RESUMO
Type 1 diabetes mellitus is believed to be due to the autoimmune destruction of ß-cells by T lymphocytes, but a single course of rituximab, a monoclonal anti-CD20 B lymphocyte Ab, can attenuate C-peptide loss over the first year of disease. The effects of B cell depletion on disease-associated T cell responses have not been studied. We compare changes in lymphocyte subsets, T cell proliferative responses to disease-associated target Ags, and C-peptide levels of participants who did (responders) or did not (nonresponders) show signs of ß-cell preservation 1 y after rituximab therapy in a placebo-controlled TrialNet trial. Rituximab decreased B lymphocyte levels after four weekly doses of mAb. T cell proliferative responses to diabetes-associated Ags were present at baseline in 75% of anti-CD20- and 82% of placebo-treated subjects and were not different over time. However, in rituximab-treated subjects with significant C-peptide preservation at 6 mo (58%), the proliferative responses to diabetes-associated total (p = 0.032), islet-specific (p = 0.048), and neuronal autoantigens (p = 0.005) increased over the 12-mo observation period. This relationship was not seen in placebo-treated patients. We conclude that in patients with type 1 diabetes mellitus, anti-B cell mAb causes increased proliferative responses to diabetes Ags and attenuated ß-cell loss. The way in which these responses affect the disease course remains unknown.
Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Fatores Imunológicos/administração & dosagem , Células Secretoras de Insulina/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Anticorpos Monoclonais Murinos/efeitos adversos , Anticorpos Monoclonais Murinos/imunologia , Autoantígenos/imunologia , Peptídeo C/imunologia , Criança , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Humanos , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/imunologia , Células Secretoras de Insulina/patologia , Masculino , Rituximab , Subpopulações de Linfócitos T/patologia , Fatores de TempoRESUMO
T-cell development requires cytokines and intimate contact with stromal cells provided exclusively by the thymus. Consequently, an in vitro model of thymocyte differentiation, fetal thymic organ culture (FTOC), has been developed. FTOC recapitulates the normal development of T-cells derived from both mouse and human progenitor populations, providing a more rapid means to study T-cell development compared with alternative in vivo approaches. Furthermore, FTOC is easily amenable to genetic manipulation using retroviral gene transfer. In this chapter, we outline the basic FTOC technique and describe several applications, including retroviral transduction of mouse thymocyte subsets and human CD34+ stem/progenitor cells.
Assuntos
Diferenciação Celular/fisiologia , Linfopoese/fisiologia , Retroviridae , Linfócitos T/fisiologia , Timo/fisiologia , Transdução Genética/métodos , Animais , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/fisiologia , Humanos , Linfopoese/genética , Camundongos , Técnicas de Cultura de Órgãos/métodos , Timo/citologiaRESUMO
The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Central Repositories, part of the National Institutes of Health (NIH), are an important resource available to researchers and the general public. The Central Repositories house samples, genetic data, phenotypic data, and study documentation from >100 NIDDK-funded clinical studies, in areas such as diabetes, digestive disease, and liver disease research. The Central Repositories also have an exceptionally rich collection of studies related to kidney disease, including the Modification of Diet in Renal Disease landmark study and recent data from the Chronic Renal Insufficiency Cohort and CKD in Children Cohort studies. The data are carefully curated and linked to the samples from the study. The NIDDK is working to make the materials and data accessible to researchers. The Data Repositories continue to improve flexible online searching tools that help researchers identify the samples or data of interest, and NIDDK has created several different paths to access the data and samples, including some funding initiatives. Over the past several years, the Central Repositories have seen steadily increasing interest and use of the stored materials. NIDDK plans to make more collections available and do more outreach and education about use of the datasets to the nephrology research community in the future to enhance the value of this resource.
