RESUMO
The inhibitory immunoregulatory receptor CTLA-4 is critical in maintaining self-tolerance, but the mechanisms of its actions have remained controversial. Here we examined the antigen specificity of tissue-infiltrating CD4(+) T cells in Ctla4(-/-) mice. After adoptive transfer, T cells isolated from tissues of Ctla4(-/-) mice showed T cell antigen receptor (TCR)-dependent accumulation in the tissues from which they were derived, which suggested reactivity to tissue-specific antigens. We identified the pancreas-specific enzyme PDIA2 as an autoantigen in Ctla4(-/-) mice. CTLA-4 expressed either on PDIA2-specific effector cells or on regulatory T cells was sufficient to control tissue destruction mediated by PDIA2-specific T cells. Our results demonstrate that both cell-intrinsic and non-cell-autonomous actions of CTLA-4 operate to maintain T cell tolerance to a self antigen.
Assuntos
Antígenos CD/imunologia , Autoantígenos/imunologia , Ativação Linfocitária/imunologia , Tolerância a Antígenos Próprios/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Antígeno CTLA-4 , Citometria de Fluxo , Camundongos , Camundongos Transgênicos , Isomerases de Dissulfetos de Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularisprotein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.
Assuntos
Borrelia burgdorferi/fisiologia , Ixodes/metabolismo , Doença de Lyme/transmissão , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Vetores Aracnídeos/microbiologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Imunidade Humoral , Inflamação/enzimologia , Inflamação/genética , Inflamação/metabolismo , Ixodes/enzimologia , Ixodes/genética , Proteínas de Membrana/metabolismo , Camundongos , Filogenia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Interferência de RNA , Proteínas Recombinantes , Alinhamento de Sequência , Spirochaetales/fisiologiaRESUMO
Autoimmune thyroiditis (AIT)-related brain damage is one of most severe extrathyroidal manifestations of AIT, but the mechanism remains unclear. In this study, we confirmed that protein disulfide-isomerase A3 (PDIA3) is expressed in both thyroid and brain tissues of mouse, and found the significantly increased serum levels of anti-PDIA3 antibody (PDIA3Ab) in classical mouse models of thyroiditis. In addition, we investigated the PDIA3-specific autoimmune reaction in thyroid and brain tissues in a mouse model with high-serum PDIA3Ab induced by immunization with recombinant PDIA3 protein. PDIA3-immunized mice had elevated serum thyrotropin and impaired learning and memory. PDIA3-expressing cells had IgG deposition, and IgG colocalized with C3 in the thyroid and brain tissues of PDIA3-immunized mice, resulting in membrane attack complex formation. Our results suggest that PDIA3 protein may be a common autoantigen shared by the thyroid and brain tissues and involve in the thyroidal and intracerebral damage through activating complement system.
Assuntos
Autoanticorpos/imunologia , Encéfalo/imunologia , Encefalite/imunologia , Doença de Hashimoto/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Glândula Tireoide/imunologia , Tireoidite Autoimune/imunologia , Animais , Apoptose/imunologia , Autoantígenos/imunologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Encéfalo/ultraestrutura , Modelos Animais de Doenças , Encefalite/patologia , Encefalite/fisiopatologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Doença de Hashimoto/patologia , Doença de Hashimoto/fisiopatologia , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto , Camundongos , Glândula Tireoide/patologia , Tireoidite Autoimune/patologia , Tireoidite Autoimune/fisiopatologiaRESUMO
BACKGROUND: Hemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA's immunogenicity, and many efforts have been made to stabilize the expressed HA proteins. METHODS: In this study, the protein disulfide isomerases (PDIs) were investigated for the ability to improve the stability of HA protein. Two members of the PDIs family, PDI and ERp57, were over-expressed or down-expressed in 293 T cells. The expression of H3 HA and PDIs were investigated by real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry. The stability of HA was investigated by western-blot under non-reducing condition. Moreover, BALB/c mice were immunized subcutaneously twice with the vaccine that contained HA proteins from the ERp57-overexpressed and conventional 293 T cells respectively to investigate the impact of ERp57 on the immunogenicity of H3N2 HA. RESULTS: The percentage of the disulfide-bonded HA trimers increased significantly in the PDIs-overexpressed 293 T cells, and ERp57 was more valid to the stability of HA than PDI. The knockdown of ERp57 by small interfering RNA significantly decreased the percentage of the disulfide-bonded HA trimers. HA proteins from ERp57-overexpressed 293 T cells stimulated the mice to generate significantly higher HA-specific IgG against H1N1 and H3N2 viruses than those from the conventional cells. The mice receiving H3 HA from ERp57-overexpressed 293 T cells showed the better resistance against H1N1 viruses and the higher survival rate than the mice receiving H3 HA from the conventional cells. CONCLUSION: ERp57 could improve the stability and immunogenicity of H3N2 HA.