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Organ-specific autoimmune diseases, such as type 1 diabetes, are believed to result from T-cell-mediated damage of the target tissue. The immune-mediated tissue injury, in turn, is known to depend on complex interactions between genetic and environmental factors. Nevertheless, the mechanisms whereby environmental factors contribute to the pathogenesis of autoimmune diseases remain elusive and represent a major untapped target to develop novel strategies for disease prevention. Given the impact of the early environment on the developing immune system, epigenetic changes induced by maternal factors during fetal life have been linked to a likelihood of developing an autoimmune disease later in life. In humans, DNA methylation is the epigenetic mechanism most extensively investigated. This review provides an overview of the critical role of DNA methylation changes induced by prenatal maternal conditions contributing to the increased risk of immune-mediated diseases on the offspring, with a particular focus on T1D. A deeper understanding of epigenetic alterations induced by environmental stressors during fetal life may be pivotal for developing targeted prevention strategies of type 1 diabetes by modifying the maternal environment.
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Diabetes Mellitus Tipo 1/genética , Desenvolvimento Fetal/genética , Impressão Genômica , Herança Materna , Animais , Metilação de DNA , Diabetes Mellitus Tipo 1/imunologia , HumanosRESUMO
While human enteroviruses are generally regarded as a lytic virus, and persistent non-cytolytic enterovirus infection in pancreatic beta cells has been suspected of playing a role in type 1 diabetes pathogenesis. However, it is still unclear how enteroviruses could exit the pancreatic beta cell in a non-lytic manner. This study aimed to investigate the role of beta cell-derived extracellular vesicles (EVs) in the non-lytic enteroviral spread and infection. Size-exclusion chromatography and antibody-based immunoaffinity purification were used to isolate EVs from echovirus 16-infected human beta EndoC-ßH1 cells. EVs were then characterized using transmission electron microscopy and Multiplex Bead-Based Flow Cytometry Assay. Virus production and release were quantified by 50% cell culture infectious dose (CCID50) assay and qRT-PCR. Our results showed that EVs from echovirus 16-infected EndoC-ßH1 cells harbor infectious viruses and promote their spread during the pre-lytic phase of infection. Furthermore, the EVs-mediated infection was not inhibited by virus-specific neutralizing antibodies. In summary, this study demonstrated that enteroviruses could exit beta cells non-lytically within infectious EVs, thereby thwarting the access of neutralizing antibodies to viral particles. These data suggest that enterovirus transmission through EVs may contribute to viral dissemination and immune evasion in persistently infected beta cells.
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AIM/HYPOTHESIS: Human enteroviral infections are suggested to be associated with type 1 diabetes. However, the mechanism by which enteroviruses can trigger disease remains unknown. The present study aims to investigate the impact of enterovirus on autophagy, a cellular process that regulates beta cell homeostasis, using the clonal beta cell line INS(832/13) and human islet cells as in vitro models. METHODS: INS(832/13) cells and human islet cells were infected with a strain of echovirus 16 (E16), originally isolated from the stool of a child who developed type 1 diabetes-associated autoantibodies. Virus production and release was determined by 50% cell culture infectious dose (CCID50) assay and FACS analysis. The occurrence of autophagy, autophagosomes, lysosomes and autolysosomes was detected by western blot, baculoviral-mediated expression of microtubule-associated protein light chain 3 (LC3)II-GFP and LysoTracker Red, and quantified by Cellomics ArrayScan. Autophagy was also monitored with a Cyto-ID detection kit. Nutrient deprivation (low glucose [2.8 mmol/l]), amino acid starvation (Earle's Balanced Salt Solution [EBSS]) and autophagy-modifying agents (rapamycin and