RESUMEN
As most autoimmune diseases, inherited predisposition to Graves' disease (GD) is polygenic with the main contributory genes being located in the HLA region. Also, as in other autoimmune diseases, family linkage, candidate gene association, and GWAS studies have identified an expanding number of predisposing genes (CTLA4, CD40, PTPN22...) and 2 of them, TG and TSHR, are thyroid specific. In spite of this expanding number of associated genes, it has been estimated that all together they account for only a 20% of the heritability of GD. TSHR is of special interest as it codes for the target of TSHR stimulating antibodies (TSAbs), which are unequivocally pathogenic and an exception in autoimmunity by being stimulating rather than neutral, blocking, or cytotoxic. This is surprising because the generation of stimulating TSHR antibodies by immunisation of laboratory animals has been remarkably difficult, suggesting an underlying mechanism that favours stimulating over neutral or blocking anti-TSHR antibodies must be operating in GD patients. Besides, after HLA, TSHR is the gene most tightly associated to GD. The TSHR polymorphisms conferring susceptibility are located in the unusually large intron 1. Two mechanisms have been already put forward to explain its association with GD. According to one, the risk alleles determine an increase in the expression of TSHR mRNA splice variants that code for a soluble form of the receptor. The wider distribution of soluble TSHR would favour its immunogenicity and the development of an autoimmune response to it. It does not explain why it becomes immunogenic, as immunogenicity and distribution are not necessarily connected, nor why the immune response focus to the production of stimulating antibodies. According to the second mechanism proposed, the risk alleles determine a lower TSHR expression in the thymus and this would favour the escape of more TSHR reactive T cells, that is, central tolerance failure. The unexpected finding that thymocytes express TSHR and that TSAbs stimulate them lead to postulate that this would accelerate their egress from the thymus and a less efficient deletion of the TSHR self-reactive T cells. It can be envisaged that these autoreactive T cells may enhance the production of TSHR-Abs in the germinal centres of the thyroid draining lymph nodes, especially of those capable of further stimulating the egress of autoreactive T cells from the thymus. This mechanism, which does not exclude the former, provides and insight of the way in which TSAbs are favoured over neutral or blocking antibodies. Finally this would explain the frequent finding of thymic hyperplasia in GD patients.
Asunto(s)
Enfermedad de Graves/genética , Receptores de Tirotropina/genética , Animales , Predisposición Genética a la Enfermedad , Humanos , Sinapsis Inmunológicas/metabolismo , Linfocitos T Reguladores/inmunología , Timo/metabolismoRESUMEN
Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3) is a genetic disorder caused by mutations in UNC13D gene, coding the granule priming factor Munc13-4 that intervenes in NK and T cell cytotoxic function. Here we report the case of a 17-month-old girl with prolonged symptomatic EBV infectious mononucleosis and clinical symptoms of hemophagocytic syndrome. In vitro functional analysis pointed to a degranulation defect. The genetic analysis of UNC13D gene identified initially a heterozygous mutation (c.753+1G>T) in the donor splice-site that resulted in exon 9 skipping (maternal allele). Mutations in other genes were considered, but additional analysis of UNC13D cDNA revealed in the paternal allele a heterozygous transition from G to A (c.2448-13G>A) at the 3' acceptor splice-site in intron 25, generating a new acceptor splice-site that leads to a frameshift and a premature STOP codon. Allele specific amplification of the cDNA confirmed the absence of a functional mRNA from the paternal allele. This case illustrates an atypical compound heterozygous UNC13D mutation affecting the RNA splicing that generates a typical FHL3 phenotype.