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Isomerases de Dissulfetos de Proteínas/genética , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Humanos , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H1N1/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Estabilidade Proteica , VacinaçãoRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV), a virulent pathogen of swine, suppresses the innate immune response and induces persistent infection. One mechanism used by viruses to evade the immune system is to cripple the antigen-processing machinery in monocyte-derived dendritic cells (MoDCs). In this study, we show that MoDCs infected by PRRSV express lower levels of the major histocompatibility complex (MHC)-peptide complex proteins TAP1 and ERp57 and are impaired in their ability to stimulate T cell proliferation and increase their production of CD83. Neutralization of sCD83 removes the inhibitory effects of PRRSV on MoDCs. When MoDCs are incubated with exogenously added sCD83 protein, TAP1 and ERp57 expression decreases and T lymphocyte activation is impaired. PRRSV nonstructural protein 1α (Nsp1α) enhances CD83 promoter activity. Mutations in the ZF domain of Nsp1α abolish its ability to activate the CD83 promoter. We generated recombinant PRRSVs with mutations in Nsp1α and the corresponding repaired PRRSVs. Viruses with Nsp1α mutations did not decrease levels of TAP1 and ERp57, impair the ability of MoDCs to stimulate T cell proliferation, or increase levels of sCD83. We show that the ZF domain of Nsp1α stimulates the secretion of CD83, which in turn inhibits MoDC function. Our study provides new insights into the mechanisms of immune suppression by PRRSV.IMPORTANCE PRRSV has a severe impact on the swine industry throughout the world. Understanding the mechanisms by which PRRSV infection suppresses the immune system is essential for a robust and sustainable swine industry. Here, we demonstrated that PRRSV infection manipulates MoDCs by interfering with their ability to produce proteins in the MHC-peptide complex. The virus also impairs the ability of MoDCs to stimulate cell proliferation, due in large part to the enhanced release of soluble CD83 from PRRSV-infected MoDCs. The viral nonstructural protein 1 (Nsp1) is responsible for upregulating CD83 promoter activity. Amino acids in the ZF domain of Nsp1α (L5-2A, rG45A, G48A, and L61-6A) are essential for CD83 promoter activation. Viruses with mutations at these sites no longer inhibit MoDC-mediated T cell proliferation. These findings provide novel insights into the mechanism by which the adaptive immune response is suppressed during PRRSV infection.
Assuntos
Antígenos CD/imunologia , Células Dendríticas/imunologia , Imunoglobulinas/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Linfócitos T/imunologia , Proteínas não Estruturais Virais/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Antígenos CD/genética , Proliferação de Células , Imunoglobulinas/genética , Glicoproteínas de Membrana/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Domínios Proteicos , Suínos , Proteínas não Estruturais Virais/genética , Antígeno CD83RESUMO
Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPß chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos HLA-DP/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Chaperonas Moleculares/imunologia , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/imunologia , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Calnexina/genética , Calnexina/imunologia , Calreticulina/genética , Calreticulina/imunologia , Linhagem Celular , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Chaperonas Moleculares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologiaRESUMO
Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin ß subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine ß3, and human ß1 and ß2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of ß3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant ß3 subunit. We further developed mouse anti-mouse ß3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbß3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbß3 endogenous PDI-like activity reduced αIIbß3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.
Assuntos
Cadeias beta de Integrinas/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Domínio Catalítico , Moléculas de Adesão Celular , Humanos , Camundongos , Proteínas do Tecido Nervoso , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Proteínas Recombinantes , Semaforinas , Trombose/prevenção & controleRESUMO
Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)-dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3-/- mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5-/- mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells.
