RESUMO
Environmental factors are the main contributors to type 1 diabetes (T1D) pathogenesis, yet they remain unidentified. Enteroviruses are proposed candidate triggers due to temporal correlations between infection and T1D autoimmunity and to detection of viral proteins in diseased islets. However, such correlations are not universal and may be relatively uncommon. Furthermore, evidence of a cause-effect relationship is lacking, as infection of non-obese diabetic mice with Coxsackie enteroviruses can either trigger or blunt disease. The proposed mechanisms are either non-antigen-specific (i.e. ß-cell destruction and release of sequestered antigens, islet inflammation) or antigen-specific (i.e. epitope mimicry, by which immune responses to enteroviruses may be diverted against homologous ß-cell antigens). The case for the latter mechanisms is even less stringent, as there is little evidence of promiscuous antigen recognition at the single T-cell level. Other infectious agents may thus be implicated. Demonstration of their role will require fulfilling the Koch's postulates, namely isolation of the agent preferentially in T1D patients, including before disease onset; and T1D induction when the agent is inoculated into mice. The same is needed for cross-reactive T cells to support epitope mimicry mechanisms. Generation of alternative (humanized) mouse models that could be challenged with candidate microbes is needed.
Assuntos
Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Infecções/imunologia , Mimetismo Molecular/fisiologia , Animais , Autoimunidade/imunologia , Meio Ambiente , Humanos , Infecções/complicações , CamundongosRESUMO
Glutamic acid decarboxylase (GAD)(65) formulated with aluminium hydroxide (GAD-alum) was effective in preserving insulin secretion in a Phase II clinical trial in children and adolescents with recent-onset type 1 diabetes. In addition, GAD-alum treated patients increased CD4(+) CD25(hi) forkhead box protein 3(+) (FoxP3(+)) cell numbers in response to in-vitro GAD(65) stimulation. We have carried out a 4-year follow-up study of 59 of the original 70 patients to investigate long-term effects on the frequency and function of regulatory T cells after GAD-alum treatment. Peripheral blood mononuclear cells were stimulated in vitro with GAD65 for 7 days and expression of regulatory T cell markers was measured by flow cytometry. Regulatory T cells (CD4(+) CD25(hi) CD127(lo)) and effector T cells (CD4(+) CD25(-) CD127(+)) were further sorted, expanded and used in suppression assays to assess regulatory T cell function after GAD-alum treatment. GAD-alum-treated patients displayed higher frequencies of in-vitro GAD(65) -induced CD4(+) CD25(+) CD127(+) as well as CD4(+) CD25(hi) CD127(lo) and CD4(+) FoxP3(+) cells compared to placebo. Moreover, GAD(65) stimulation induced a population of CD4(hi) cells consisting mainly of CD25(+) CD127(+) , which was specific of GAD-alum-treated patients (16 of 25 versus one of 25 in placebo). Assessment of suppressive function in expanded regulatory T cells revealed no difference between GAD-alum- and placebo-treated individuals. Regulatory T cell frequency did not correlate with C-peptide secretion throughout the study. In conclusion, GAD-alum treatment induced both GAD(65) -reactive CD25(+) CD127(+) and CD25(hi) CD127(lo) cells, but no difference in regulatory T cell function 4 years after GAD-alum treatment.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Glutamato Descarboxilase/administração & dosagem , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adolescente , Compostos de Alúmen/administração & dosagem , Autoantígenos/administração & dosagem , Criança , Diabetes Mellitus Tipo 1/enzimologia , Feminino , Seguimentos , Glutamato Descarboxilase/imunologia , Humanos , Terapia de Imunossupressão , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Ativação Linfocitária , Masculino , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Decades of research efforts aimed at upgrading type 1 diabetes (T1DM) treatment did not harvest much success besides improving insulin therapy, which remains the standard of care since 1922. Immunological strategies targeting autoimmune mechanisms, rather than their metabolic consequences, are highly demanded. A dealt of preclinical studies in animal models offered some promises, which were however not maintained once translated into human. All these immune intervention trials evaluated metabolic and clinical endpoints, namely C-peptide secretion, HbA(1c) and insulin requirements. While critical, we argue that these endpoints are insufficient and should be complemented with immune surrogate endpoints, i.e. biomarkers reflecting the immune modifications induced by such treatments. This is even more critical when clinical expectations are not met, in order to sort out the reasons of such failure, i.e. whether immune changes are not accomplished or whether, despite being accomplished, they are insufficient to translate into clinical benefits. Furthermore, these ancillary analyses may give precious indications to design further trials, i.e. to enroll patients with the best odds to respond to therapy and to follow-up their response.