chloroquine) were used in control experiments. Insulin secretion and the expression of autophagy-related (Atg) genes and genes involved in autophagosome-lysosome fusion were determined. RESULTS: E16-infected INS(832/13) cells displayed an accumulation of autophagosomes, compared with non-treated (NT) cells (grown in complete RPMI1640 containing 11.1 mmol/l glucose) (32.1 ± 1.7 vs 21.0 ± 1.2 µm2/cell; p = 0.05). This was accompanied by increased LC3II ratio both in E16-infected cells grown in low glucose (LG) (2.8 mmol/l) (0.42 ± 0.03 vs 0.11 ± 0.04 (arbitrary units [a.u.]); p < 0.0001) and grown in media containing 11.1 mmol/l glucose (0.37 ± 0.016 vs 0.05 ± 0.02 (a.u.); p < 0.0001). Additionally, p62 accumulated in cells after E16 infection when grown in LG (1.23 ± 0.31 vs 0.36 ± 0.12 (a.u.); p = 0.012) and grown in media containing 11.1 mmol/l glucose (1.79 ± 0.39 vs 0.66 ± 0.15 (a.u.); p = 0.0078). mRNA levels of genes involved in autophagosome formation and autophagosome-lysosome fusion remained unchanged in E16-infected cells, except Atg7, which was significantly increased when autophagy was induced by E16 infection, in combination with LG (1.48 ± 0.08-fold; p = 0.02) and at 11.1 mmol/l glucose (1.26 ± 0.2-fold; p = 0.001), compared with NT controls. Moreover, autophagosomes accumulated in E16-infected cells to the same extent as when cells were treated with the lysosomal inhibitor, chloroquine, clearly indicating that autophagosome turnover was blocked. Upon infection, there was an increased viral titre in the cell culture supernatant and a marked reduction in glucose-stimulated insulin secretion (112.9 ± 24.4 vs 209.8 ± 24.4 ng [mg protein]-1 h-1; p = 0.006), compared with uninfected controls, but cellular viability remained unaffected. Importantly, and in agreement with the observations for INS(832/13) cells, E16 infection impaired autophagic flux in primary human islet cells (46.5 ± 1.6 vs 34.4 ± 2.1 µm2/cell; p = 0.01). CONCLUSIONS/INTERPRETATION: Enteroviruses disrupt beta cell autophagy by impairing the later stages of the autophagic pathway, without influencing expression of key genes involved in core autophagy machinery. This results in increased viral replication, non-lytic viral spread and accumulation of autophagic structures, all of which may contribute to beta cell demise and type 1 diabetes. Graphical abstract.
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Autofagia/fisiologia , Ilhotas Pancreáticas/metabolismo , Pâncreas/fisiologia , Autofagia/genética , Western Blotting , Feminino , Humanos , Masculino , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
The number of the X chromosome-linked genes has been previously suggested to influence immune responses and the development of autoimmune diseases. In the present study, we aimed at evaluating the level of expression of CD40L (an X-linked gene involved in adaptive immunity) and TLR7 (an X-linked gene involved in innate immunity) in a variety of different karyotypes. Those included males, females and patients with X chromosome aneuploidy. Healthy females (46, XX; n = 10) and healthy males (46, XY; n = 10) were compared to females with Turner syndrome (TS) (45, X; n = 11) and males with Klinefelter syndrome (KS) (47, XXY; n = 5). Stimulation of peripheral blood mononuclear cells (PBMCs) with PMA and ionomycin resulted in higher percentage of CD3 + CD40L+ T cells (P < 0.001) and higher level expression of CD40L in T cell (P < 0.001) in female and KS patients compared with male and TS patients. TLR7-mediated IFN-alpha production by HLADR + CD3- CD19- cells was significantly upregulated in healthy women compared with healthy males, TS and KS patients (P < 0.001). TLR7 agonist-stimulated PBMCs from healthy females and KS patients expressed significantly higher levels of TLR7 mRNA than those from male and TS patients (P < 0.05). The increased expression of the X-linked genes TLR7 and CD40L in healthy females and KS patients suggests that the presence of two X chromosomes plays a major role in enhancing both innate and adaptive immune responses. These results may contribute to the explanation of sex-based differences in immune biology and the sex bias in predisposition to autoimmune diseases.