Assuntos
Plaquetas/imunologia , Complemento C3/genética , Complemento C5/genética , Hemostasia/imunologia , Trombose/imunologia , Veia Cava Inferior/imunologia , Animais , Plaquetas/patologia , Ativação do Complemento , Complemento C3/metabolismo , Complemento C5/metabolismo , Armadilhas Extracelulares/genética , Armadilhas Extracelulares/imunologia , Fibrina/genética , Fibrina/imunologia , Expressão Gênica , Humanos , Hidrolases/genética , Hidrolases/imunologia , Imunidade Inata , Leucócitos/imunologia , Leucócitos/patologia , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/patologia , Fosfatidilserinas/metabolismo , Ativação Plaquetária/imunologia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Proteína-Arginina Desiminase do Tipo 4 , Tromboplastina/genética , Tromboplastina/imunologia , Trombose/sangue , Trombose/genética , Trombose/patologia , Veia Cava Inferior/metabolismo , Veia Cava Inferior/patologiaRESUMO
Changes in the T cell surface redox environment regulate critical cell functions, such as cell migration, viral entry and cytokine production. Cell surface protein disulfide isomerase (PDI) contributes to the regulation of T cell surface redox status. Cell surface PDI can be released into the extracellular milieu or can be internalized by T cells. We have found that galectin-9, a soluble lectin expressed by T cells, endothelial cells and dendritic cells, binds to and retains PDI on the cell surface. While endogenous galectin-9 is not required for basal cell surface PDI expression, exogenous galectin-9 mediated retention of cell surface PDI shifted the disulfide/thiol equilibrium on the T cell surface. O-glycans on PDI are required for galectin-9 binding, and PDI recognition appears to be specific for galectin-9, as galectin-1 and galectin-3 do not bind PDI. Galectin-9 is widely expressed by immune and endothelial cells in inflamed tissues, suggesting that T cells would be exposed to abundant galectin-9, in cis and in trans, in infectious or autoimmune conditions.
Assuntos
Membrana Celular/metabolismo , Galectina 1/metabolismo , Galectinas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Linfócitos T/metabolismo , Sítios de Ligação , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Galectina 1/genética , Galectina 3/genética , Galectina 3/metabolismo , Galectinas/antagonistas & inibidores , Galectinas/genética , Galectinas/farmacologia , Expressão Gênica , Regulação da Expressão Gênica , Glicosilação , Humanos , Modelos Moleculares , Oxirredução , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Evidence continues to increase linking autoimmunity and other complex diseases to the chemicals commonly found in our environment. Bisphenol A (BPA) is a synthetic monomer used widely in many forms, from food containers to toys, medical products and many others. The potential for BPA to participate as a triggering agent for autoimmune diseases is likely due to its known immunological influences. The goal of this research was to determine if immune reactivity to BPA has any correlation with neurological antibodies. BPA binds to a target enzyme called protein disulfide isomerase (PDI). Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are neuronal antigens that are target sites for neuroinflammation and neuroautoimmunity. We determined the co-occurrence of anti-MBP and anti-MOG antibodies with antibodies made against BPA bound to human serum albumin in 100 healthy human subjects. Correlation between BPA to PDI, BPA to MOG, BPA to MBP, PDI to MBP and PDI to MOG were all highly statistically significant (P < 0.0001). The outcome of our study suggests that immune reactivity to BPA-human serum albumin and PDI has a high degree of statistical significance with substantial correlation with both MBP and MOG antibody levels. This suggests that BPA may be a trigger for the production of antibodies against PDI, MBP and MOG. Immune reactivity to BPA bound to human tissue proteins may be a contributing factor to neurological autoimmune disorders. Further research is needed to determine the exact relationship of these antibodies with neuroautoimmunities. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd.
Assuntos
Anticorpos Bloqueadores/biossíntese , Anticorpos/metabolismo , Compostos Benzidrílicos/antagonistas & inibidores , Compostos Benzidrílicos/imunologia , Neurônios/imunologia , Fenóis/antagonistas & inibidores , Fenóis/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Adolescente , Adulto , Idoso , Anticorpos/farmacologia , Anticorpos Bloqueadores/análise , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/imunologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Humanos , Pessoa de Meia-Idade , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Glicoproteína Mielina-Oligodendrócito/biossíntese , Glicoproteína Mielina-Oligodendrócito/genética , Doenças do Sistema Nervoso/induzido quimicamente , Doenças do Sistema Nervoso/imunologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Adulto JovemRESUMO
In situ proteome labeling was carried out with 9 drug-like probes in live mammalian cells, with the corresponding cellular targets captured on microarrays and simultaneously screened using a diverse set of antibodies, revealing potential on- and off-targets.
