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1.
Diabetologia ; 66(3): 551-566, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36508037

RESUMO

AIMS/HYPOTHESIS: B cells play an important role in driving the development of type 1 diabetes; however, it remains unclear how they contribute to local beta cell destruction during disease progression. Here, we use gene expression profiling of B cell subsets identified in inflamed pancreatic tissue to explore their primary functional role during the progression of autoimmune diabetes. METHODS: Transcriptional profiling was performed on FACS-sorted B cell subsets isolated from pancreatic islets and the pancreatic lymph nodes of NOD mice. RESULTS: B cells are highly modified by the inflamed pancreatic tissue and can be distinguished by their transcriptional profile from those in the lymph nodes. We identified both a discrete and a core shared gene expression profile in islet CD19+CD138- and CD19+CD138+ B cell subsets, the latter of which is known to have enriched autoreactivity during diabetes development. On localisation to pancreatic islets, compared with CD138- B cells, CD138+ B cells overexpress genes associated with adhesion molecules and growth factors. Their shared signature consists of gene expression changes related to the differentiation of antibody-secreting cells and gene regulatory networks associated with IFN signalling pathways, proinflammatory cytokines and Toll-like receptor (TLR) activation. Finally, abundant TLR7 expression was detected in islet B cells and was enhanced specifically in CD138+ B cells. CONCLUSIONS/INTERPRETATION: Our study provides a detailed transcriptional analysis of islet B cells. Specific gene signatures and interaction networks have been identified that point towards a functional role for B cells in driving autoimmune diabetes.


Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Camundongos , Animais , Diabetes Mellitus Tipo 1/metabolismo , Camundongos Endogâmicos NOD , Pâncreas/metabolismo , Ilhotas Pancreáticas/metabolismo , Perfilação da Expressão Gênica
2.
Diabet Med ; 40(12): e15228, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37735524

RESUMO

AIMS: This review aims to introduce research in the pancreas to a broader audience. The pancreas is a heterocrine gland residing deep within our abdominal cavity. It is the home to our islets, which play a pivotal role in regulating metabolic homeostasis. Due to its structure and location, it is an impossible organ to study, in molecular detail, in living humans, and yet, understanding the pancreas is critical if we aim to characterise the immunopathology of type 1 diabetes (T1D) and one day prevent the triggering of the autoimmune attack associated with ß-cell demise. METHODS: Over a 100 years ago, we began studying pancreatic histology using cadaveric samples and clever adaptations to microscopes. As histologists, some may say nothing much has changed. Nevertheless, our microscopes can now interrogate multiple proteins at molecular resolution. Images of pancreas sections are no longer constrained to a single field of view and can capture a thousands and thousands of cells. AI-image-analysis packages can analyse these massive data sets offering breakthrough findings. CONCLUSION: This narrative review will provide an overview of pancreatic anatomy, and the importance of research focused on the pancreas in T1D. It will range from histological breakthroughs to briefly discussing the challenges associated with characterising the organ. I shall briefly introduce a selection of the available global biobanks and touch on the distinct pancreatic endotypes that differ immunologically and in ß-cell behaviour. Finally, I will introduce the idea of developing a collaborative tool aimed at developing a cohesive framework for characterising heterogeneity and stratifying endotypes in T1D more readily.


Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Pâncreas/patologia , Ilhotas Pancreáticas/metabolismo
3.
Diabet Med ; 40(9): e15155, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37246834