Asunto(s)
Infecciones por Virus de Epstein-Barr/complicaciones , Linfohistiocitosis Hemofagocítica/genética , Proteínas de la Membrana/genética , Mutación , Secuencia de Bases , Codón sin Sentido , Análisis Mutacional de ADN , Femenino , Mutación del Sistema de Lectura , Heterocigoto , Humanos , Lactante , Linfohistiocitosis Hemofagocítica/etiología , Proteínas de la Membrana/química , Modelos Moleculares , Mutación Puntual , Estructura Terciaria de Proteína , Sitios de Empalme de ARN/genéticaRESUMEN
Celiac disease (CD) is a complex autoimmune disorder caused by ingestion of gluten in genetically susceptible individuals. Different genetic risk factors have been identified, but virtually all patients are human leukocyte antigen (HLA)-DQ2 and/or HLA-DQ8 positive. We describe a new, fast, accurate and simple real-time polymerase chain reaction (PCR)-based assay for the genotyping and homozygosity analysis of the CD-related HLA alleles. The assay overcomes the major limitations of protocols currently in use, allowing HLA-DQ2/DQ8 genotyping by using only three real-time PCR reactions. For the appraisal of DQ2 homozygosity, only one more reaction is needed. These reactions are easily automated and suitable for large screening studies in diagnostic procedures, as it is demonstrated by their successful application in our HLA diagnostic laboratory. Finally, we assessed the clinical relevance of this real-time PCR-based assay by studying a cohort of fully characterized patients. As expected, all CD patients had at least one of the CD-associated alleles, and the highest CD risk was indicated by the presence of the HLA-DQ2.5 heterodimer (HLA-DQA1*05-DQB1*02) with HLA-DQB1*02 in homozygosity.
Asunto(s)
Alelos , Enfermedad Celíaca/genética , Antígenos HLA-DQ/genética , Cadenas beta de HLA-DQ/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Enfermedad Celíaca/epidemiología , Femenino , Homocigoto , Humanos , Masculino , Factores de Riesgo , España/epidemiologíaRESUMEN
BACKGROUND: The use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (Good Manufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy. METHODS: Tolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-α, IL-ß and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced. RESULTS: Differences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-γ) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-γ production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells. CONCLUSIONS: Our results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.
Asunto(s)
Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Antiinflamatorios/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colecalciferol/farmacología , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Dexametasona/farmacología , Factores de Transcripción Forkhead/metabolismo , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-10/biosíntesis , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Fenotipo , Sirolimus/farmacología , Células TH1/citología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
BACKGROUND: The identification of major immunogenic peptides in multiple sclerosis (MS) is of great importance for the development of antigen-specific therapies. Cellular reactivity against a selected mix of seven myelin peptides was evaluated in vitro. The evolution of this reactivity over time and its correlation with clinical variables was also analysed. MATERIAL AND METHODS: Forty-two patients with MS, 15 with other demyelinating diseases and 40 healthy donors (HD) were studied. Cell proliferation was measured by 3[H] thymidine incorporation into samples obtained at 0, 3, 6 and 12months of MS patient follow-up. RESULTS: A positive reaction to the peptide mix was detected in 31 of the 42 patients (74%), 12 of the 40 HD (30%) and 6 of the 15 (40%) patients with other demyelinating diseases. Patients with positive proliferation had greater disability (EDSS score, 3 [1-5.5] vs. 1.0[1-2], P=0.021), higher number of relapses (7±4.1 vs. 3±1.2, P<0.001) and shorter time since the last relapse (9±7.5 vs. 32±12.3months, P=0.036). After 12months of follow-up, cell reactivity was maintained in 33 patients (78%). CONCLUSION: A high percentage of patients exhibit a significant and maintained reactivity to myelin peptides over time. Therefore, this mix may be useful as a source of antigen in the development of protocols aimed at inducing specific tolerance in MS.
Asunto(s)
Proliferación Celular , Epítopos de Linfocito T/inmunología , Inmunoterapia/métodos , Activación de Linfocitos/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Proteínas de la Mielina/uso terapéutico , Fragmentos de Péptidos/fisiología , Adulto , Modulación Antigénica/inmunología , Femenino , Humanos , Tolerancia Inmunológica , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/patología , Esclerosis Múltiple Recurrente-Remitente/terapia , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Dendritic cells (DCs) are powerful antigen-presenting cells capable of maintaining peripheral tolerance. The possibility to generate tolerogenic DCs opens new therapeutic approaches in the prevention or remission of autoimmunity. There is currently no treatment inducing long-term tolerance and remission in type 1 diabetes (T1D), a disease caused by autoimmunity towards beta cells. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow islet regeneration. Apoptotic cells--a source of autoantigens--are cleared rapidly by macrophages and DCs through an immunologically silent process that contributes to maintaining tolerance. Our aims were to prevent T1D and to evaluate the re-establishment of peripheral tolerance using autologous DCs pulsed in vitro with apoptotic bodies from beta cells. Immature DCs derived from bone marrow of non-obese diabetic (NOD) mice were obtained and pulsed with antigen-specific apoptotic bodies from the beta cell line NIT-1. Those DCs that phagocytosed apoptotic cells diminished the expression of co-stimulatory molecules CD40 and CD86 and reduced secretion of proinflammatory cytokines. Moreover, these cells were resistant to increase the expression of co-stimulatory molecules after lipopolysaccharide activation. The administration of these cells to NOD transgenic mice expressing interferon-beta in their insulin-producing cells, a model of accelerated autoimmune diabetes, decreased diabetes incidence significantly and correlated positively with insulitis reduction. DCs pulsed with apoptotic cells that express disease-associated antigens constitutes a promising strategy to prevent T1D.