Assuntos
Sondas Moleculares/análise , Sondas Moleculares/metabolismo , Análise Serial de Proteínas , Proteoma/análise , Proteoma/metabolismo , Anticorpos/imunologia , Células Hep G2 , Humanos , Sondas Moleculares/imunologia , Estrutura Molecular , Isomerases de Dissulfetos de Proteínas/análise , Isomerases de Dissulfetos de Proteínas/imunologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Quinases/análise , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Proteoma/imunologiaRESUMO
The TAP translocates peptide Ags into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. MHC class I acquires its peptide cargo in the peptide loading complex, an oligomeric complex that the chaperone tapasin organizes by bridging TAP to MHC class I and recruiting accessory molecules such as ERp57 and calreticulin. Three tapasin binding sites on TAP have been described, two of which are located in the N-terminal domains of TAP1 and TAP2. The third binding site is present in the core transmembrane (TM) domain of TAP1 and is used only by the unassembled subunits. Tapasin is required to promote TAP stability, but through which binding site(s) it is acting is unknown. In particular, the role of tapasin binding to the core TM domain of TAP1 single chains is mysterious because this interaction is lost upon TAP2 association. In this study, we map the respective binding site in TAP1 to the polar face of the amphipathic TM helix TM9 and identify key residues that are essential to establish the interaction. We find that this interaction is dispensable for the peptide transport function but essential to achieve full stability of human TAP1. The interaction is also required for proper heterodimerization of the transporter. Based on similar results obtained using TAP mutants that lack tapasin binding to either N-terminal domain, we conclude that all three tapasin-binding sites in TAP cooperate to achieve high transporter stability and efficient heterodimerization.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Multimerização Proteica/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação , Calreticulina/química , Calreticulina/genética , Calreticulina/imunologia , Calreticulina/metabolismo , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
The pathogenesis of glaucoma, a common neurodegenerative disease, involves an immunologic component. Changes in the natural autoantibody profile of glaucoma patients were detected, showing not only up-regulated but also down-regulated immunoreactivities. In recent studies we were able to demonstrate that the antibody changes have a large influence on protein profiles of neuroretinal cells. Furthermore we could demonstrate neuroprotective potential of one of the down-regulated antibodies (γ-synuclein antibody). Anti-GFAP antibody is another antibody found down-regulated in glaucoma patients. Since GFAP expression is intensified in glaucomatous retina, the aim of this study was to detect the effect of GFAP antibodies on neuroretinal cells. This is realized with a viability-test as well as proteomic analysis of cells incubated with GFAP antibodies. Furthermore, possible interaction partners of the GFAP antibody in neuroretinal cells were identified by western blot, mass spectrometry and indirect immunofluorescence staining. We found that the GFAP antibody is able to protect cells from oxidative stress, which is due to changed protein expressions of the actin cytoskeleton. Furthermore we detected a cross-reaction of the antibody to endoplasmic reticulum resident protein 57 on the cell membrane, which seems to lead to a changed signaling in the cells triggering the protective effects.
Assuntos
Autoanticorpos/fisiologia , Glaucoma/genética , Glaucoma/imunologia , Proteína Glial Fibrilar Ácida/imunologia , Estresse Oxidativo , Isomerases de Dissulfetos de Proteínas/imunologia , Células Ganglionares da Retina/imunologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Autoimunidade , Células Cultivadas , Reações Cruzadas , Regulação para Baixo , Proteínas do Olho/metabolismo , Humanos , Isomerases de Dissulfetos de Proteínas/genéticaRESUMO
In Leishmania species, protein disulfide isomerase (PDI) - a redox chaperone is primarily associated with virulence and survival. The precise mechanism, especially in relation to redox changes and its effects on immunological responses in visceral leishmaniasis (VL) is not completely understood as yet. Therefore, we purified a recombinant PDI from Leishmania donovani (r-LdPDI) which was of â¼15 kDa molecular size and examined its effects on immunological responses in peripheral blood (PBMC) of human VL cases. For these studies, alanine was tested as an inhibitor and was used in parallel to all experiments. This protein was identified to have a direct correlation with parasite growth which significantly increased number of promastigotes as well as axenic amastigotes after 96 h of culture. Our experiments examining the immunological response against r-LdPDI also indicate the activation of pro-L. donovani dictated immunological responses in VL. The stimulation of PBMC with r-LdPDI induced lactate dehydrogenase (LDH) activities and up regulated interleukin-10 (IL-10) production but not the HLA-DR expression, Nitric oxide (NO) release and IFN-γ production indicating a pivoted role for r-LdPDI in causing a strong immunosuppression in a susceptible host. Further, we observed that an addition of alanine in L. donovani culture offers a significant inhibition in growth of parasite and helps in reconstitution of protective immune response in VL cases. Therefore, we demonstrate a future cross talk on use of alanine which can reduce the activities of PDI of L. donovani, eliminating the parasite induced immunosuppression and inducing collateral host protective response in VL.