Assuntos
Autoanticorpos/sangue , Peptídeo C/sangue , Diabetes Mellitus Tipo 1/terapia , Hemoglobinas Glicadas/metabolismo , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Animais , Biomarcadores/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , Imunoterapia , Insulina/sangue , Masculino , Camundongos , Modelos Animais , Valor Preditivo dos Testes , Prognóstico , Reprodutibilidade dos TestesRESUMO
AIMS/HYPOTHESIS: Anti-zinc transporter (ZnT)8 autoantibodies are commonly detected in type 1 diabetic patients. We hypothesised that ZnT8 is also recognised by CD8(+) T cells and aimed to identify HLA-A2 (A*02:01)-restricted epitope targets. METHODS: Candidate epitopes were selected by ZnT8 plasmid DNA immunisation of HLA-A2/DQ8 transgenic mice and tested for T cell recognition in peripheral blood mononuclear cells of type 1 diabetic, type 2 diabetic and healthy participants by IFN-γ enzyme-linked immunospot. RESULTS: White HLA-A2(+) adults (83%) and children (60%) with type 1 diabetes displayed ZnT8-reactive CD8(+) T cells that recognised a single ZnT8(186-194) (VAANIVLTV) epitope. This ZnT8(186-194)-reactive fraction accounted for 50% to 53% of total ZnT8-specific CD8(+) T cells. Another sequence, ZnT8(153-161) (VVTGVLVYL), was recognised in 20% and 25% of type 1 diabetic adults and children, respectively. Both epitopes were type 1 diabetes-specific, being marginally recognised by type 2 diabetic and healthy participants (7-12% for ZnT8(186-194), 0% for ZnT8(153-161)). CONCLUSIONS/INTERPRETATION: ZnT8-reactive CD8(+) T cells are predominantly directed against the ZnT8(186-194) epitope and are detected in a majority of type 1 diabetic patients. The exceptional immunodominance of ZnT8(186-194) may point to common environmental triggers precipitating beta cell autoimmunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte de Cátions/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Adolescente , Adulto , Animais , Autoanticorpos/genética , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte de Cátions/genética , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/genética , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Feminino , Antígeno HLA-A2/genética , Humanos , Lactente , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Transportador 8 de ZincoRESUMO
BACKGROUND: Identification of T-cell reactivity to ß-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). METHODS: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545). We also compared the readout of TMrs and PMrs with a CD8+ T-cell interferon-γ enzyme-linked immunospot assay. RESULTS: Despite the minute frequencies of autoreactive cells detected by TMrs/PMrs, most (73-77%) T1D patients had responses to one or more of the epitopes used. All four epitopes were recognized by T1D patients, with a prevalence ranging from 5 to 25%. TMrs and PMrs detected more positive responses to the ß-cell epitopes than CD8+ T-cell interferon-γ enzyme-linked immunospot. However, concordance between positive responses to TMrs and PMrs was limited. CONCLUSIONS: Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Insulina/imunologia , Precursores de Proteínas/imunologia , Adolescente , Adulto , ELISPOT , Glutamato Descarboxilase/imunologia , Antígenos HLA-A/imunologia , Humanos , Interferon gama/imunologiaRESUMO
