transfer also their production of IL-10. With OVA as model Ag, when naive T cells were cocultured in vitro with B cells pretreated with OVA conjugated to CTB (OVA/CTB) Ag-specific CD4(+) Foxp3 regulatory T (Treg) cells increased >50-fold. These cells effectively suppressed CD25(-)CD4(+) effector T (Teff) cells in secondary cultures. Adoptive transfer of OVA/CTB-treated B cells to mice subsequently immunized with OVA in CFA induced increase in Foxp3 Treg cells together with suppression and depletion of Teff cells. Likewise, adoptive transfer of B cells pulsed with myelin oligodendrocyte glycoprotein peptide(35-55) (MOGp) conjugated to CTB increased the number of Treg cells, suppressed MOGp-specific T cell proliferation and IL-17 and IFN-γ production, and prevented the development of experimental autoimmune encephalomyelitis. Similar effects were seen when B cells were given "therapeutically" to mice with early-stage experimental autoimmune encephalomyelitis. Our results suggest that B cells pulsed in vitro with relevant Ag/CTB conjugates may be used in cell therapy to induce Ag-specific suppression of autoimmune disease.
Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis.
Source
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, Disciplina de Imunologia, Rua Botucatu, 862, 4º andar, São Paulo, 04023-900, Brazil.
Abstract
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.
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