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Imunidade Adaptativa/genética , Ligante de CD40/biossíntese , Ligante de CD40/genética , Cromossomos Humanos X/genética , Dosagem de Genes/genética , Imunidade Inata/genética , Receptor 7 Toll-Like/biossíntese , Receptor 7 Toll-Like/genética , Imunidade Adaptativa/imunologia , Antígenos CD19/biossíntese , Complexo CD3/biossíntese , Células Cultivadas , Variações do Número de Cópias de DNA/genética , Feminino , Humanos , Imunidade Inata/imunologia , Interferon-alfa/biossíntese , Ionomicina/farmacologia , Síndrome de Klinefelter/genética , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Ácidos Polimetacrílicos/farmacologia , RNA Mensageiro/biossíntese , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Síndrome de Turner/genéticaRESUMO
Human enteroviruses (HEV), especially coxsackievirus serotype B (CVB) and echovirus (E), have been associated with diseases of both the exocrine and endocrine pancreas, but so far evidence on HEV infection in human pancreas has been reported only in islets and ductal cells. This study aimed to investigate the capability of echovirus strains to infect human exocrine and endocrine pancreatic cells. Infection of explanted human islets and exocrine cells with seven field strains of E6 caused cytopathic effect, virus titer increase and production of HEV protein VP1 in both cell types. Virus particles were found in islets and acinar cells infected with E6. No cytopathic effect or infectious progeny production was observed in exocrine cells exposed to the beta cell-tropic strains of E16 and E30. Endocrine cells responded to E6, E16 and E30 by upregulating the transcription of interferon-induced with helicase C domain 1 (IF1H1), 2'-5'-oligoadenylate synthetase 1 (OAS1), interferon-ß (IFN-ß), chemokine (C-X-C motif) ligand 10 (CXCL10) and chemokine (C-C motif) ligand 5 (CCL5). Echovirus 6, but not E16 or E30, led to increased transcription of these genes in exocrine cells. These data demonstrate for the first time that human exocrine cells represent a target for E6 infection and suggest that certain HEV serotypes can replicate in human pancreatic exocrine cells, while the pancreatic endocrine cells are permissive to a wider range of HEV.
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Echovirus 6 Humano/imunologia , Imunidade Inata , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/virologia , Pâncreas Exócrino/imunologia , Pâncreas Exócrino/virologia , Efeito Citopatogênico Viral , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/biossíntese , Técnicas de Cultura de Órgãos , Carga Viral , Proteínas Estruturais Virais/análiseRESUMO
In an earlier study, infection of human pancreatic islets with epidemic strains of echovirus (E4, E16, E30), with proven but differently ability to induce islet autoimmunity, resulted either in a severe damage (i.e., E16 and E30) or proceeded without visible changes in infected islets (i.e., E4). In this study, the ability of these strains to replicate in beta cells and the consequence of such an infection for beta cell lysis and beta cell function was studied in the pancreatic beta cell lines INS-1, MIN6, and NIT-1. The strains of E16 and E30 did replicate in INS1, MIN6, and NIT1 cells and resulted in a pronounced cytopathic effect within 3 days following infection. By contrast, E4 replicated in all examined insulinoma cells with no apparent cell destruction. The insulin release in response to high glucose stimulation was hampered in all infected cells (P < 0.05) when no evidence of cytolysis was present; however, the adverse effect of E16 and E30 on insulin secretion appeared to be higher than that of the E4 strain. The differential effects of echovirus infection on cell lysis, and beta cell function in the rodent insulinoma INS1, MIN6, and NIT 1 cells reflect those previously obtained in primary human islets and support the notion that the insulin-producing beta cells can harbor a non-cytopathic viral infection.
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Efeito Citopatogênico Viral , Enterovirus Humano B/fisiologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/virologia , Insulina/metabolismo , Replicação Viral , Morte Celular , Linhagem Celular , Enterovirus Humano B/patogenicidade , Glucose/farmacologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/patologia , InsulinomaRESUMO
Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.