BACKGROUND: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study. METHODS: A tetramer-based assay was used in five participating centres to measure T-cell reactivities to DR0401-restricted epitopes. Three participating centres concurrently performed ELISPOT or immunoblot assays. Each centre used blind-coded, centrally distributed peptide and tetramer reagents. RESULTS: All participating centres detected responses to auto-antigens and the positive control antigen, and in some cases cloned the corresponding T cells. However, response rates varied among centres. In total, 74% of patients were positive for at least one islet epitope. The most commonly recognized epitope was GAD270-285. Only a minority of the patients tested by tetramer and ELISPOT were concordant for both assays. CONCLUSIONS: This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Adulto , ELISPOT , Humanos , Proinsulina/imunologiaRESUMO
BACKGROUND: Type 1 diabetes (T1D) is a cell-mediated autoimmune disease characterized by destruction of the pancreatic islet cells. The use of cryopreserved cells is preferable to the use of freshly isolated cells to monitor clinical trials to decrease assay and laboratory variability. METHODS: The T-Cell Workshop Committee of the Immunology of Diabetes Society compared two widely accepted T-cell freezing protocols (warm and cold) to freshly isolated peripheral blood mononuclear cells from patients with T1D and controls in terms of recovery, viability, cell subset composition, and performance in functional assays currently in use in T1D-related research. Nine laboratories participated in the study with four different functional assays included. RESULTS: The cold freezing method yielded higher recovery and viability compared with the warm freezing method. Irrespective of freezing protocol, B cells and CD8+ T cells were enriched, monocyte fraction decreased, and islet antigen-reactive responses were lower in frozen versus fresh cells. However, these results need to take in to account that the overall response to islet autoantigens was low in some assays. CONCLUSIONS: In the current study, none of the tested T-cell functional assays performed well using frozen samples. More research is required to identify a freezing method and a T-cell functional assay that will produce responses in patients with T1D comparable to responses using fresh peripheral blood mononuclear cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Criopreservação/métodos , Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Sobrevivência Celular , Humanos , Proinsulina/imunologiaRESUMO
Autoimmune T cell responses directed against insulin-producing ß cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D 'immune staging' and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen-specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials.
Assuntos
Preservação de Sangue/métodos , Separação Celular/métodos , Criopreservação/métodos , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Superfície/imunologia , Doenças Autoimunes/imunologia , Preservação de Sangue/normas , Separação Celular/normas , Ensaios Clínicos como Assunto , Criopreservação/normas , Humanos , Infecções/imunologia , Camundongos , Neoplasias/imunologiaRESUMO
Type 1 diabetes (T1D) is an autoimmune disease caused by the T cell-mediated destruction of the pancreatic insulin-producing beta cells. Currently there are no widely accepted and standardized assays available to analyse the function of autoreactive T cells involved in T1D. The development of such an assay would greatly aid efforts to understand the pathogenesis of T1D and is also urgently required to guide the development of antigen-based therapies intended to prevent, or cure, T1D. Here we describe some of the assays used currently to detect autoreactive T cells in human blood and review critically their strengths and weaknesses. The challenges and future prospects for the T cell assays are discussed.