Asunto(s)
Apoptosis/inmunología , Autoantígenos/inmunología , Células Dendríticas , Diabetes Mellitus Tipo 1/prevención & control , Tolerancia Inmunológica/inmunología , Inmunoterapia/métodos , Células Secretoras de Insulina/inmunología , Animales , Autoantígenos/administración & dosificación , Células Cultivadas , Vesículas Citoplasmáticas/inmunología , Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Endocitosis , Epítopos , Femenino , Células Secretoras de Insulina/patología , Interferón beta/genética , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Organismos Libres de Patógenos EspecíficosRESUMEN
Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing beta cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non-classical class I human leucocyte antigen and leucocyte immunoglobulin-like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C-C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long-standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Perfilación de la Expresión Génica , Islotes Pancreáticos/metabolismo , Páncreas/metabolismo , Páncreas/patología , Adolescente , Adulto , Antígenos CD/análisis , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Recuento de Células , Diabetes Mellitus Tipo 1/inmunología , Regulación hacia Abajo/genética , Femenino , Expresión Génica/genética , Células Secretoras de Glucagón/metabolismo , Antígenos HLA/genética , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunidad Innata/genética , Inflamación/genética , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/patología , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Asociadas a Pancreatitis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/genética , Adulto Joven , Antígenos HLA-ERESUMEN
Lung transplantation (LT) has become an accepted therapy for selected patients with advanced lung disease. One of the main limitations to successful LT is rejection of the transplanted organ where chemokines are pivotal mediators. Here, we test the relationship between copy number variation (CNV) in the CCL4L chemokine gene and rejection risk in LT patients (n=161). Patients with no acute rejection showed a significantly lower mean number of CCL4L copies than patients that showed acute rejection (1.66 vs 1.96, P=0.014), with an even greater number of gene copies seen in patients with more than one episode of acute rejection (1.66 vs 2.30, P=0.001). Additionally, patients with > or =2 CCL4L copies had a significantly higher risk of acute rejection compared with patients that had 0-1 CCL4L copies (odds ratio 2.65; 95% confidence interval, 1.33-5.28; P=0.0046). A combined analysis of CCL4L CNV and the rs4796195 CCL4L single nucleotide polymorphism demonstrated that the effect of CCL4L copy number in acute rejection is mainly because of the number of copies of the CCL4L1 allelic variant. This finding constitutes the first report of CNV as a correlate factor in allograft rejection.
Asunto(s)
Quimiocina CCL4/genética , Rechazo de Injerto/genética , Trasplante de Pulmón , Enfermedad Aguda , Enfermedad Crónica , Femenino , Dosificación de Gen/genética , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The recent description of a large amount of copy number variation (CNV) in the human genome has extended the concept of genome diversity. In this study we integrate the analysis of CNV and single nucleotide polymorphisms (SNPs) in the human CCL4L chemokine gene. CCL4L is a nonallelic copy of CCL4/MIP-1beta chemokine and displays a CNV that also includes the CCL3L gene, a nonallelic copy of CCL3/MIP-1alpha. This CNV and two functionally relevant CCL4L SNPs (rs4796195 and rs3744595) have been recently associated to HIV pathology in three independent studies. We have quantified the CCL4L copy number and genotyped both SNPs in samples from HGDP-CEPH Diversity Panel. A strong correlation between CCL4L CNV and one of the SNPs analyzed is found, whereas no significant linkage disequilibrium is found between the two SNPs despite their close distance (647 bp), suggesting a recent appearance of the second SNP when the diversity in the first one and CNV had already been generated. The present study points out that in genes with CNV, it may be a key issue to combine the assessment of gene copy number with the genotyping of relevant SNPs to understand the phenotypic impact of genome variation in the immune response.