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Genômica , Glucose , Transcriptoma/fisiologia , 5'-Nucleotidase/biossíntese , 5'-Nucleotidase/genética , Linhagem Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Glucose/genética , Glucose/metabolismo , Humanos , Ilhotas Pancreáticas , Masculino , Edição de RNA/fisiologia , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Tetraspaninas/biossíntese , Tetraspaninas/genética , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/genética , Quinases Ativadas por p21/biossíntese , Quinases Ativadas por p21/genéticaRESUMO
Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.
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Enterovirus Humano B/imunologia , Expressão Gênica/imunologia , Imunidade Inata/genética , Ilhotas Pancreáticas/imunologia , Células Cultivadas , Infecções por Echovirus/imunologia , Infecções por Echovirus/virologia , Enterovirus Humano B/genética , Epidemias , Genes Virais , Interações Hospedeiro-Patógeno , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/virologia , FilogeniaRESUMO
INTRODUCTION: An association between pollen count (Poaceae) and symptoms is well known, but to a lesser degree the importance of priming and lag effects. Also, threshold levels for changes in symptom severity need to be validated. The present study aims to investigate the relationship between pollen counts, symptoms and health related quality of life (HRQL), and to validate thresholds levels, useful in public pollen warnings. MATERIAL AND METHODS: Children aged 7-18 with grass pollen allergy filled out a symptom diary during the pollen season for nose, eyes and lung symptoms, as well as a HRQL questionnaire every week. Pollen counts were monitored using a volumetric spore trap. RESULTS: 89 (91%) of the included 98 children completed the study. There was a clear association between pollen count, symptom severity and HRQL during the whole pollen season, but no difference in this respect between early and late pollen season. There was a lag effect of 1-3 days after pollen exposure except for lung symptoms. We found only two threshold levels, at 30 and 80 pollen grains/m(3) for the total symptom score, not three as is used today. The nose and eyes reacted to low doses, but for the lung symptoms, symptom strength did hardly change until 50 pollen grains/m(3). CONCLUSION: Grass pollen has an effect on symptoms and HRQL, lasting up to 5 days after exposure. Symptoms from the lungs appear to have higher threshold levels than the eyes and the nose. Overall symptom severity does not appear to change during the course of season. Threshold levels need to be revised. We suggest a traffic light model for public pollen warnings directed to children, where green signifies "no problem", yellow signifies "can be problems, especially if you are highly sensitive" and red signifies "alert - take action".
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BACKGROUND: The vast array and quantity of longitudinal samples collected in The Environmental Determinants of Diabetes in the Young study present a series of challenges in terms of quality control procedures and data validity. To address this, pilot studies have been conducted to standardize and enhance both biospecimen collection and sample obtainment in terms of autoantibody collection, stool sample preservation, RNA, biomarker stability, metabolic biomarkers and T-cell viability. RESEARCH DESIGN AND METHODS: The Environmental Determinants of Diabetes in the Young is a multicentre, international prospective study (n = 8677) designed to identify environmental triggers of type 1 diabetes (T1D) in genetically at-risk children from ages 3 months until 15 years. The study is conducted through six primary clinical centres located in four countries. RESULTS: As of May 2012, over three million biological samples and 250 million total data points have been collected, which will be analysed to assess autoimmunity status, presence of inflammatory biomarkers, genetic factors, exposure to infectious agents, dietary biomarkers and other potentially important environmental exposures in relation to autoimmunity and progression to T1D. CONCLUSIONS: Detailed procedures were utilized to standardize both data harmonization and management when handling a large quantity of longitudinal samples obtained from multiple locations. In addition, a description of the available specimens is provided that serve as an invaluable repository for the elucidation of determinants in T1D focusing on autoantibody concordance and harmonization, transglutaminase autoantibody, inflammatory biomarkers (T-cells), genetic proficiency testing, RNA lab internal quality control testing, infectious agents (monitoring cross-contamination, virus preservation and nasal swab collection validity) and HbA1c testing.