Assuntos
Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Testes Imunológicos/métodos , Ilhotas Pancreáticas/imunologia , Linfócitos T/imunologia , Humanos , Ativação Linfocitária , Linfócitos T/citologiaRESUMO
AIMS/HYPOTHESIS: Displaying immunomodulatory capacities, mesenchymal stem cells (MSCs) are considered as beneficial agents for autoimmune diseases. The aim of this study was to examine the ability of MSCs to prevent autoimmune diabetes in the NOD mouse model. METHODS: Prevention of spontaneous insulitis or of diabetes was evaluated after a single i.v. injection of MSCs in 4-week-old female NOD mice, or following the co-injection of MSCs and diabetogenic T cells in irradiated male NOD recipients, respectively. The frequency of CD4(+)FOXP3(+) cells and Foxp3 mRNA levels in the spleen of male NOD recipients were also quantified. In vivo cell homing was assessed by monitoring 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T cells or MSCs. In vitro, cell proliferation and cytokine production were assessed by adding graded doses of irradiated MSCs to insulin B9-23 peptide-specific T cell lines in the presence of irradiated splenocytes pulsed with the peptide. RESULTS: MSCs reduced the capacity of diabetogenic T cells to infiltrate pancreatic islets and to transfer diabetes. This protective effect was not associated with the modification of diabetogenic T cell homing, but correlated with a preferential migration of MSCs to pancreatic lymph nodes. While injection of diabetogenic T cells resulted in a decrease in levels of FOXP3(+) regulatory T cells, this decrease was inhibited by MSC co-transfer. Moreover, MSCs were able to suppress both allogeneic and insulin-specific proliferative responses in vitro. This suppressive effect was associated with the induction of IL10-secreting FOXP3(+) T cells. CONCLUSIONS/INTERPRETATION: MSCs prevent autoimmune beta cell destruction and subsequent diabetes by inducing regulatory T cells. MSCs may thus offer a novel cell-based approach for the prevention of autoimmune diabetes and for islet cell transplantation.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Estado Pré-Diabético/terapia , Linfócitos T Reguladores/citologia , Animais , Movimento Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Células Secretoras de Insulina/imunologia , Interleucina-10/metabolismo , Teste de Cultura Mista de Linfócitos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Estado Pré-Diabético/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
A 30-year-old female presented with uncontrolled hypertension due to arteriovenous malformation in the upper third of the right kidney, which worsened during pregnancy. The arteriovenous malformation was detected by color-coded Doppler sonography, confirmed by angiography, and the fistula was sealed by superselective arterial embolization with metallic coils. Superselective embolization is the most effective and safe treatment for this rare and complex pathology.
Assuntos
Fístula Arteriovenosa/complicações , Fístula Arteriovenosa/terapia , Embolização Terapêutica , Hipertensão/terapia , Complicações Cardiovasculares na Gravidez/terapia , Artéria Renal/anormalidades , Veias Renais/anormalidades , Adulto , Feminino , Humanos , Hipertensão/etiologia , GravidezRESUMO
OBJECTIVE: To determine whether CD38 loss in benign and malignant prostatic disease is related to human leukocyte antigen (HLA)-DR up-regulation, by assessing the histopathology of the prostate and the effect of androgen deprivation. MATERIALS AND METHODS: Serial sections of frozen fetal (eight), infant (six), normal adult (10), benign hyperplastic (BPH, 24), and primary (10) and hormone-treated (11) carcinomatous human prostatic tissues were analysed by immunohistology for anti-CD38 and HLA-DR antigens. RESULTS: In BPH samples there was a significant correlation between CD38 loss (mean 21% of acini) and HLA-DR up-regulation (mean 20%; P < 0.001). Moreover, 76% of all CD38-negative acini in BPH had HLA-DR up-regulation in the same prostate epithelial cells, predominantly in atrophic and cystic glands, and in cells with retained secretions (74%). In contrast to the uniform expression in normal adult prostate, CD38 was negative or partly expressed in fetal acini (mean 19%) and almost completely negative in acini of the early infant period (mean 0.7%). In contrast to BPH, cancer cells did not selectively up-regulate HLA-DR when CD38 was lost. In patients with cancer treated by androgen deprivation, cancer cells were CD38-negative. CONCLUSIONS: The absence of CD38 and presence of HLA-DR expression in prostatic epithelium is consistent in BPH and tissue surrounding tumour, and strongly related to gland atrophy. This is particularly interesting as HLA-DR triggering can induce apoptosis of cells, whereas CD38 prevents it. A permissive role for androgens to maintain full CD38 expression in epithelial cells is suggested.