Assuntos
Pesquisa Biomédica/métodos , Bases de Dados Factuais , National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) , Nefrologia/métodos , Acesso à Informação , Comportamento Cooperativo , Mineração de Dados , Humanos , Disseminação de Informação , Comunicação Interdisciplinar , Cooperação Internacional , Estados UnidosRESUMO
We report the results of an independent laboratory's tests of novel agents to prevent or reverse type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse, BioBreeding diabetes prone (BBDP) rat, and multiple autoimmune disease prone (MAD) rat models. Methods were developed to better mimic human clinical trials, including: prescreening, randomization, blinding, and improved glycemic care of the animals. Agents were suggested by the research community in an open call for proposals, and selected for testing by an NIDDK appointed independent review panel. Agents selected for testing to prevent diabetes at later stages of progression in a rodent model were a STAT4 antagonist (DT22669), alpha1 anti-trypsin (Aralast NP), celastrol (a natural product with anti-inflammatory properties), and a Macrophage Inflammatory Factor inhibitor (ISO-092). Agents tested for reversal of established T1D in rodent models were: alpha1 anti-trypsin (Aralast NP), tolerogenic peptides (Tregitopes), and a long-acting formulation of GLP-1 (PGC-GLP-1). None of these agents were seen to prevent or reverse type 1 diabetes, while the positive control interventions were effective: anti-CD3 treatment provided disease reversal in the NOD mouse, dexamethasone prevented T1D induction in the MAD rat, and cyclosporin prevented T1D in the BBDP rat. For some tested agents, details of previous formulation, delivery, or dosing, as well as laboratory procedure, availability of reagents and experimental design, could have impacted our ability to confirm prior reports of efficacy in preclinical animal models. In addition, the testing protocols utilized here provided detection of effects in a range commonly used in placebo controlled clinical trials (for example, 50% effect size), and thus may have been underpowered to observe more limited effects. That said, we believe the results compiled here, showing good control and repeatability, confirm the feasibility of screening diverse test agents in an independent laboratory.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/prevenção & controle , Hipoglicemiantes/uso terapêutico , Triterpenos/farmacologia , Animais , Anticorpos/farmacologia , Glicemia/efeitos dos fármacos , Complexo CD3/metabolismo , Diabetes Mellitus Tipo 1/sangue , Modelos Animais de Doenças , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Humanos , Hipoglicemiantes/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Triterpenos Pentacíclicos , Ratos , Indução de Remissão , Triterpenos/uso terapêuticoRESUMO
Standardized protocols for maintaining near-normal glycemic levels in diabetic rodent models for testing therapeutic agents to treat disease are unavailable. We developed protocols for 2 common models of spontaneous type 1 diabetes, the BioBreeding diabetes-prone (BBDP) rat and nonobese diabetic (NOD) mouse. Insulin formulation, dose level, timing of dose administration, and delivery method were examined and adjusted so that glycemic levels remained within a normal range and fluctuation throughout feeding and resting cycles was minimized. Protamine zinc formulations provided the longest activity, regardless of the source of insulin. Glycemic control with few fluctuations was achieved in diabetic BBDP rats through twice-daily administration of protamine zinc insulin, and results were similar regardless of whether BBDP rats were acutely or chronically diabetic at initiation of treatment. In contrast, glycemic control could not be attained in NOD mice through administration of insulin twice daily. However, glycemic control was achieved in mice through daily administration of 0.25 U insulin through osmotic pumps. Whereas twice-daily injections of protamine zinc insulin provided glycemic control with only minor fluctuations in BBDP rats, mice required continuous delivery of insulin to prevent wide glycemic excursions. Use of these standard protocols likely will aid in the testing of agents to prevent or reverse diabetes.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Modelos Animais de Doenças , Insulina Isófana/administração & dosagem , Insulina Isófana/uso terapêutico , Análise de Variância , Animais , Glicemia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Esquema de Medicação , Índice Glicêmico , Insulina Isófana/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Ratos , Ratos Endogâmicos BBRESUMO