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Bancos de Espécimes Biológicos/organização & administração , Bancos de Espécimes Biológicos/normas , Diabetes Mellitus Tipo 1/patologia , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Adolescente , Autoanticorpos/sangue , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/genética , Fezes/virologia , Humanos , Lactente , Recém-Nascido , Estudos Longitudinais , Controle de Qualidade , RNA Mensageiro/análiseRESUMO
Type 1 diabetes is characterized by autoimmune destruction of pancreatic ß-cells in genetically susceptible individuals. Triggers of islet autoimmunity, time course, and the precise mechanisms responsible for the progressive ß-cell failure are not completely understood. The recent escalation of obesity in affluent countries has been suggested to contribute to the increased incidence of type 1 diabetes. Understanding the link between metabolism and immune tolerance could lead to the identification of new markers for the monitoring of disease onset and progression. We studied several immune cell subsets and factors with high metabolic impact as markers associated with disease progression in high-risk subjects and type 1 diabetic patients at onset and at 12 and 24 months after diagnosis. A multiple correlation matrix among different parameters was evaluated statistically and assessed visually on two-dimensional graphs. Markers to predict residual ß-cell function up to 1 year after diagnosis were identified in multivariate logistic regression models. The meta-immunological profile changed significantly over time in patients, and a specific signature that was associated with worsening disease was identified. A multivariate logistic regression model measuring age, BMI, fasting C-peptide, number of circulating CD3(+)CD16(+)CD56(+) cells, and the percentage of CD1c(+)CD19(-)CD14(-)CD303(-) type 1 myeloid dendritic cells at disease onset had a significant predictive value. The identification of a specific meta-immunological profile associated with disease status may contribute to our understanding of the basis of diabetes progression.
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Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Monitorização Imunológica/métodos , Linfócitos T/imunologia , Biomarcadores , Criança , Progressão da Doença , Citometria de Fluxo , Humanos , Células Secretoras de Insulina/imunologia , PrognósticoRESUMO
INTRODUCTION: Respiratory allergic disorders like rhinitis and asthma are common conditions that not only affect target organs, but complicate the daily life of affected children and adolescents. OBJECTIVES: The aim of this study was to investigate the QoL (quality of life) in children with grass pollen allergy in and out of grass pollen season. METHODS: We used the Pediatric Allergic Disease Quality of Life Questionnaire (PADQLQ), a disease-specific questionnaire including both asthma and rhinitis symptoms. We also used the DISABKIDS (a European project which aims at enhancing the quality of life and the independence of children with chronic health conditions and their families) questionnaire, a generic questionnaire covering non-organ-specific effects of disease. RESULTS: Ninety-eight children 718 years old with grass pollen allergy were included. Eighty-nine children (91%) completed the study. The QoL was significantly decreased during pollen season assessed both with DISABKIDS and PADQLQ. The correlation between the questionnaires was 0.73. Not only the physical domain score (P = 0.00093) but also the emotional domain score (P = 0.034) was significantly lowered. Children with multiple manifestations (asthma and rhinitis) had lower QoL than children with rhinitis alone (P = 0.01). Multiple regression analysis showed a highly significant impact on QoL for symptoms from nose, eyes and lungs. They were equally important (standardized coefficient 047, 0.47 and 0.46, respectively). CONCLUSION: The QoL in children and adolescents with respiratory allergy deteriorates during pollen season. This was shown both with generic (DISABKIDS) and disease-specific instrument (PADQLQ).
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Asma/fisiopatologia , Rinite Alérgica Sazonal/fisiopatologia , Adolescente , Alérgenos/imunologia , Asma/diagnóstico , Asma/psicologia , Criança , Feminino , Humanos , Masculino , Qualidade de Vida , Rinite Alérgica Sazonal/diagnóstico , Rinite Alérgica Sazonal/psicologia , Inquéritos e Questionários , SuéciaRESUMO
A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time consuming. Here, we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in-utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify interindividual DNA methylation variation that is present both at birth and 3 yr later. These findings suggest that disease-relevant epigenetic variation could be detected at birth, i.e., before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of interindividual epigenomic variation in a range of common human diseases.