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Antígenos HLA/metabolismo , Próstata/embriologia , Hiperplasia Prostática/imunologia , ADP-Ribosil Ciclase 1 , Adolescente , Adulto , Androgênios/metabolismo , Atrofia/metabolismo , Criança , Pré-Escolar , Humanos , Immunoblotting , Lactente , Recém-Nascido , Masculino , Glicoproteínas de Membrana , Próstata/patologia , Prostatite/metabolismo , Regulação para CimaRESUMO
AIMS/HYPOTHESIS: Serum anti-CD38 autoantibodies (aAbs) have been reported in 17 to 19% of patients with long-standing Type I (insulin-dependent) diabetes mellitus and Type II (non-insulin-dependent) diabetes mellitus. Whether these aAbs are also found in new-onset Type I diabetes and in Latent Autoimmune Diabetes in Adults (LADA) is not known, as is their relationship with conventional islet aAbs. METHODS: These issues were addressed by studying new-onset Type I and LADA diabetic cohorts with a recently developed anti-CD38 enzymatic immuno-assay. RESULTS: Anti-CD38 aAb prevalence among new-onset Type I patients (3.8%) was lower than previously found in long-standing Type I diabetes (11.7%, as defined with the 97.5 percentile cutoff; p=0.01), suggesting a late appearance of these aAbs. Among LADA patients, 14.9% were anti-CD38(+). Anti-CD38 were only associated with anti-GAD aAbs in new-onset Type I diabetes. Although the CD38 target molecule was expressed in human pancreatic islets, anti-CD38 aAbs did not contribute to the islet cell antibody (ICA) immunofluorescence reactivity. All the positive sera analysed for Ca(2+) release were found to mobilise it. In agreement with these agonistic features, anti-CD38(+) new-onset Type I patients showed higher fasting C-peptide values as compared to negative counterparts; the association was stronger when the analysis was limited to the agonistic anti-CD38(+) sera. A similar trend was found among LADA patients. CONCLUSION/INTERPRETATION: Anti-CD38 aAbs are distinct markers of islet autoimmunity which are more prevalent in long-standing disease, as opposed to the other known islet aAbs. Their in vitro agonistic properties could be operating in vivo as well, as they identify sub-groups of patients with higher residual beta-cell function.
Assuntos
ADP-Ribosil Ciclase/imunologia , Antígenos CD/imunologia , Autoanticorpos/análise , Diabetes Mellitus Tipo 1/imunologia , ADP-Ribosil Ciclase 1 , Adolescente , Idoso , Peptídeo C/sangue , Cálcio/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: Search for an ideal responder T-lymphocyte source for adoptive T-lymphocyte therapy in renal cell carcinoma (RCC). METHODS: Cytotoxic T-lymphocyte (CTL) activity of (a) normal, tumor-distant, renal T lymphocytes, (b) tumor-infiltrating T lymphocytes and (c) peripheral blood T lymphocytes against autologous tumor epithelial cells (EC) of 10 patients with organ-confined, primary RCC was analyzed in a primary CTL assay. Freshly enriched T lymphocytes were cultured with or without autologous, mitomycin-C-treated normal or tumor EC in the presence or absence of antigen-presenting cells (APC) for 7 days. RESULTS: Both tissue T-lymphocyte populations displayed a similar CD4:CD8 ratio (1:1). Elevated CD62L coexpression of CD4+ T lymphocytes in normal, tumor-distant, renal tissue resulted in a significantly higher transient T-cell activation than that seen in renal tumor tissue (46 vs. 27%; p = 0.002). All trials to induce significant lysis of autologous, renal tumor EC in tumor-infiltrating and peripheral blood T lymphocytes failed. Only when normal, tumor-distant, renal T lymphocytes were stimulated by autologous APC and tumor EC was significant autologous tumor EC lysis obtained (mean 14%; p<0.05). Costimulation by anti-CD3 (mean 21%; p<0.05) or interleukin-2 (mean 31%; p<0.05) further increased tumor EC lysis significantly. CONCLUSIONS: Increased turnover of T lymphocytes in normal, tumor-distant, renal tissue was associated with a higher yield of pre-CTL which can be transformed into a functionally active effector T-cell pool by stimulation via antigen plus APC. Thus, tumor-distant renal tissue has to be included in the tissue-sampling procedure for adoptive immunotherapy.
