RESUMO
Tissue transglutaminase (tTG) belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. There is a strong evidence that tTG is involved in pathology, such as neurodegenerative diseases, cancer, and celiac disease. To study physiopathological implication of tTG, a sandwich immunoassay has been developed with a new monoclonal antibody for the capture and polyclonal antibody both generated in house. Using this ready to use assay, the tTG protein level can be measured in human tissue homogenates and cells extracts easily in about 4 h. The limit of detection is 1.7 ng/ml; the coefficients of intra- and inter-assay variations range from 1 to 2 % and from 7 to 10 %, respectively. The assay is specific to tTG, and no cross reactivity with TG1, TG3, TG6, TG7, or factor XIIIa was observed. Finally, in the addition to the tTG activity assay previously developed, this assay should be a valuable tool to increase our knowledge of the tTG involvement in physiological and pathological states.
Assuntos
Anticorpos Monoclonais/química , Ensaio de Imunoadsorção Enzimática/normas , Proteínas de Ligação ao GTP/isolamento & purificação , Fígado/enzimologia , Neurônios/enzimologia , Transglutaminases/isolamento & purificação , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Cobaias , Células HEK293 , Humanos , Isoenzimas/isolamento & purificação , Fígado/química , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/química , Variações Dependentes do Observador , Proteína 2 Glutamina gama-Glutamiltransferase , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Autoantibodies detected after kidney transplantation may contribute to chronic rejection. We and others have previously described the organization of immune effectors into functional intragraft tertiary lymphoid tissue, a site where breakdown of B-cell tolerance may occur. To test this, we performed a comprehensive analysis of 26 chronically rejected kidney grafts. Antibodies were screened by indirect immunofluorescence on HEp2 cells, a procedure that detects antibodies to intracellular antigens, and monkey kidney sections, which detects kidney tissue autoantigens. The incidence of anti-HEp2 autoantibodies was significantly higher in graft explant culture supernatants than in patient sera. Reactivity against monkey kidney sections was detected in almost half of culture supernatants with anti-HEp2 autoantibodies. A local enrichment in T helper 17 and B-cell-activating factor (CD257) correlated with intragraft production of anti-HEp2 antibodies. A decrease in Tregs and a symmetric increase of activated OX40 (CD134)-expressing CD4+ T cells were found in grafts in which anti-kidney autoantibodies were produced. Thus, a stepwise breakdown of B-cell tolerance occurs within the graft during chronic rejection. Hence, the intragraft microenvironment interferes with peripheral deletion of autoreactive immature B cells that, in turn, produce antibodies against intracellular autoantigens. When intragraft immune regulation is insufficient, spreading of the local response against kidney autoantigens is favored.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim/imunologia , Rim/imunologia , Tolerância ao Transplante/imunologia , Adolescente , Adulto , Idoso , Animais , Autoanticorpos/sangue , Fator Ativador de Células B/imunologia , Fator Ativador de Células B/metabolismo , Contagem de Linfócito CD4 , Linhagem Celular Tumoral , Pré-Escolar , Citocinas/metabolismo , Feminino , Rejeição de Enxerto/sangue , Haplorrinos , Humanos , Masculino , Pessoa de Meia-Idade , Receptores OX40/metabolismo , Linfócitos T Reguladores , Células Th17 , Adulto JovemRESUMO
To evaluate the influence of intragraft inflammatory infiltrate on the course of chronic rejection, 11 human renal grafts, detransplanted for terminal failure, were analyzed. Samples were divided into two groups according to their graft survival (> or < or = 8 y). In both groups, the main cell population infiltrating the graft interstitia was T lymphocytes. The extent of the lymphocytic infiltration and the distribution of naive and memory, CD4(+) and CD8(+) T cells, were similar in both groups. Although all types of Th polarization profiles can lead to terminal chronic rejection, a correlation between shorter graft survival and the presence of Th17 cells that produce IL-17 and IL-21 was observed. In contrast, grafts infiltrated by regulatory T cells survived significantly longer. The correlation between the expressions of activation-induced cytidine deaminase (the key enzyme of the germinal center reaction) and IL-21 suggests that Th17 could exert their deleterious effect by promoting lymphoid neogenesis, namely, the organization of inflammatory effectors into ectopic germinal centers in which a local humoral immune response is elicited. Further studies will determine whether Th17 infiltration can be used as a prognosis tool and whether the Th17 subset constitutes a therapeutic target for slowing down chronic rejection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Movimento Celular/imunologia , Rejeição de Enxerto/imunologia , Interleucina-17/biossíntese , Tecido Linfoide/imunologia , Adolescente , Adulto , Idoso , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular/imunologia , Pré-Escolar , Doença Crônica , Progressão da Doença , Feminino , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Humanos , Mediadores da Inflamação/fisiologia , Interleucina-17/fisiologia , Transplante de Rim/imunologia , Transplante de Rim/patologia , Tecido Linfoide/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: HIV-1 uses cellular co-factors for virion formation and release. The virus is able to incorporate into the viral particles host cellular proteins, such as tetraspanins which could serve to facilitate HIV-1 egress. Here, we investigated the implication of several tetraspanins on HIV-1 formation and release in chronically infected T-lymphoblastic cells, a model that permits the study of the late steps of HIV-1 replication. RESULTS: Our data revealed that HIV-1 Gag and Env structural proteins co-localized with tetraspanins in the form of clusters. Co-immunoprecipitation experiments showed that Gag proteins interact, directly or indirectly, with CD81, and less with CD82, in tetraspanin-enriched microdomains composed of CD81/CD82/CD63. In addition, when HIV-1 producing cells were treated with anti-CD81 antibodies, or upon CD81 silencing by RNA interference, HIV-1 release was significantly impaired, and its infectivity was modulated. Finally, CD81 downregulation resulted in Gag redistribution at the cell surface. CONCLUSION: Our findings not only extend the notion that HIV-1 assembly can occur on tetraspanin-enriched microdomains in T cells, but also highlight a critical role for the tetraspanin CD81 on the late steps of HIV replication.