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Alelos , Metilação de DNA , Epigênese Genética , Feminino , Loci Gênicos , Variação Genética , Genoma Humano , Testes Hematológicos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Estudos Longitudinais , Masculino , Análise de Sequência de DNARESUMO
Coeliac disease and type 1 diabetes are autoimmune diseases that may share the same initiating environmental factors. In this study, the occurrence of type 1 diabetes associated autoantibodies (GADA and IA-2A) and tissue transglutaminase autoantibodies (TGA) was determined in patients with confirmed viral infections and no signs of type 1 diabetes or coeliac disease. Serum samples from 82 Cuban patients tested positive for PCR and IgG specific to enterovirus (HEV, serotype echovirus 16, 20 samples), Epstein-Barr virus (EBV, 20 samples), cytomegalovirus (CMV, 21 samples), and hepatitis C virus (HCV, 21 samples); and sera from 164 controls negative serologically to EBV, CMV, HCV, and echovirus 16 were enrolled in the study. All subjects were screened for GADA, IA-2A, and TGA. The prevalence of TGA in patients infected with HEV, EBV, CMV, or HCV was 55% (11/20), 25% (5/20), 9.5% (2/21), and 9.5% (2/21), respectively. GADA and IA-2A were found in 15% (3/20) and 25% (5/20) of patients infected with HEV. None of the patients infected by EBV, CMV, and HCV had GADA or IA-2A. All children infected with HEV who were positive for type 1 diabetes-associated autoantibodies were also TGA-positive. None of the sera from uninfected subjects were positive for GADA, IA-2A or TGA. In conclusion, TGA can develop during infection with HEV, EBV, CMV, or HCV, while the emergence of islet cell related autoantibodies is restricted to HEV infections. The findings suggest that HEV may be a shared environmental factor for the development of islet and gut-related autoimmunity.
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Autoanticorpos/sangue , Doença Celíaca/imunologia , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Proteínas Tirosina Fosfatases/imunologia , Viroses/complicações , Adolescente , Adulto , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Cuba , Feminino , Humanos , Lactente , Masculino , Estudos Soroepidemiológicos , Adulto JovemRESUMO
BACKGROUND: High levels of soluble cytotoxic T-lymphocyte antigen 4 (soluble CTLA-4), an alternative splice form of the regulatory T-cell (Treg) associated CTLA-4 gene, have been associated with type 1 diabetes (T1D) and other autoimmune diseases, such as Grave's disease and myasthenia gravis. At the same time, studies have shown soluble CTLA-4 to inhibit T-cell activation through B7 binding. This study aimed to investigate the role of soluble CTLA-4 in relation to full-length CTLA-4 and other Treg-associated markers in T1D children and in individuals with high or low risk of developing the disease. METHODS: T1D children were studied at 4 days, 1 and 2 years after diagnosis in comparison to individuals with high or low risk of developing the disease. Isolated peripheral blood mononuclear cells were stimulated with the T1D-associated glutamic acid decarboxylase 65 and phytohaemagglutinin. Subsequently, soluble CTLA-4, full-length CTLA-4, FOXP3 and TGF-ß mRNA transcription were quantified and protein concentrations of soluble CTLA-4 were measured in culture supernatant and sera. RESULTS AND CONCLUSIONS: Low protein concentrations of circulating soluble CTLA-4 and a positive correlation between soluble CTLA-4 mRNA and protein were seen in T1D, in parallel with a negative correlation in healthy subjects. Further, low levels of mitogen-induced soluble CTLA-4 were accompanied by low C-peptide levels. Interestingly, low mitogen-induced soluble CTLA-4 mRNA and low TGF-ß mRNA expression were seen in high risk individuals, suggesting an alteration in activation and down-regulating immune mechanisms during the pre-diabetic phase.