Assuntos
Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T Citotóxicos/imunologia , Células Apresentadoras de Antígenos/imunologia , Apoptose , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/terapia , Humanos , Imunoterapia , Neoplasias Renais/genética , Neoplasias Renais/terapia , Células Tumorais CultivadasRESUMO
Insulin secretion is one of the functions mediated by CD38, a nonlineage pleiotropic cell surface receptor. The molecule is the target of an autoimmune response, because serum autoantibodies (aAbs) to CD38 have been detected in diabetic patients. In the healthy Caucasian population, the CD38 gene is bi-allelic (86% CD38*B and 14% CD38*A), whereas an Arg140Trp mutation has been identified in Japanese diabetic patients. We investigated the relationship between CD38 and diabetes in Caucasian patients by characterizing anti-CD38 aAbs in terms of prevalence and function (agonistic/nonagonistic activity) and by exploring the potential influence of the CD38 genetic background. A novel enzymatic immunoassay, using recombinant soluble CD38 as the target antigen, was developed for the analysis of anti-CD38 aAb titers. Sera from 19.15% of type 1 and 16.67% of type 2 diabetic patients were positive. The majority of anti-CD38 aAbs (57.14%) displayed agonistic properties, i.e., they demonstrated the capability to trigger Ca2+ release in lymphocytic cell lines. In agreement with these functional features, the presence of anti-CD38 aAbs in type 2 diabetic patients was associated with significantly higher levels of fasting plasma C-peptide and insulin, as compared with anti-CD38-counterparts. No diabetic subject carrying the Arg140Trp mutation and no preferential association between diabetes or aAb status and the CD38*A allele was found in the study population. These results show the significance of anti-CD38 aAbs as a new diagnostic marker of beta-cell autoimmunity in diabetes. Moreover, the prevalent agonistic activity of these aAbs suggests that they could mediate relevant effects on target cells by means of Ca2+ mobilization.
Assuntos
Antígenos CD , Antígenos de Diferenciação/imunologia , Autoanticorpos/análise , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , NAD+ Nucleosidase/imunologia , População Branca/genética , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Idoso , Antígenos de Diferenciação/genética , Autoanticorpos/fisiologia , Cálcio/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-Idade , NAD+ Nucleosidase/genética , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Human CD38 is a signal transduction molecule, and, concurrently, an ectoenzyme catalyzing the synthesis and degradation of cyclic ADP-ribose (cADPR), a potent Ca2+ mobilizer. One facet of CD38 that has not yet been addressed is its role in NK cells. To this end, the events triggered by CD38 ligation with agonistic mAb were analyzed on freshly purified human NK cells. Ligation was followed by (i) a significant rise in the intracellular level of Ca2+, (ii) increased expression of HLA class II and CD25, and (iii) tyrosine phosphorylation of discrete cytoplasmic substrates. The phosphorylation cascade involved CD3-zeta and FcepsilonRIgamma chains, zeta-associated protein (ZAP)-70 and the proto-oncogene product c-Cbl. NK effector functions were then analyzed: CD38 signaling was able (iv) to induce release of IFN-gamma and, more prominently, of granulocyte macrophage colony stimulating factor, as assessed by measuring both mRNA and protein products; and, lastly, (v) to induce cytolytic effector functions on target cells after IL-2 activation, as shown both by cytotoxicity assays and ultrastructural changes. The tyrosine-phosphorylated substrates and all the effects mediated by CD38 were similar to those observed following triggering via CD16 (FcgammaRIIIA); moreover, Ca2+ mobilization via CD38 no longer operated in NK-derived cell lines lacking CD16. These results suggest that the activation signals transduced by CD38 in NK cells elicit relevant cellular events. The effects are similar to those elicited via CD16 and possibly rely on common signaling pathways.