OBJECTIVE: In patients with long-standing type 1 diabetes, we investigated whether improved beta-cell function can be achieved by combining intensive insulin therapy with agents that may 1) promote beta-cell growth and/or limit beta-cell apoptosis and 2) weaken the anti-beta-cell autoimmunity. RESEARCH DESIGN AND METHODS: For this study, 20 individuals (mean age 39.5 +/- 11.1 years) with long-standing type 1 diabetes (21.3 +/- 10.7 years) were enrolled in this prospective open-label crossover trial. After achieving optimal blood glucose control, 16 subjects were randomized to exenatide with or without daclizumab. Endogenous insulin production was determined by repeatedly measuring serum C-peptide. RESULTS: In 85% of individuals with long-standing type 1 diabetes who were screened for participation in this trial, C-peptide levels >or=0.05 ng/ml (0.02 nmol/l) were found. Residual beta-cells responded to physiological (mixed-meal) and pharmacological (arginine) stimuli. During exenatide treatment, patients lost 4.1 +/- 2.9 kg body wt and insulin requirements declined significantly (total daily dose on exenatide 0.48 +/- 0.11 vs. 0.55 +/- 0.13 units x kg(-1) x day(-1) without exenatide; P = 0.0062). No signs of further activation of the underlying autoimmune disease were observed. Exenatide delayed gastric emptying, suppressed endogenous incretin levels, but did not increase C-peptide secretion. CONCLUSIONS: In long-standing type 1 diabetes, which remains an active autoimmune disease even decades after its onset, surviving beta-cells secrete insulin in a physiologically regulated manner. However, the combination of intensified insulin therapy, exenatide, and daclizumab did not induce improved function of these remaining beta-cells.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Imunoglobulina G/uso terapêutico , Imunossupressores/uso terapêutico , Células Secretoras de Insulina/metabolismo , Peptídeos/uso terapêutico , Peçonhas/uso terapêutico , Adulto , Idade de Início , Anticorpos Monoclonais Humanizados , Autoimunidade/efeitos dos fármacos , Estudos Cross-Over , Daclizumabe , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Quimioterapia Combinada , Exenatida , Feminino , Hemoglobinas Glicadas/metabolismo , Antígenos HLA-DR/análise , Cadeias HLA-DRB1 , Humanos , Insulina/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/imunologia , Masculino , Projetos de Pesquisa , Inquéritos e Questionários , Estados Unidos , Adulto JovemRESUMO
OBJECTIVE: Type 1 diabetes results from an immunemediated destruction of beta-cells, likely to be mediated by T lymphocytes, but the sensitivity, specificity, and other measures of validity of existing assays for islet autoreactive T-cells are not well established. Such assays are vital for monitoring responses to interventions that may modulate disease progression. RESEARCH DESIGN AND METHODS: We studied the ability of cellular assays to discriminate responses in patients with type 1 diabetes and normal control subjects in a randomized blinded study in the U.S. and U.K. We evaluated the reproducibility of these measurements overall and to individual analytes from repeat collections. RESULTS: Responses in the cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays could differentiate patients from control subjects with odds ratios of 21.7, 3.44, and 3.36, respectively, with sensitivity and specificity as high as 74 and 88%. The class II tetramer and U.S. ELISPOT assays performed less well. Despite the significant association of the responses with type 1 diabetes, the reproducibility of the measured responses, both overall and individual analytes, was relatively low. Positive samples from normal control subjects (i.e., false positives) were generally isolated to single assays. CONCLUSIONS: The cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays can distinguish responses from patients with type 1 diabetes and healthy control subjects. The limited reproducibility of the measurements overall and of responses to individual analytes may reflect the difficulty in detection of low frequency of antigen-specific T-cells or variability in their appearance in peripheral blood.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Reprodutibilidade dos Testes , Linfócitos T/imunologia , Adolescente , Adulto , Autoanticorpos/análise , Criança , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Feminino , Vetores Genéticos , Antígenos HLA-DR/genética , Teste de Histocompatibilidade , Humanos , Imunidade Celular , Immunoblotting , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Ativação Linfocitária , Masculino , Valores de Referência , Adulto JovemRESUMO
The membrane-spanning 4A (MS4A) family of proteins includes CD20, Fc epsilonRIbeta, and HTm4, whose genes are grouped in a chromosomal location that is associated with increased susceptibility to allergy and atopic asthma. One family member, Chandra/MS4a4B, was reported to be expressed in T helper 1 (Th1) T cells but not Th2 T cells. In the present study, Ms4a4b was isolated in a screen of genes differentially expressed during thymocyte development. MS4a4B was detected in immature CD4- CD8- CD44+ CD25- thymocytes, turned off during further stages of thymocyte development and reexpressed in mature single-positive thymocytes. MS4a4B expression was found in naive CD8+ and CD4+ peripheral T cells and natural killer (NK) cells but not in B cells. MS4a4B is expressed at the cell surface with its C-terminus located in the cytoplasm. When expressed in a T-cell hybridoma by retroviral vector, MS4a4B protein constitutively associated with lipid raft microdomains, whereas in primary T cells endogenous MS4a4B protein became enriched in rafts after T-cell activation. Overexpression of MS4a4B in primary CD4+ T-cell blasts enhanced T-cell receptor (TCR)-induced Th1 cytokine production. These results suggest that MS4a4B expression is tightly regulated during T-cell development and that MS4a4B expression promotes Th1 function and/or differentiation.