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Antígeno CTLA-4/sangue , Diabetes Mellitus Tipo 1/imunologia , Expressão Gênica , Isoformas de Proteínas/imunologia , Adolescente , Autoimunidade , Biomarcadores , Células Sanguíneas , Peptídeo C/metabolismo , Antígeno CTLA-4/biossíntese , Antígeno CTLA-4/química , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Feminino , Fatores de Transcrição Forkhead/biossíntese , Humanos , Estudos Longitudinais , Masculino , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/imunologia , Transcrição Gênica , Fator de Crescimento Transformador beta/biossínteseRESUMO
The transcription factor T-cell factor 7-like 2 (TCF7L2) confers type 2 diabetes risk mainly through impaired insulin secretion, perturbed incretin effect and reduced beta-cell survival. The aim of this study was to identify the molecular mechanism through which TCF7L2 influences beta-cell survival. TCF7L2 target genes in INS-1 cells were identified using Chromatin Immunoprecipitation. Validation of targets was obtained by: siRNA silencing, real-time quantitative polymerase chain reaction, electrophoretic mobility shift assay, luciferase reporter assays and western blot. Apoptosis rate was measured by DNA degradation and caspase-3 content. Islet viability was estimated by measuring metabolic rate. TCF7L2 binds to 3646 gene promoters in INS-1 cells in high or low glucose, including Tp53, Pten, Uggt1, Adamts9 and Fto. SiRNA-mediated reduction in TCF7L2 activity resulted in increased apoptosis and increased expression of Tp53, which resulted in elevated p53 protein activity and an increased expression of the p53 target gene Tp53inp1 (encoding p53-induced-nuclear-protein 1). Reversing the increase in p53INP1 protein expression, seen after Tcf7l2 silencing, protected INS-1 cells from Tcf7l2 depletion-induced apoptosis. This result was replicated in primary rat islets. The risk T-allele of rs7903146 is associated with increased TCF7L2 mRNA expression and transcriptional activity. On the other hand, in vitro silencing of TCF7L2 lead to increased apoptosis. One possibility is that the risk T-allele increases expression of an inhibitory TCF7L2 isoform with lower transcriptional activity. These results identify the p53-p53INP1 pathway as a molecular mechanism through which TCF7L2 may affect beta-cell survival and established a molecular link between Tcf7l2 and two type 2 diabetes-associated genes, Tp53inp1 and Adamts9.
Assuntos
Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Proteínas de Choque Térmico/metabolismo , Células Secretoras de Insulina/citologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Sobrevivência Celular , Diabetes Mellitus Tipo 2/genética , Regulação da Expressão Gênica , Humanos , Células Secretoras de Insulina/metabolismo , Proteínas Nucleares , Ratos , Ratos Wistar , Transdução de Sinais , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The pioneer microbiota of the neonate may affect future actions of the immune system. This study aimed to map the pioneer microbiota in healthy neonates vaginally born at term. A subgroup of neonates born large for GA (LGA) was compared with the neonates appropriate for GA (AGA). Fecal samples were collected, within 48 h after birth, from 79 neonates. Quantitative PCR was used for enumeration of Lactobacillus, a subgroup of Lactobacillus common in the vagina, Bifidobacterium, Enterococcus, Enterobacteriaceae, and the Bacteroides fragilis group. Cloning and sequencing were applied for subgroups of neonates born LGA or AGA. Lactobacillus was detected in all neonates, whereas other bacterial groups were detected only in 14 to 30% of the subjects. The prevalence of Gram-negative Proteobacteria was higher in neonates born LGA, whereas Gram-positive Firmicutes was more prevalent in neonates born AGA (p < 0.001). This study contributed to increased knowledge of the pioneer microbiota and indicates that neonates born LGA had significantly different microbiota compared with those born AGA. As the early microbiota can be important for maturation of the immune system, the outcome from this study may be relevant in the care of pregnant woman and newborns.