Asunto(s)
Quimiocina CCL4/genética , Dosificación de Gen , Variación Genética , Genética de Población , Polimorfismo de Nucleótido Simple , África del Sur del Sahara , Alelos , Américas , Pueblo Asiatico/genética , Frecuencia de los Genes , Heterogeneidad Genética , Genoma Humano , Heterocigoto , Humanos , OceaníaRESUMEN
Autoimmune thyroid diseases (AITD) are considered as prototypic organ-specific autoimmune diseases, yet their underlying aetiology remains poorly understood. Among the various pathophysiological mechanisms considered, a failure of central tolerance has received little attention. Here we present evidence in favour of dysregulated thymic function playing a role in AITD. Flow-cytometric analyses conducted in peripheral blood lymphocytes from 58 AITD patients and 48 age- and-sex-matched controls showed that AITD patients have significantly higher blood levels of CD4(+)CD45RA(+), CD4(+)CD31(+) and CD4/CD8 double-positive T lymphocytes, all markers of recent thymic emigrants (RTE). In addition, the alpha-signal joint T cell receptor excision circles (TRECs) content (a molecular marker of RTEs) was higher in the group of AITD patients older than 35 years than in age-matched controls. This was independent from peripheral T cell expansion as assessed by relative telomere length. Comparisons of TREC levels in peripheral blood lymphocytes and intrathyroidal lymphocytes in paired samples showed higher levels within the thyroid during the initial 30 months of the disease, indicating an influx of RTE into the thyroid during the initial stages of AITD. Additionally, a lack of correlation between TREC levels and forkhead box P3 expression suggests that the intrathyroidal RTE are not natural regulatory T cells. These results uncover a hitherto unknown correlation between altered thymic T cell export, the composition of intrathyroidal T cells and autoimmune pathology.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos T/metabolismo , Timo/inmunología , Enfermedades de la Tiroides/inmunología , Adulto , Factores de Edad , Anciano , Enfermedades Autoinmunes/sangre , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Autotolerancia , España , Linfocitos T/inmunología , Telómero/patología , Timo/fisiopatología , Enfermedades de la Tiroides/sangre , Adulto JovenRESUMEN
The destruction of beta cells by the islet infiltrating lymphocytes causes type 1 diabetes. Transgenic mice models expressing interferon (IFN)-beta in beta cells, in the non-obese diabetic (NOD) strain and in a diabetes-free, major histocompatibility complex-matched, homologous strain, the non-obese resistant (NOR) mice, developed accelerated type 1 diabetes after 3 weeks of age. Our aim was to determine if natural killer (NK) cells could affect the acceleration of the disease. We determined the amount of NK cells in the pancreas, spleen and lymph nodes from NOD rat insulin promoter (RIP)-IFN-beta mice. Pancreatic cytokines were assessed by quantitative real-time polymerase chain reaction and protein arrays. To confirm the relevance of NK cells in the acceleration of autoimmune diabetes this subset was depleted with anti-asialo GM1 antibodies. An increase of intrapancreatic NK cells characterized the accelerated onset of diabetes both in NOD and NOR RIP-IFN-beta transgenic models. Cytokines involved in NK function and migration were found to be hyperexpressed in the pancreas from accelerated diabetic mice. Interestingly, the depletion of NK cells in vivo abolished completely the acceleration of diabetes. NK cells connect innate to adaptive immunity and might play a role in autoimmunity. We report here that NK cells are required critically in the pancreas for accelerated diabetes. This model links inflammation to acceleration of beta cell-specific autoimmunity mediated by NK cells.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Interferón beta/inmunología , Células Asesinas Naturales/inmunología , Animales , Linfocitos B/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Gangliósido G(M1)/inmunología , Islotes Pancreáticos/inmunología , Ganglios Linfáticos/inmunología , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos NODRESUMEN
BACKGROUND AND OBJECTIVE: Statin-associated autoimmune myopathy (SAAM) with anti-HMGCR antibodies has recently been described. Several specific immunoassays are in use to detect HMGCR antibodies. In the course of systematic autoantibody screening we recognized a new distinct IFL staining pattern on rat liver sections that regularly coincided with anti-HMGCR antibodies. In this study we investigated whether this new IFL pattern is specifically associated to statin-associated autoimmune myopathy and corresponds to anti-HMGCR antibodies. PATIENTS AND METHODS: Twenty-three patients positive for anti-HMGCR antibodies (14 diagnosed with SAAM) were investigated for anti-HMGCR antibodies by two ELISA assays and confirmed by immmunoblot. HMGCR associated liver IFL pattern (HALIP) was detected by indirect IFL and the reactivity against HMGCR was confirmed by immunoabsorption using purified human HMGCR antigen. 