Assuntos
Alanina/farmacologia , Leishmania donovani/enzimologia , Leishmaniose Visceral/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Adolescente , Adulto , Feminino , Humanos , Imunidade Celular/imunologia , Fatores Imunológicos/imunologia , Terapia de Imunossupressão , Interferon gama/biossíntese , Interleucina-10/biossíntese , L-Lactato Desidrogenase , Macrófagos/imunologia , Macrófagos/parasitologia , Masculino , Óxido Nítrico/metabolismo , Adulto JovemRESUMO
STUDY QUESTION: Is there a non-invasive biomarker for the diagnosis of testicular inflammatory lesions? SUMMARY ANSWER: In sera from infertile azoospermic patients with histologically confirmed low-grade testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60) were found. WHAT IS KNOWN ALREADY: Infection and inflammation of the genital tract are supposed to be responsible for up to 15% of cases among infertile males. However, specific seminal or serological markers are not available to assess subacute or chronic inflammatory conditions in the testis. STUDY DESIGN, SIZE, DURATION: This study consisted of the identification of autoantibodies for testicular antigens in sera of patients with low-grade testicular inflammation, validation of candidates, development of an ELISA for the most promising target antigen and measurement of autoantibodies titers in healthy normozoospermic men (n = 20); male blood donors (n = 14); men with impaired semen quality without (n = 14) or with (n = 26) symptoms of genital tract infection/inflammation; azoospermic men with histologically confirmed testicular inflammatory lesions (n = 16); men after pharmacotherapy of genital tract infection/inflammation (n = 15) and men with acute epididymo-orchitis (n = 30). PARTICIPANTS/MATERIALS, SETTING, METHODS: Proteins in lysates of normal testicular tissue were separated by high-resolution 2D gel electrophoresis and probed with sera of 13 patients with histologically confirmed chronic testicular inflammation. There were 14 proteins that immunoreacted with a majority of these sera and could be identified by mass spectrometry. Of these 14 proteins, disulfide isomerase family A, member 3 (ER-60), transferrin and chaperonin containing TCP1 complex, subunit 5 (epsilon) (CCT5) were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed. MAIN RESULTS AND THE ROLE OF CHANCE: The newly established ELISA detected significantly elevated titers of autoantibodies against ER-60 in the sera from infertile men with histologically confirmed chronic testicular inflammation (median 8.6; P < 0.01) compared with the control groups. Moreover, elevated levels of anti-ER-60 titers were detected in patients suffering from acute epididymo-orchitis (median 3.3; P < 0.05) as compared with healthy normozoospermic men (median 2.13; P < 0.001), male blood donors with unknown fertility status (median 2.72; P < 0.01), patients with impaired semen quality but no infection/inflammation (median 2.59; P < 0.001) and patients with symptoms of genital tract infections and/or inflammation (median 2.18; P < 0.001). Significantly lower levels of anti-ER-60 antibodies were measured in sera from patients after application of anti-inflammatory pharmacotherapy (median 1.9; P < 0.01) compared with those with histologically confirmed chronic testicular inflammation. The cut-off value of the assay was set to 6.6 U/ml based on a calculated sensitivity of 100% and a specificity of 81.2%. LIMITATIONS, REASONS FOR CAUTION: The results obtained in this study showed statistically significant elevated titers of ER-60 antibodies in sera from patients with histologically confirmed testicular inflammatory lesions and from a few patients with acute epididymo-orchitis. However, the number of serum samples tested was limited. Severe testicular damage seen in azoospermic patients could represent a bias towards ER-60 reactivity, while the assay does not allow for different etiologies of the lesions to be distinguished. Due to ethical reasons, the prevalence of testicular inflammatory lesions among controls and non-azoospermic men cannot be studied at the histological level. WIDER IMPLICATIONS OF THE FINDINGS: Measurement of ER-60 autoantibody titers in serum could be a novel non-invasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant of the Deutsche Forschungsgemeinschaft (ME 1323/4-4) and the Translational Science Fund (Wirtschafts-und Strukturbank Hessen-WI Bank). M.F., A.P., W.W., H.-C.S. and A.M. are supported by the LOEWE focus group 'MIBIE' (Male infertility during infection and inflammation). The ER-60 ELISA is protected by a patent to the Justus-Liebig-University of Giessen with A.M. and M.F. as inventors (patent no. DE 10 2008 053 503). T.Z. as employee of the DRG Company was responsible for the ELISA development.