RESUMO

AIMS: Morphological studies of pancreas samples obtained from young people with recent-onset type 1 diabetes have revealed distinct patterns of immune cell infiltration of the pancreatic islets suggestive of two age-associated type 1 diabetes endotypes that differ by inflammatory responses and rates of disease progression. The objective of this study was to investigate whether these proposed disease endotypes are associated with pathological differences in immune cell activation and cytokine secretion by applying multiplexed gene expression analysis to pancreatic tissue from recent-onset type 1 diabetes cases. METHODS: RNA was extracted from samples of fixed, paraffin-embedded pancreas tissue from type 1 diabetes cases characterised by endotype and from controls without diabetes. Expression levels of 750 genes associated with autoimmune inflammation were determined by hybridisation to a panel of capture and reporter probes and these were counted as a measure of gene expression. Normalised counts were analysed for differences in expression between 29 type 1 diabetes cases and 7 controls without diabetes, and between the two type 1 diabetes endotypes. RESULTS: Ten inflammation-associated genes, including INS, were significantly under-expressed in both endotypes and 48 genes were more highly expressed. A different set of 13 genes associated with the development, activation and migration of lymphocytes was uniquely overexpressed in the pancreas of people developing diabetes at younger age. CONCLUSIONS: The results provide evidence that histologically defined type 1 diabetes endotypes differ in their immunopathology and identify inflammatory pathways specifically involved in disease developing at a young age, essential for a better understanding of disease heterogeneity.


Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Humanos , Adolescente , Diabetes Mellitus Tipo 1/metabolismo , Pâncreas/patologia , Ilhotas Pancreáticas/metabolismo , Inflamação/metabolismo , Diferenciação Celular
4.
Diabetologia ; 63(6): 1258-1267, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32172310

RESUMO

AIMS/HYPOTHESIS: It is unclear whether type 1 diabetes is a single disease or if endotypes exist. Our aim was to use a unique collection of pancreas samples recovered soon after disease onset to resolve this issue. METHODS: Immunohistological analysis was used to determine the distribution of proinsulin and insulin in the islets of pancreas samples recovered soon after type 1 diabetes onset (<2 years) from young people diagnosed at age <7 years, 7-12 years and ≥13 years. The patterns were correlated with the insulitis profiles in the inflamed islets of the same groups of individuals. C-peptide levels and the proinsulin:C-peptide ratio were measured in the circulation of a cohort of living patients with longer duration of disease but who were diagnosed in these same age ranges. RESULTS: Distinct patterns of proinsulin localisation were seen in the islets of people with recent-onset type 1 diabetes, which differed markedly between children diagnosed at <7 years and those diagnosed at ≥13 years. Proinsulin processing was aberrant in most residual insulin-containing islets of the younger group but this was much less evident in the group ≥13 years (p < 0.0001). Among all individuals (including children in the middle [7-12 years] range) aberrant proinsulin processing correlated with the assigned immune cell profiles defined by analysis of the lymphocyte composition of islet infiltrates. C-peptide levels were much lower in individuals diagnosed at <7 years than in those diagnosed at ≥13 years (median <3 pmol/l, IQR <3 to <3 vs 34.5 pmol/l, IQR <3-151; p < 0.0001), while the median proinsulin:C-peptide ratio was increased in those with age of onset <7 years compared with people diagnosed aged ≥13 years (0.18, IQR 0.10-0.31) vs 0.01, IQR 0.009-0.10 pmol/l; p < 0.0001). CONCLUSIONS/INTERPRETATION: Among those with type 1 diabetes diagnosed under the age of 30 years, there are histologically distinct endotypes that correlate with age at diagnosis. Recognition of such differences should inform the design of future immunotherapeutic interventions designed to arrest disease progression.


Assuntos
Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/metabolismo , Insulina/sangue , Insulina/metabolismo , Pâncreas/metabolismo , Proinsulina/sangue , Proinsulina/metabolismo , Adolescente , Fatores Etários , Criança , Diabetes Mellitus Tipo 1/diagnóstico , Humanos , Masculino
5.
Diabet Med ; 41(8): e15406, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39030905
6.
Diabetologia ; 61(11): 2344-2355, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30074059

RESUMO

AIMS/HYPOTHESIS: The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells. METHODS: Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets. RESULTS: An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking. CONCLUSIONS/INTERPRETATION: The SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection.