Assuntos
Trato Gastrointestinal/microbiologia , Recém-Nascido , Metagenoma/genética , Vagina/microbiologia , Bactérias/genética , Peso ao Nascer , Parto Obstétrico , Fezes/microbiologia , Feminino , Idade Gestacional , Humanos , Masculino , Projetos Piloto , Gravidez , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND: The Goto-Kakizaki (GK) rat is a well-studied non-obese spontaneous type 2 diabetes (T2D) animal model characterized by impaired glucose-stimulated insulin secretion (GSIS) in the pancreatic beta cells. MicroRNAs (miRNAs) are short regulatory RNAs involved in many fundamental biological processes. We aim to identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats and investigate both their short- and long term glucose-dependence during glucose-stimulatory conditions. METHODOLOGY/PRINCIPAL FINDINGS: Global profiling of 348 miRNAs in the islets of GK rats and Wistar controls (females, 60 days, Nâ=â6 for both sets) using locked nucleic acid (LNA)-based microarrays allowed for the clear separation of the two groups. Significant analysis of microarrays (SAM) identified 30 differentially-expressed miRNAs, 24 of which are predominantly upregulated in the GK rat islets. Monitoring of qPCR-validated miRNAs during GSIS experiments on isolated islets showed disparate expression trajectories between GK and controls indicating distinct short- and long-term glucose dependence. We specifically found expression of rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 to be regulated by hyperglycaemia. The putative targets of upregulated miRNAs in the GK, filtered with glucose-regulated mRNAs, were found to be enriched for insulin-secretion genes known to be downregulated in T2D patients. Finally, the binding of rno-miR-335 to a fragment of the 3'UTR of one of known down-regulated exocytotic genes in GK islets, Stxbp1 was shown by luciferase assay. CONCLUSIONS/SIGNIFICANCE: The perturbed miRNA network found in the GK rat islets is indicative of a system-wide impairment in the regulation of genes important for the normal functions of pancreatic islets, particularly in processes involving insulin secretion during glucose stimulatory conditions. Our findings suggest that the reduced insulin secretion observed in the GK rat may be partly due to upregulated miRNA expression leading to decreased production of key proteins of the insulin exocytotic machinery.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , MicroRNAs/genética , Animais , Perfilação da Expressão Gênica , Técnicas In Vitro , Reação em Cadeia da Polimerase , Ratos , Ratos WistarRESUMO
AIM: The aim of the study was to translate and validate the PADQLQ (Pediatric Allergic Disease Quality of Life Questionnaire), a disease-specific quality of life questionnaire for the assessment of quality of life in children with pollen allergy. METHODS: The PADQLQ was translated into Swedish according to guidelines. Children aged 7-18 with grass pollen allergy were included. Quality of life was assessed in parallel with ordinary symptom scales (VAS) before, during and after the pollen season. RESULTS: A total of 98 children were included and 89 (91%) completed the study. The results for PADQLQ showed good cross-sectional and longitudinal validity. The retrospective estimation after the season showed good consensus with the assessment during pollen season. CONCLUSION: Quality of life in children assessed with the PADQLQ (Pediatric Allergic Disease Quality of Life Questionnaire) is a reliable strategy for evaluating the burden of disease in children with pollen allergy and for the evaluation of treatment.
Assuntos
Qualidade de Vida , Rinite Alérgica Sazonal , Inquéritos e Questionários , Adolescente , Criança , Humanos , Idioma , Estudos Retrospectivos , Rinite Alérgica Sazonal/diagnóstico , SuéciaRESUMO
Many microRNAs (miRNAs) are known to be cell-type specific and are implicated in development of diseases. We investigated the global expression pattern of miRNAs in human pancreatic islets compared to liver and skeletal muscle, using bead-based technology and quantitative RT-PCR. In addition to the known islet-specific miR-375, we also found enrichment of miR-127-3p, miR-184, miR-195 and miR-493∗ in the pancreatic islets. The expression of miR-375, miR-127-3p, miR-184 and the liver-enriched miR-122 is positively correlated to insulin biosynthesis, while the expression of miR-127-3p and miR-184 is negatively correlated to glucose-stimulated insulin secretion (GSIS). These correlations were absent in islets of glucose intolerant donors (HbA1c ≥ 6.1). We suggest that the presence of an islet-specific miRNA network, which consists of at least miR-375, miR-127-3p and miR-184, potentially involved in insulin secretion. Our results provide new insight into miRNA-mediated regulation of insulin secretion in healthy and glucose intolerant subjects.