Assuntos
Antígenos CD , Antígenos de Diferenciação/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Complexo CD3/análise , Cálcio/metabolismo , Citotoxicidade Imunológica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Interferon gama/análise , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Glicoproteínas de Membrana , NAD+ Nucleosidase/imunologia , Fosforilação , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/análise , RNA Mensageiro/análise , Receptores de IgE/análise , Receptores de IgG/imunologia , Receptores de Interleucina-2/análise , Transdução de Sinais , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70RESUMO
CD38, a nonlineage-restricted surface glycoprotein, is an ecto-enzyme (ADP ribosyl cyclase/cADPR hydrolase/EC 3.2.2.6) that regulates cytoplasmic Ca2+ and cell-cell interactions. The molecule also delivers trans-membrane signals, despite a structural ineptitude to the scope. To reconcile these issues in a unitarian model, we compared the effects of CD38 signaling in circulating and residential T lymphocytes, the latter represented by those colonizing the intestinal lamina propria. Results are as follows: 1) LP T cells express an enzymatically active form of CD38, characterized by a modified ratio between cyclase and hydrolase functions; 2) LP T cells do not mobilize Ca2+ upon CD38 ligation, as seen in PB T cells (this condition is due to a lack in activation of PLC- g, constantly observed in PB T lymphocytes); 3) The early steps of CD38 signaling involve activation of lck, syk, and LAT; 4) Late events include synthesis and release of IL-2, IL-4, IL-5, IL-10, IFN-g and GM-CSF; 5) The uniqueness of the CD38 pathway in LP T cells is not caused by impaired interactions with the CD31 ligand. The differences observed concern the signaling machinery that CD38 exploits for its own use and not the interplay with its ligand.
Assuntos
Antígenos CD , Antígenos de Diferenciação/metabolismo , Cálcio/metabolismo , Mucosa/metabolismo , NAD+ Nucleosidase/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Anticorpos Monoclonais/metabolismo , Antígenos de Diferenciação/genética , Western Blotting , Separação Celular , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Isoenzimas/metabolismo , Células Jurkat , Glicoproteínas de Membrana , Modelos Biológicos , Mucosa/citologia , Complexos Multienzimáticos , NAD+ Nucleosidase/genética , Fosfolipase C gama , Fosforilação , Testes de Precipitina , Fosfolipases Tipo C/metabolismoRESUMO
During the last three decades, research focused on cancer treatment has led to the development of many cytotoxic agents. Despite the fact that these efforts have significantly improved the prognosis of certain malignancies such as some lymphomas, leukemias and testicular carcinomas, other tumors such as ovarian, lung and metastatic breast cancer are still associated with a poor prognosis. An innovative approach has recently emerged, thanks to a better understanding of tumor cell biology and many efforts are aimed at finding compounds capable of restoring a more differentiated phenotype to tumor cells, thereby reducing the tumor's aggressiveness and ultimately reverting it to its normal counterpart [1, 2]. Retinoids are the prototype of this new therapeutical approach called "differentiation therapy".
Assuntos
Anticarcinógenos/uso terapêutico , Neoplasias da Mama/prevenção & controle , Retinoides/uso terapêutico , Animais , Feminino , Humanos , RatosAssuntos
Antígenos CD , Antígenos de Diferenciação/fisiologia , Leucócitos/fisiologia , Complexos Multienzimáticos/fisiologia , NAD+ Nucleosidase/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Transdução de Sinais , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adesão Celular , Humanos , Glicoproteínas de MembranaRESUMO
This work analyzes the hypothesis that human CD38 may cooperate with MHC Class II by acting as coreceptor in a superantigen-induced activation. The initial evidence is that CD38 ligation by specific monoclonal antibodies inhibits superantigen-induced T lymphocyte proliferation. Inhibitory effects become apparent after engagement of CD38 expressed by monocytes, whereas ligation of CD38 expressed by T lymphocytes does not apparently affect activation. The inhibition requires a cell-to-cell interaction, followed by the relevant transmembrane signaling that is reproduced by CD38 ligation. Indeed, CD38 ligation on monocytes induces tyrosine phosphorylation of several intracellular proteins including the protooncogene product c-cbl and the fgr and hck tyrosine kinases. The receptorial nature of the CD38-mediated events is confirmed by the observation of an intracellular calcium flux in monocytes secondary to CD38 ligation. These effects are additive with the similar events elicited by MHC Class II ligation, a likely indication that CD38 and MHC Class II share a common activation pathway. This conclusion is strengthened by results of comodulation experiments, indicating that CD38 and MHC Class II display lateral associations on monocytes. These results attribute to CD38 expressed by monocytes a role in the transduction of signal(s) involved in superantigen-induced activation, operating in synergy with MHC Class II.