Assuntos
Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Isoformas de Proteínas/metabolismo , Adolescente , Adulto , Western Blotting , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Pâncreas/patologia , Adulto Jovem
7.
Curr Diab Rep ; 18(11): 115, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30259209

RESUMO

PURPOSE OF REVIEW: To explore the impact of age on type 1 diabetes (T1D) pathogenesis. RECENT FINDINGS: Children progress more rapidly from autoantibody positivity to T1D and have lower C-peptide levels compared to adults. In histological analysis of post-mortem pancreata, younger age of diagnosis is associated with reduced numbers of insulin containing islets and a hyper-immune CD20hi infiltrate. Moreover compared to adults, children exhibit decreased immune regulatory function and increased engagement and trafficking of autoreactive CD8+ T cells, and age-related differences in ß cell vulnerability may also contribute to the more aggressive immune phenotype observed in children. To account for some of these differences, HLA and non-HLA genetic loci that influence multiple disease characteristics, including age of onset, are being increasingly characterized. The exception of T1D as an autoimmune disease more prevalent in children than adults results from a combination of immune, metabolic, and genetic factors. Age-related differences in T1D pathology have important implications for better tailoring of immunotherapies.


Assuntos
Diabetes Mellitus Tipo 1/patologia , Progressão da Doença , Fatores Etários , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Sistema Imunitário/patologia
8.
Diabetologia ; 59(11): 2448-2458, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27506584

RESUMO

AIMS/HYPOTHESIS: Human pancreatic beta cells may be complicit in their own demise in type 1 diabetes, but how this occurs remains unclear. One potentially contributing factor is hyperexpression of HLA class I antigens. This was first described approximately 30 years ago, but has never been fully characterised and was recently challenged as artefactual. Therefore, we investigated HLA class I expression at the protein and RNA levels in pancreases from three cohorts of patients with type 1 diabetes. The principal aims were to consider whether HLA class I hyperexpression is artefactual and, if not, to determine the factors driving it. METHODS: Pancreas samples from type 1 diabetes patients with residual insulin-containing islets (n = 26) from the Network for Pancreatic Organ donors with Diabetes (nPOD), Diabetes Virus Detection study (DiViD) and UK recent-onset type 1 diabetes collections were immunostained for HLA class I isoforms, signal transducer and activator of transcription 1 (STAT1), NLR family CARD domain containing 5 (NLRC5) and islet hormones. RNA was extracted from islets isolated by laser-capture microdissection from nPOD and DiViD samples and analysed using gene-expression arrays. RESULTS: Hyperexpression of HLA class I was observed in the insulin-containing islets of type 1 diabetes patients from all three tissue collections, and was confirmed at both the RNA and protein levels. The expression of ß2-microglobulin (a second component required for the generation of functional HLA class I complexes) was also elevated. Both 'classical' HLA class I isoforms (i.e. HLA-ABC) as well as a 'non-classical' HLA molecule, HLA-F, were hyperexpressed in insulin-containing islets. This hyperexpression did not correlate with detectable upregulation of the transcriptional regulator NLRC5. However, it was strongly associated with increased STAT1 expression in all three cohorts. Islet hyperexpression of HLA class I molecules occurred in the insulin-containing islets of patients with recent-onset type 1 diabetes and was also detectable in many patients with disease duration of up to 11 years, declining thereafter. CONCLUSIONS/INTERPRETATION: Islet cell HLA class I hyperexpression is not an artefact, but is a hallmark in the immunopathogenesis of type 1 diabetes. The response is closely associated with elevated expression of STAT1 and, together, these occur uniquely in patients with type 1 diabetes, thereby contributing to their selective susceptibility to autoimmune-mediated destruction.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Ilhotas Pancreáticas/metabolismo , Diabetes Mellitus Tipo 1/patologia , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Pâncreas/metabolismo , Fator de Transcrição STAT1/metabolismo
9.
Diabetologia ; 57(2): 392-401, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24190581

RESUMO

AIMS/HYPOTHESIS: Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. METHODS: Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. RESULTS: Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. CONCLUSIONS/INTERPRETATION: When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.