90 patients with other autoimmune diseases and 45 non-autoimmune statin treated patients were studied as controls. RESULTS: 21 out of 23 (91%) anti-HMGCR positive patients were HALIP positive. The staining was completely and specifically removed by immunoabsorption with human purified HMGCR. None of the control sera from autoimmune patients or non-autoimmune statin treated subjects was positive for HALIP. Statistical concordance between HALIP and anti-HMGCR antibody specific tests was 98.7%, kappa 0.95. CONCLUSIONS: A new and distinct IFL staining pattern (HALIP) is associated to HMGCR associated myopathy. Absorption and concordance studies indicate that the antigen recognized in the liver by HALIP is HMGCR or a closely related protein. Awareness of this new pattern can help to detect HMGCR autoantibodies in statin treated patients tested for autoimmune serology.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/etiología , Ensayo de Inmunoadsorción Enzimática/métodos , Hidroximetilglutaril-CoA Reductasas/inmunología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Enfermedades Musculares/etiología , Enfermedades Autoinmunes/inmunología , Humanos , Persona de Mediana EdadRESUMEN
To establish whether there is a correlation between the autoimmune response to the islets and beta-cell function during the initial stages of type I (insulin-dependent) diabetes, and islet cell antibody (ICA) titer and C-peptide levels (fasting and glucagon stimulated) were determined in 39 newly diagnosed patients at onset of diabetes and every 3-6 mo for 2 yr. ICAs were detected in 74% of the patients, and beta-cell function was detected in 84% of the patients at onset. The ICA+ and ICA- groups had similar C-peptide values at diagnosis and at 3 mo, but from 6 mo on, the ICA+ group consistently showed a tendency to lose C-peptide secretory capacity more quickly when assessed by fasting and glucagon-stimulated C-peptide levels (ICA+ vs. ICA- fasting C-peptide levels at 18 and 24 mo, P = .013 and .017, respectively; ICA+ vs. ICA- glucagon-stimulated C-peptide levels at 6, 18, and 24 mo, P = .023, .007, and .028, respectively). The initial ICA titer had the highest predictive value on the outcome of beta-cell function (P = .04), and patients with complement-fixing ICAs did not behave differently from the general ICA+ group. This correlation between beta-cell function and ICA titer supports the role of autoimmunity in the pathogenesis of type I diabetes and has important implications for the design of immunotherapy trials.
Asunto(s)
Autoanticuerpos/análisis , Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Adolescente , Adulto , Péptido C/análisis , Niño , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Estudios de Seguimiento , Glucagón/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/fisiología , Masculino , Células Parietales Gástricas/inmunología , Glándula Tiroides/inmunologíaRESUMEN
Understanding how T lymphocytes recognize beta-cell autoantigens is essential for the elucidation of the pathogenesis of insulin-dependent diabetes mellitus. The increased and ectopic expression of HLA class I and II molecules detected in human beta-cells may facilitate this interaction. T-lymphocyte recognition of surface antigens also involves adhesion accessory molecules: intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 3 (LFA-3). These molecules not only allow cell contact but can also provide costimulatory signals for T-lymphocyte activation. Levels of ICAM-1 and LFA-3 expression in normal human islet cells and regulation of their expression by cytokines interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and IL-6 have been studied by two-color immunofluorescence staining of pancreatic cryostat sections and fluorescence-activated cell sorter analysis. Neither ICAM-1 nor LFA-3 could be demonstrated in sections or in fresh cell preparations, but after 18 h of culture, beta-, alpha-, and delta-cells expressed spontaneously moderate levels of ICAM-1 (but not LFA-3). IFN-gamma and TNF-alpha alone or in combination strongly enhanced this spontaneous expression of ICAM-1 in a time- and/or dose-dependent and additive manner but had no effect on LFA-3. An SV40-transformed islet cell line showed high basal levels of both ICAM-1 and LFA-3, but the response to cytokines followed the same pattern as primary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antígenos de Superficie/genética , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/genética , Regulación de la Expresión Génica/fisiología , Islotes Pancreáticos/química , Glicoproteínas de Membrana/genética , Antígenos de Superficie/fisiología , Antígenos CD58 , Moléculas de Adhesión Celular/fisiología , Transformación Celular Viral/genética , Transformación Celular Viral/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos HLA/genética , Antígenos HLA/fisiología , Humanos , Molécula 1 de Adhesión Intercelular , Interferón gamma/farmacología , Interleucina-1/farmacología , Interleucina-6/farmacología , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Glicoproteínas de Membrana/fisiología , Virus 40 de los Simios/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Since their demonstration in 1975, ICSAs have been proposed as serological markers and pathogenic elements in IDDM. ICSAs are detected in the sera of most newly diagnosed IDDM patients by indirect IFL that uses viable preparations of rat islet or insulinoma cells as substrate, but they also can be detected by using human insulinoma or fetal islet cells. We have tried to demonstrate ICSAs in the sera of 31 newly diagnosed diabetic patients, including 6 positive samples on human fetal islet cells, which used their natural target for the first time: normal human islet cells. In spite of using different types of preparations of these cells (i.e., freshly dispersed cell suspensions, monolayer cultures, or dispersed islets after culture), ICSAs could not be detected by IFL under the UV microscope, nor by flow cytometry. In contrast, 9 of 29 of the sera gave a positive staining on the RIN rat insulinoma cells. In an attempt to establish whether the putative ICSA autoantigen is present in the surface of human islet cells in the diabetic pancreas, the insulitis microenvironment was emulated by exposing the islets to three types of stress: 1) cytokines (IFN-gamma and TNF-alpha); 2) heat shock; and 3) hyperglycemia. However, diabetic sera failed again to recognize membrane antigens on the islet cells after either of these treatments. Neither were islet cells from a newly diagnosed diabetic patient stained by its autologous serum (ICA titer > 80 JDF U). These results suggest that ICSA autoantigen is not expressed in the membrane of human islet cells and therefore raises doubts about their proposed pathogenic role.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Adolescente , Anticuerpos Monoclonales , Biomarcadores/sangre , Membrana Celular/inmunología , Células Cultivadas , Diabetes Mellitus Tipo 1/sangre , Femenino , Humanos , Inmunoglobulina G , Masculino , Valores de ReferenciaRESUMEN
We have used a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol to examine the expression of cytokines in the pancreases and islets of patients with type I diabetes. We detect a significant increase in the level of expression of interferon (IFN)-alpha in the pancreases of the diabetic patients as compared with the control pancreases. In contrast, IFN-beta was detected at comparable levels in both groups, while IFN-gamma was detected in three of four control pancreases and one of four pancreases from the diabetic individuals. The IFN-alpha cDNAs generated by the RT-PCR were cloned and sequenced to determine which alpha-subtypes were being expressed. We found that the repertoire of subtypes was quite limited in any one individual (diabetic or not), although each individual was different with respect to the pattern of subtypes expressed. We also examined these pancreases for the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-2, IL-4, and IL-6. We found no detectable expression of TNF-alpha or IL-2 in any pancreases, and the expression of the other cytokines was variable, with no pattern emerging from the comparison of the diabetic and nondiabetic individuals. We conclude that, of the cytokines examined, only IFN-alpha was significantly increased in the diabetic patients, a result that is consistent with the possibility that this cytokine is directly involved in the development of type I diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Interferones/biosíntesis , Islotes Pancreáticos/metabolismo , Adolescente , Adulto , Secuencia de Bases , Niño , Preescolar , Cartilla de ADN , Femenino , Humanos , Lactante , Interferón-alfa/biosíntesis , Interferón beta/biosíntesis , Interferón gamma/biosíntesis , Interleucina-1/biosíntesis , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Interleucina-6/biosíntesis , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
A possible role of transporter associated with antigen processing (TAP)-1 in the pathogenesis of IDDM has been investigated by examining the level of TAP-1 expression in the islets of IDDM pancreas and by studying in vitro the effect of interferon (IFN)-gamma, IFN-alpha, and tumor necrosis factor-alpha in TAP-1 expression by cultured islet cells. A remarkable hyperexpression of TAP-1 has been found in the endocrine cells (beta and non-beta) of IDDM islets, which constitutes first evidence of hyperexpression of this molecule in the target organ of an autoimmune disease. TAP-1 hyperexpression correlated clearly with HLA class I hyperexpression but only very partially with HLA class II ectopic expression. IFN-gamma and IFN-alpha, both cytokines putatively implicated in IDDM pathogenesis, were capable of inducing TAP-1 protein (as assessed by immunofluorescence flow cytometry) and message (by Northern blot analysis and reverse transcription polymerase chain reaction). These findings suggest that under