Assuntos
Antígenos CD/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Linfócitos T/virologia , Replicação Viral/fisiologia , Linhagem Celular , Regulação para Baixo , HIV-1/patogenicidade , Imunoprecipitação , Proteínas de Membrana/metabolismo , Interferência de RNA , Tetraspanina 28 , Proteínas Virais/metabolismo , Vírion/isolamento & purificação , Vírion/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Chronic rejection is one of the main causes of late allograft failure and no therapy is currently available to prevent efficiently its development. Improving the comprehension of the mechanisms involved in the pathophysiology of chronic rejection is a mandatory step to propose innovative therapies that would prolong grafts' survival. Using the rat aortic interposition model of chronic vascular rejection, we have demonstrated that the intragraft inflammatory infiltrate progressively organized itself into a functional ectopic lymphoid tissue (tertiary lymphoid organ) supporting the local synthesis of alloantibody. Thus, during chronic rejection the graft is at the same time the target and the site of elaboration of the humoral allo-immune response. This hypothesis has been confirmed in the clinical setting by the analysis of human grafts (kidneys, hearts and lungs) removed for terminal failure due to chronic rejection. This lymphoid neogenesis process, previously identified in other chronic inflammatory diseases, occurs with a strikingly high frequency in chronically rejected grafts, suggesting that an additional mechanism synergizes to initiate the development of tertiary lymphoid organs during chronic rejection. We propose that the defective lymphatic drainage of chronically rejected organs triggers lymphoid neogenesis and we discuss the complex crosstalk between lymphoid neogenesis and lymphangiogenesis that takes place during chronic rejection.
Assuntos
Rejeição de Enxerto/fisiopatologia , Linfangiogênese/fisiologia , Tecido Linfoide/fisiopatologia , Animais , Aorta Abdominal/imunologia , Aorta Abdominal/transplante , Bioprótese/efeitos adversos , Prótese Vascular/efeitos adversos , Coristoma/etiologia , Coristoma/imunologia , Coristoma/patologia , Doença Crônica , Rejeição de Enxerto/imunologia , Humanos , Complicações Intraoperatórias/imunologia , Complicações Intraoperatórias/fisiopatologia , Isoanticorpos/biossíntese , Vasos Linfáticos/lesões , Vasos Linfáticos/fisiopatologia , Ratos , Transplante Homólogo/efeitos adversos , Transplante Homólogo/imunologiaRESUMO
Owing to ordered, stage-specific T-cell receptor (TCR) gene rearrangements and cell division during T-cell development, small cohorts of "half-sibling" T cells sharing an ancestral TCR VDJbeta rearrangement but expressing different TCR alpha-locus rearrangements may be selected into the mature T-cell repertoire. We wondered whether different alphabetaTCRs expressed by T cells from the same ancestral VDJbeta cohort might be capable of recognizing the same foreign peptide-major histocompatibility complex complex (pMHC). By a combined flow cytometric and single-cell polymerase chain reaction (PCR) approach to analyze TCRs selected by the previously defined foreign antigen, pCW3170-179/H-2Kd, we were able to identify cohorts of half-sibling antigen-specific CD8 T cells after their expansion in immunized mice. We amplified residual DJbeta rearrangements as clonal markers to confirm that the shared VDJbeta sequences represent ancestral rearrangements rather than identical but independent ones. An intriguing explanation of our findings would be that only a very limited repertoire of TCR alpha-chains is selected to pair with a given TCR beta-chain during T-cell development.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linhagem da Célula/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Antígenos HLA-C/imunologia , Peptídeos/imunologia , Animais , Linhagem da Célula/imunologia , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T/genética , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/imunologia , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/imunologia , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Antígenos HLA-C/genética , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos DBARESUMO
Cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein whose roles are still widely discussed, particularly in the field of immunology. Using TgA20- and Tg33-transgenic mice overexpressing PrP(C), we investigated the consequences of this overexpression on T cell development. In both models, overexpression of PrP(C) induces strong alterations at different steps of T cell maturation. On TgA20 mice, we observed that these alterations are cell autonomous and lead to a decrease of alphabeta T cells and a concomitant increase of gammadelta T cell numbers. PrP(C) has been shown to bind and chelate copper and, interestingly, under a copper supplementation diet, TgA20 mice presented a partial restoration of the alphabeta T cell development, suggesting that PrP(C) overexpression, by chelating copper, generates an antioxidant context differentially impacting on alphabeta and gammadelta T cell lineage.