Assuntos
Anticorpos Monoclonais/metabolismo , Proteínas do Capsídeo/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Infecções por Enterovirus/metabolismo , Enterovirus/metabolismo , Pâncreas/metabolismo , Antígenos Virais/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Reações Cruzadas , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/virologia , Infecções por Enterovirus/complicações , Infecções por Enterovirus/imunologia , Feminino , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/metabolismo , Masculino , Pâncreas/imunologia , Pâncreas/virologia , Reprodutibilidade dos Testes , Replicação Viral
10.
J Virol ; 87(13): 7646-54, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23637411

RESUMO

Type III interferons (IFNs), also called lambda interferons (IFN-λ), comprise three isoforms, IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B). Only limited information is available on their expression and biological functions in humans. Type I and type II IFNs protect human pancreatic islets against coxsackievirus infection, and this is important since such viruses have been proposed to play a role in the development of human type 1 diabetes. Here we investigated whether type III IFN is expressed during infection of human islet cells with coxsackievirus and if type III IFN regulates permissiveness to such infections. We show that human islets respond to a coxsackievirus serotype B3 (CVB3) infection by inducing the expression of type III IFNs. We also demonstrate that islet endocrine cells from nondiabetic individuals express the type III IFN receptor subunits IFN-λR1 and IL-10R2. Pancreatic alpha cells express both receptor subunits, while pancreatic beta cells express only IL-10R2. Type III IFN stimulation elicited a biological response in human islets as indicated by the upregulated expression of antiviral genes as well as pattern recognition receptors. We also show that type III IFN significantly reduces CVB3 replication. Our studies reveal that type III IFNs are expressed during CVB3 infection and that the expression of the type III IFN receptor by the human pancreatic islet allows this group of IFNs to regulate the islets' permissiveness to infection. Our novel observations suggest that type III IFNs may regulate viral replication and thereby contribute to reduced tissue damage and promote islet cell survival during coxsackievirus infection.


Assuntos
Infecções por Coxsackievirus/imunologia , Interleucinas/imunologia , Ilhotas Pancreáticas/metabolismo , Receptores de Interferon/imunologia , Replicação Viral/imunologia , Idoso , Análise de Variância , Feminino , Imunofluorescência , Humanos , Immunoblotting , Imuno-Histoquímica , Interferons , Ilhotas Pancreáticas/virologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real
11.
IUBMB Life ; 66(11): 723-34, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25504835

RESUMO

Type 1 diabetes mellitus (T1DM) is caused by the selective deletion of pancreatic ß-cells in response to an assault mounted within the pancreas by infiltrating immune cells. However, this apparently clear and focussed annunciation conceals a stark reality in which the cellular and molecular events leading to ß-cell loss remain poorly understood in humans. This reflects the difficulty of studying these processes in living individuals and the fact that, using pathological specimens, islet inflammation has been analysed in fewer than 200 recent-onset cases of T1DM worldwide, over the past century. Nevertheless, insights have been gained and the composition of the islet infiltrate is being disclosed. This is shown to be primarily lymphocytic in nature, with populations of both CD8+ and CD4+ T cells displaying an autoreactivity against specific islet antigenic peptides. The T cells are often accompanied by influent CD20+ B cells, although new data imply that the proportions of these individual cell types vary and that patients fall into at least two distinct categories having either a hyper-immune (CD20Hi) or a pauci-immune (CD20Lo) phenotype. The overall rate of ß-cell decline appears to correlate with these two phenotypes such that hyper-immune patients lose ß-cells more quickly and tend to develop disease at an earlier age than those with the pauci-immune profile. In this article, we review the evidence which underpins our current understanding of the aetiology of T1DM and highlight both the established features as well as areas of on-going ambiguity and debate.


Assuntos
Antígenos CD20/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Inflamação/imunologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/fisiopatologia , Subpopulações de Linfócitos T/imunologia , Diabetes Mellitus Tipo 1/imunologia , Humanos , Ilhotas Pancreáticas/imunologia
12.
Front Immunol ; 14: 1276255, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37908349