Assuntos
Autoanticorpos/análise , Infertilidade Masculina/diagnóstico , Inflamação/diagnóstico , Isomerases de Dissulfetos de Proteínas/imunologia , Testículo/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Azoospermia/diagnóstico , Azoospermia/imunologia , Azoospermia/patologia , Biomarcadores/análise , Humanos , Infertilidade Masculina/imunologia , Infertilidade Masculina/patologia , Inflamação/imunologia , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Análise do Sêmen , Adulto JovemRESUMO
The excretory/secretory (ES) proteins of schistosomes play important roles in modulating host immune systems and are regarded as potential vaccine candidates and drug targets. Protein disulfide isomerase (PDI) is an essential enzyme that is involved in disulfide bond formation and rearrangement. In the present study, SjPDI, a 52.8 kDa protein previously identified in a proteomics analysis as one of the ES proteins of Schistosoma japonicum, was cloned and characterized. Western blot analysis showed that recombinant SjPDI (rSjPDI) was recognized by serum from rabbits vaccinated with schistosome worm antigen. Worm protein extracts and ES protein extracts from S. japonicum could react with anti-rSjPDI mouse serum. Real-time PCR analysis indicated that SjPDI was expressed at all developmental stages tested, and a high expression level was detected in 42-day-old male worms. Immunofluorescence analysis revealed that SjPDI was mainly distributed on the tegument and parenchyma of S. japonicum worms. An enzyme-linked immunosorbent assay (ELISA) demonstrated that rSjPDI could induce a high level of rSjPDI-specific IgG antibodies. The biological activity of purified rSjPDI was confirmed by isomerization and antioxidative activity assays. The 35.32%, 26.19% reduction in the worm burden and 33.17%, 31.7% lower liver egg count were obtained in mice vaccinated with rSjPDI compared with the blank control group in two independent trials. Our preliminary results suggest that rSjPDI plays an important role in the development of the schistosome and is a potential vaccine candidate for schistosomiasis.
Assuntos
Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Isomerases de Dissulfetos de Proteínas/genética , Schistosoma japonicum/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Western Blotting , Feminino , Imunização/métodos , Imunoglobulina G/sangue , Fígado/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Filogenia , Isomerases de Dissulfetos de Proteínas/imunologia , Isomerases de Dissulfetos de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Coelhos , Distribuição Aleatória , Schistosoma japonicum/classificação , Schistosoma japonicum/genética , Schistosoma japonicum/imunologia , Alinhamento de Sequência , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
In this study we sought to better understand the role of the glycoprotein quality control machinery in the assembly of MHC class I molecules with high-affinity peptides. The lectin-like chaperone calreticulin (CRT) and the thiol oxidoreductase ERp57 participate in the final step of this process as part of the peptide-loading complex (PLC). We provide evidence for an MHC class I/CRT intermediate before PLC engagement and examine the nature of that chaperone interaction in detail. To investigate the mechanism of peptide loading and roles of individual components, we reconstituted a PLC subcomplex, excluding the Transporter Associated with Antigen Processing, from purified, recombinant proteins. ERp57 disulfide linked to the class I-specific chaperone tapasin and CRT were the minimal PLC components required for MHC class I association and peptide loading. Mutations disrupting the interaction of CRT with ERp57 or the class I glycan completely eliminated PLC activity in vitro. By using the purified system, we also provide direct evidence for a role for UDP-glucose:glycoprotein glucosyltransferase 1 in MHC class I assembly. The recombinant Drosophila enzyme reglucosylated MHC class I molecules associated with suboptimal ligands and allowed PLC reengagement and high-affinity peptide exchange. Collectively, the data indicate that CRT in the PLC enhances weak tapasin/class I interactions in a manner that is glycan-dependent and regulated by UDP-glucose:glycoprotein glucosyltransferase 1.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Polissacarídeos/imunologia , Animais , Calreticulina/genética , Calreticulina/imunologia , Calreticulina/metabolismo , Linhagem Celular , Drosophila melanogaster , Glucosiltransferases/genética , Glucosiltransferases/imunologia , Glucosiltransferases/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Polissacarídeos/genética , Polissacarídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/imunologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