Assuntos
Regulação da Expressão Gênica/imunologia , Microdomínios da Membrana/imunologia , Proteínas de Membrana/genética , Células Th1/metabolismo , Animais , Antígenos CD20 , Perfilação da Expressão Gênica , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Linfócitos T/metabolismo , Timo/metabolismo , TransfecçãoRESUMO
The TLX1/HOX11 homeobox gene is frequently activated in T-cell acute lymphoblastic leukaemia (T-ALL) by the t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations or by as yet unknown transcriptional mechanisms in the absence of 10q24 cytogenetic abnormalities. Almost all TLX1(+) T-ALLs exhibit a CD4(+)CD8(+) double-positive (DP) phenotype. To investigate the role of TLX1 as an initiating oncogene in T-ALL pathogenesis, we assessed the consequences of retroviral vector-directed TLX1 expression during the differentiation of murine and human thymocytes in fetal thymic organ cultures. Interestingly, enforced expression of TLX1 disrupted the differentiation of murine fetal liver precursors and human cord blood CD34(+) stem/progenitor cells prior to the DP thymocyte stage. Although differentiation arrest was associated with an increased percentage of apoptotic thymocytes, it could only be partially bypassed by coexpression of transgenic BCL2. Mutation of the invariant asparagine residue at position 51 of the homeodomain - which is required for efficient DNA binding - released the block, consistent with the notion that TLX1 inhibits thymocyte differentiation and promotes T-cell oncogenesis by functioning as a transcription factor. The relevance of these findings is discussed in the context of activating NOTCH1 mutations and the other genetic lesions implicated in the multistep transformation process of TLX1(+) T-ALL.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Homeodomínio/genética , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas Proto-Oncogênicas/genética , Timo/imunologia , Animais , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Genes Homeobox , Genes bcl-2/imunologia , Vetores Genéticos , Proteínas de Homeodomínio/metabolismo , Humanos , Imunofenotipagem , Leucemia-Linfoma de Células T do Adulto/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Cultura de Órgãos , Proteínas Proto-Oncogênicas/metabolismo , Retroviridae/genética , Transdução GenéticaRESUMO
The pre-TCR promotes thymocyte development in the alphabeta lineage. Productive rearrangement of the TCRbeta locus triggers the assembly of the pre-TCR, which includes the pTalpha chain and CD3 epsilongammadeltazeta subunits. This complex receptor signals the up-regulation of CD4 and CD8 expression, thymocyte proliferation/survival, and the cessation of TCRbeta rearrangements (allelic exclusion). In this study, we investigate the function of two conserved tyrosine residues located in the TCRbeta chain transmembrane region of the pre-TCR. We show that replacement of both tyrosines with alanine and expression of the mutant receptor in RAG-1(null) thymocytes prevents surface expression and abolishes pre-TCR function relative to wild-type receptor. Replacement of both tyrosines with phenylalanines (YF double mutant) generates a complex phenotype in which thymocyte survival and proliferation are severely disrupted, differentiation is moderately disrupted, and allelic exclusion is unaffected. We further show that the YF double mutant receptor is expressed on the cell surface and associates with pTalpha and CD3epsilon at the same level as does wild-type TCRbeta, while association of the YF double mutant with CD3zeta is slightly reduced relative to wild type. These data demonstrate that pre-TCR signaling pathways leading to proliferation and survival, differentiation, and allelic exclusion are differently sensitive to subtle mutation-induced alterations in pre-TCR structure.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Linfócitos T/imunologia , Tirosina/fisiologia , Sequência de Aminoácidos , Animais , Diferenciação Celular , Sobrevivência Celular , Citometria de Fluxo , Genes RAG-1 , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Homologia de Sequência de Aminoácidos , Timo/imunologiaRESUMO
Natural killer (NK) inhibitory receptors, which recognize major histocompatability complex (MHC) proteins in humans, are known as killer Ig-like receptors (KIRs) and are encoded by a multi-gene immunoglobulin (Ig) superfamily. In a screen for genes differentially expressed in the mouse thymus, we discovered the first close rodent homologue of the NK receptor KIR family, which we named KIR- Like (Kirl). KIRL1 shares 40% amino acid identity with primate KIR family members, with the majority of the homology contained within the Ig-like ectodomains. KIRL1 is more similar to the KIRs than to any other known member of the Ig domain-containing leukocyte receptor superfamily. This highly significant homology suggests that the KIR family did not arise independently in primates, as has been previously suggested, but rather evolved from a primordial gene already present in the common rodent/primate ancestor. KIRL1 lacks the cytoplasmic protein motifs that mediate inhibition in KIRs (immunoregulatory tyrosine inhibiting motif, ITIM); KIRL1 also lacks the transmembrane activation signature (a conserved K residue involved in association with the immunoregulatory tyrosine activating motif-containing DAP12 molecule) found in some KIRs. Nevertheless, we hypothesize that Kirl1 is functional, for the following reasons: (1) Kirl1 mRNA is expressed at high levels in immature thymocytes; (2) Kirl1 is regulated during thymocyte development; (3) KIRL1 protein is detected in thymus. We also show that the mouse genome contains a closely related, transcribed gene, which we name Kirl2. Kirl2 encodes a KIR-like molecule with three Ig-like domains and also lacks tyrosine-based immunoregulatory motifs in its cytoplasmic region.