RESUMO

Gold nanoparticles (GNPs) have been used in the development of novel therapies as a way of delivery of both stimulatory and tolerogenic peptide cargoes. Here we report that intradermal injection of GNPs loaded with the proinsulin peptide C19-A3, in patients with type 1 diabetes, results in recruitment and retention of immune cells in the skin. These include large numbers of clonally expanded T-cells sharing the same paired T-cell receptors (TCRs) with activated phenotypes, half of which, when the TCRs were re-expressed in a cell-based system, were confirmed to be specific for either GNP or proinsulin. All the identified gold-specific clones were CD8+, whilst proinsulin-specific clones were both CD8+ and CD4+. Proinsulin-specific CD8+ clones had a distinctive cytotoxic phenotype with overexpression of granulysin (GNLY) and KIR receptors. Clonally expanded antigen-specific T cells remained in situ for months to years, with a spectrum of tissue resident memory and effector memory phenotypes. As the T-cell response is divided between targeting the gold core and the antigenic cargo, this offers a route to improving resident memory T-cells formation in response to vaccines. In addition, our scRNAseq data indicate that focusing on clonally expanded skin infiltrating T-cells recruited to intradermally injected antigen is a highly efficient method to enrich and identify antigen-specific cells. This approach has the potential to be used to monitor the intradermal delivery of antigens and nanoparticles for immune modulation in humans.


Assuntos
Diabetes Mellitus Tipo 1 , Nanopartículas Metálicas , Humanos , Autoantígenos , Proinsulina/genética , Ouro , Injeções Intradérmicas , Análise da Expressão Gênica de Célula Única , Peptídeos/genética , Receptores de Antígenos de Linfócitos T/genética
13.
Front Endocrinol (Lausanne) ; 13: 881997, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35957810

RESUMO

Aims/hypothesis: The Diabetes Virus Detection (DiViD) study has suggested the presence of low-grade enteroviral infection in pancreatic tissue collected from six of six live adult patients newly diagnosed with type 1 diabetes. The present study aimed to compare the gene and protein expression of selected virally induced pathogen recognition receptors and interferon stimulated genes in islets from these newly diagnosed type 1 diabetes (DiViD) subjects vs age-matched non-diabetic (ND) controls. Methods: RNA was extracted from laser-captured islets and Affymetrix Human Gene 2.0 ST arrays used to obtain gene expression profiles. Lists of differentially expressed genes were subjected to a data-mining pipeline searching for enrichment of canonical pathways, KEGG pathways, Gene Ontologies, transcription factor binding sites and other upstream regulators. In addition, the presence and localisation of specific viral response proteins (PKR, MxA and MDA5) were examined by combined immunofluorescent labelling in sections of pancreatic tissue. Results: The data analysis and data mining process revealed a significant enrichment of gene ontologies covering viral reproduction and infectious cycles; peptide translation, elongation and initiation, as well as oxidoreductase activity. Enrichment was identified in the KEGG pathways for oxidative phosphorylation; ribosomal and metabolic activity; antigen processing and presentation and in canonical pathways for mitochondrial dysfunction, oxidative phosphorylation and EIF2 signaling. Protein Kinase R (PKR) expression did not differ between newly diagnosed type 1 diabetes and ND islets at the level of total RNA, but a small subset of ß-cells displayed markedly increased PKR protein levels. These PKR+ ß-cells correspond to those previously shown to contain the viral protein, VP1. RNA encoding MDA5 was increased significantly in newly diagnosed type 1 diabetes islets, and immunostaining of MDA5 protein was seen in α- and certain ß-cells in both newly diagnosed type 1 diabetes and ND islets, but the expression was increased in ß-cells in type 1 diabetes. In addition, an uncharacterised subset of synaptophysin positive, but islet hormone negative, cells expressed intense MDA5 staining and these were more prevalent in DiViD cases. MxA RNA was upregulated in newly diagnosed type 1 diabetes vs ND islets and MxA protein was detected exclusively in newly diagnosed type 1 diabetes ß-cells. Conclusion/interpretation: The gene expression signatures reveal that pathways associated with cellular stress and increased immunological activity are enhanced in islets from newly diagnosed type 1 diabetes patients compared to controls. The increases in viral response proteins seen in ß-cells in newly diagnosed type 1 diabetes provide clear evidence for the activation of IFN signalling pathways. As such, these data strengthen the hypothesis that an enteroviral infection of islet ß-cells contributes to the pathogenesis of type 1 diabetes.


Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Adulto , Antivirais , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , RNA
14.
Diabetes ; 71(7): 1591-1596, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35499624

RESUMO

C-peptide declines in type 1 diabetes, although many long-duration patients retain low, but detectable levels. Histological analyses confirm that ß-cells can remain following type 1 diabetes onset. We explored the trends observed in C-peptide decline in the UK Genetic Resource Investigating Diabetes (UK GRID) cohort (N = 4,079), with ß-cell loss in pancreas donors from the network for Pancreatic Organ donors with Diabetes (nPOD) biobank and the Exeter Archival Diabetes Biobank (EADB) (combined N = 235), stratified by recently reported age at diagnosis endotypes (<7, 7-12, ≥13 years) across increasing diabetes durations. The proportion of individuals with detectable C-peptide declined beyond the first year after diagnosis, but this was most marked in the youngest age group (<1-year duration: age <7 years: 18 of 20 [90%], 7-12 years: 107 of 110 [97%], ≥13 years: 58 of 61 [95%] vs. 1-5 years postdiagnosis: <7 years: 172 of 522 [33%], 7-12 years: 604 of 995 [61%], ≥13 years: 225 of 289 [78%]). A similar profile was observed in ß-cell loss, with those diagnosed at younger ages experiencing more rapid loss of islets containing insulin-positive (insulin+) ß-cells <1 year postdiagnosis: age <7 years: 23 of 26 (88%), 7-12 years: 32 of 33 (97%), ≥13 years: 22 of 25 (88%) vs. 1-5 years postdiagnosis: <7 years: 1 of 12 (8.3%), 7-12 years: 7 of 13 (54%), ≥13 years: 7 of 8 (88%). These data should be considered in the planning and interpretation of intervention trials designed to promote ß-cell retention and function.


Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Adolescente , Peptídeo C , Criança , Diabetes Mellitus Tipo 1/genética , Humanos , Lactente , Células Secretoras de Insulina/patologia , Pâncreas/patologia , Doadores de Tecidos
15.
Front Endocrinol (Lausanne) ; 12: 617437, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33716971

RESUMO

Significant progress has been made in understanding the phenotypes of circulating immune cell sub-populations in human type 1 diabetes but much less is known about the equivalent populations that infiltrate the islets to cause beta-cell loss. In particular, considerable uncertainties remain about the phenotype and role of B-lymphocytes in the pancreas. This gap in understanding reflects both the difficulty in accessing the gland to study islet inflammation during disease progression and the fact that the number and proportion of islet-associated B-lymphocytes varies significantly according to the disease endotype. In very young children (especially those <7 years at onset) pancreatic islets are infiltrated by both CD8+ T- and CD20+ B-lymphocytes in roughly equal proportions but it is widely held that the CD8+ T-lymphocytes are responsible for driving beta-cell toxicity. By contrast, the role played by B-lymphocytes remains enigmatic. This is compounded by the fact that, in older children and teenagers (those ≥13 years at diagnosis) the proportion of B-lymphocytes found in association with inflamed islets is much reduced by comparison with those who are younger at diagnosis (reflecting two endotypes of disease) whereas CD8+ T-lymphocytes form the predominant population in both groups. In the present paper, we review the current state of understanding and develop a proposal to stimulate further discussion of the roles played by islet-associated B-lymphocytes in human type 1 diabetes. We cite evidence indicating that sites of direct contact can be found between CD8+ and CD20+-lymphocytes in and around inflamed islets and propose that such interactions may be important in determining the efficiency of beta cell killing.


Assuntos
Linfócitos B/imunologia , Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Pâncreas/imunologia , Humanos
16.
Diabetes ; 70(5): 1038-1050, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33947721