The loading of antigen-derived peptides onto MHC class I molecules for presentation to cytotoxic T cells is a key process in adaptive immune defense. Loading of MHC I is achieved by a sophisticated machinery, the peptide-loading complex (PLC), which is organized around the transporter associated with antigen processing (TAP) with the help of several auxiliary proteins. As an essential adapter protein recruiting MHC I molecules to TAP, tapasin catalyzes peptide loading of MHC I. However, the exact stoichiometry and basic molecular architecture of TAP and tapasin within the PLC remains elusive. Here, we demonstrate that two tapasin molecules are assembled in the PLC, with one tapasin bound to each TAP subunit. However, one tapasin molecule bound either to TAP1 or TAP2 is sufficient for efficient MHC I antigen presentation. By specifically blocking the interaction between tapasin-MHC I complexes and the translocation complex TAP, the MHC I surface expression is impaired to the same extent as with soluble tapasin. Thus, the proximity of the peptide supplier TAP to the acceptor MHC I is crucial for antigen processing. In summary, the human PLC consists maximally of 2× tapasin-ERp57/MHC I per TAP complex, but one tapasin-ERp57/MHC I in the PLC is essential and sufficient for antigen processing.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Proteínas de Membrana Transportadoras/imunologia , Peptídeos/imunologia , Imunidade Adaptativa/imunologia , Animais , Antígenos/imunologia , Sítios de Ligação/imunologia , Citometria de Fluxo , Células HEK293 , Antígenos HLA-A/química , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Antígenos HLA-B/química , Antígenos HLA-B/imunologia , Antígenos HLA-B/metabolismo , Antígenos HLA-C/química , Antígenos HLA-C/imunologia , Antígenos HLA-C/metabolismo , Células HeLa , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Immunoblotting , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Imunológicos , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica/imunologia , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/imunologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/imunologia , Células Sf9 , Linfócitos T Citotóxicos/imunologiaRESUMO
AIM: to observe the correlation between anti-NS-1 and anti PDI antibodies against platelets function disorder on secondary dengue infection. METHODS: 50 patients with secondary DV infection according to WHO criteria were observed by a cross sectional study. Patient's blood was collected on day 3, 5 and 7 after fever onset. Platelets aggregation test was done to prove the possibility of platelets dysfunction. Anti-NS-1 and anti-PDI antibodies were determined by solid phase ELISA. RESULTS: the inhibition of platelets aggregation was increased among day of observation. Means value of inhibition on day 3 is 46.6%, day 5 is 52.5% and day 7 is 56%. There is a significant difference (p<0.05) of inhibition of platelet aggregation value between days of observation. The antibodies against NS-1 DV and PDI were detected in all 50 sera with the positive rate of 90% develop NS-1 antibodies and 72% of PDI antibodies, on day 3 of symptoms. The highest OD of NS-1 antibodies is detected on the day 3 and decreased on day 7. The OD of PDI antibodies was increased on day 3 and still increasing on day 7. There is a significant correlation between anti NS-1 and PDI antibodies (r=0.386-0.490), while the differences of OD between observation days are not significant (p>0.05). CONCLUSION: the kinetics profile of NS1 and PDI antibodies responses, which were detected by the third day of symptoms. Dengue patients' sera inhibited platelets aggregation. NS-1 antibodies and PDI antibodies might have a role on the platelets aggregation dysfunction; however, there is no correlation between them. It is possible that other mechanism involve in the inhibition of platelets aggregation.
Assuntos
Anticorpos Antivirais/farmacologia , Vírus da Dengue/imunologia , Dengue/imunologia , Agregação Plaquetária/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/imunologia , Proteínas não Estruturais Virais/imunologia , Análise de Variância , Anticorpos Antivirais/sangue , Estudos Transversais , Humanos , Fatores de TempoRESUMO
BACKGROUND: The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. RESULTS: We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. CONCLUSIONS: Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.