the influence of cytokines (most probably IFN-alpha) beta-cells may express in their surface a high density of HLA class I-peptide complexes that may facilitate their recognition and lysis by low-affinity CD8+ T-cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Citocinas/farmacología , Diabetes Mellitus Tipo 1/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Expresión Génica/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Adolescente , Adulto , Anciano , Secuencia de Bases , Línea Celular , Células Cultivadas , Cartilla de ADN , Diabetes Mellitus Tipo 1/inmunología , Femenino , Citometría de Flujo , Expresión Génica/inmunología , Humanos , Interferón-alfa/farmacología , Interferón gamma/farmacología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteoglicanos/biosíntesis , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Pancreatic islet transplantation has been proposed as an attractive option for the treatment of type I diabetes. Transplantation into different sites has been investigated, among them those that are immuno-logically privileged (e.g., thymus, uterus, brain, anterior eye chamber, and testicle). Because of their characteristics, seminal vesicles could be considered as immunologically privileged organs, but there is no worldwide experience that can confirm it. The purpose of the present study is to assess the viability and functionality of islet transplantation into seminal vesicles of diabetic rats. One hundred ninety inbred adult male syngeneic Lewis rats were used as donors (n = 72), receptors (n = 36), and controls(n = 11). Diabetes was chemically induced through a single intraperitoneal injection of streptozotocin. Groups of 1200 purified islets were introduced in the right seminal vesicle of diabetic rats. Diabetic control rats were sham transplanted. Body weight and glycemia were monitored every 2 d. Of transplanted rats, 16.7% achieved a good function due to islet engraftment, while 30.6% achieved a partially good response, and 52.7% were considered as nonresponding. This is the first report about islet transplantation into seminal vesicles of diabetic animals. Our results indicate that islet transplantation into rat seminal vesicles is technically possible, and that islets can function normally after engraftment into the wall of the seminal vesicle.
Asunto(s)
Diabetes Mellitus Experimental/cirugía , Supervivencia de Injerto , Trasplante de Islotes Pancreáticos/métodos , Vesículas Seminales/cirugía , Animales , Glucemia , Peso Corporal , Diabetes Mellitus Experimental/sangre , Hiperglucemia/sangre , Hiperglucemia/cirugía , Masculino , Ratas , Ratas Endogámicas LewRESUMEN
A model for the possible pathogenesis of insulin-dependent diabetes mellitus (IDDM) is presented. It is partly based on studies of autoimmune thyroiditis and founded on the idea that inappropriate class II molecule expression in insulin-producing beta-cells of pancreatic islets could cause an organ-specific autoimmune disorder. The model is supported by results of several IDDM case studies as well as by the critical evaluation of the known effects of various lymphokines and lymphotoxins on endocrine target tissues. The possible roles of islet capillary endothelial cells and islet cell antibodies in the pathogenesis of IDDM are discussed.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Genes MHC Clase II , Humanos , Modelos BiológicosRESUMEN
Cytokines are important modulators of immunological reactions, but it has been postulated that they might act on other unrelated epithelial cells. We studied the effects of recombinant interferon-gamma (rIFN gamma) and recombinant tumor necrosis factor-alpha (rTNF alpha) on normal human thyroid cells. We found that the combination of these two cytokines enhanced HLA class II molecule expression on these cells compared with the effect of rIFN gamma alone. This was proven by both immunofluorescence as well as a more sensitive and quantitative RIA. rTNF alpha alone had no effect on HLA class II molecule induction on the same thyrocytes, suggesting a synergistic rather than an additive action in combination with rIFN gamma. The addition of 600 U/ml rTNF alpha to low dose rIFN gamma (10 U/mL) enhanced class II expression by 50%, as quantified by RIA. We also demonstrated that normal thyrocytes possess distinct receptors for the two cytokines and that rTNF alpha probably augments IFN gamma binding, since it increased when the cells were first incubated with rTNF alpha. This increased binding provides an explanation for the synergistic action of rTNF alpha in enhancing class II molecule expression by rIFN gamma. We conclude that the presence of receptors for these cytokines on human thyroid cells gives a direct demonstration of their potential biological action on cells normally not involved in the immunological circuit. The phenomenon might also explain their direct or indirect involvement in vivo, such as in influencing inappropriate HLA class II molecule expression in epithelial cells affected by autoimmunity.