RESUMO

Analysis of data from clinical cohorts, and more recently from human pancreatic tissue, indicates that reduced prohormone processing is an early and persistent finding in type 1 diabetes. In this article, we review the current state of knowledge regarding alterations in islet prohormone expression and processing in type 1 diabetes and consider the clinical impact of these findings. Lingering questions, including pathologic etiologies and consequences of altered prohormone expression and secretion in type 1 diabetes, and the natural history of circulating prohormone production in health and disease, are considered. Finally, key next steps required to move forward in this area are outlined, including longitudinal testing of relevant clinical populations, studies that probe the genetics of altered prohormone processing, the need for combined functional and histologic testing of human pancreatic tissues, continued interrogation of the intersection between prohormone processing and autoimmunity, and optimal approaches for analysis. Successful resolution of these questions may offer the potential to use altered prohormone processing as a biomarker to inform therapeutic strategies aimed at personalized intervention during the natural history of type 1 diabetes and as a pathogenic anchor for identification of potential disease-specific endotypes.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Diabetes Mellitus Tipo 1/imunologia , Humanos , Células Secretoras de Insulina/imunologia , Ilhotas Pancreáticas/metabolismo , Proinsulina/metabolismo
18.
Diabetes ; 68(5): 988-1001, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30833470

RESUMO

Type 1 diabetes studies consistently generate data showing islet ß-cell dysfunction and T cell-mediated anti-ß-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the ß-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single ß-cells. Consistent with immunohistological studies, ß-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These ß-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin+CD68- ß-cells, specifically found in islets with lymphocytic infiltrates. ß-Cell surface expression of HLA Class II was detected on a portion of CD45-insulin+ ß-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic ß-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. ß-Cell expression of Class II molecules suggests that ß-cells may interact directly with islet-infiltrating CD4+ T cells and may play an immunopathogenic role.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Apresentação de Antígeno/imunologia , Autoimunidade/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Humanos , Insulina/metabolismo
19.
Diabetes Care ; 41(7): 1486-1492, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880650

RESUMO

OBJECTIVE: The decline in C-peptide in the 5 years after diagnosis of type 1 diabetes has been well studied, but little is known about the longer-term trajectory. We aimed to examine the association between log-transformed C-peptide levels and the duration of diabetes up to 40 years after diagnosis. RESEARCH DESIGN AND METHODS: We assessed the pattern of association between urinary C-peptide/creatinine ratio (UCPCR) and duration of diabetes in cross-sectional data from 1,549 individuals with type 1 diabetes using nonlinear regression approaches. Findings were replicated in longitudinal follow-up data for both UCPCR (n = 161 individuals, 326 observations) and plasma C-peptide (n = 93 individuals, 473 observations). RESULTS: We identified two clear phases of C-peptide decline: an initial exponential fall over 7 years (47% decrease/year [95% CI -51, -43]) followed by a stable period thereafter (+0.07%/year [-1.3, +1.5]). The two phases had similar durations and slopes in patients above and below the median age at diagnosis (10.8 years), although levels were lower in the younger patients irrespective of duration. Patterns were consistent in both longitudinal UCPCR (n = 162; ≤7 years duration: -48%/year [-55, -38]; >7 years duration -0.1% [-4.1, +3.9]) and plasma C-peptide (n = 93; >7 years duration only: -2.6% [-6.7, +1.5]). CONCLUSIONS: These data support two clear phases of C-peptide decline: an initial exponential fall over a 7-year period, followed by a prolonged stabilization where C-peptide levels no longer decline. Understanding the pathophysiological and immunological differences between these two phases will give crucial insights into understanding ß-cell survival.


Assuntos
Peptídeo C/sangue , Diabetes Mellitus Tipo 1/sangue , Adolescente , Adulto , Análise Química do Sangue , Criança , Estudos Transversais , Progressão da Doença , Regulação para Baixo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Adulto Jovem
20.
EBioMedicine ; 36: 367-375, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30269996

RESUMO

BACKGROUND: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated. METHODS: The expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-ßH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib. FINDINGS: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1ß, induced PDL1 expression in vitro in human islet cells and EndoC-ßH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs - a proposed therapeutic strategy for T1D - or IRF1 prevented PDL1 induction. INTERPRETATION: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1.


Assuntos
Antígeno B7-H1/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Regulação da Expressão Gênica , Fator Regulador 1 de Interferon/metabolismo , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Ilhotas Pancreáticas/metabolismo , Adolescente , Adulto , Biomarcadores , Linhagem Celular , Criança , Pré-Escolar , Humanos , Células Secretoras de Insulina/metabolismo , Pessoa de Meia-Idade , Adulto Jovem
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