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Enteroviruses can infect various human organs, causing diseases such as meningitis, the common cold, hand-foot-and-mouth disease, myocarditis, pancreatitis, hepatitis, poliomyelitis, sepsis, and type 1 diabetes. Currently, there are no approved treatments for enterovirus infections. In this study, we identified a synergistic combination of orally available, safe-in-man pleconaril, AG7404, and mindeudesivir, that at non-toxic concentrations effectively inhibited enterovirus replication in human cell and organoid cultures. Importantly, the cocktail did not alter glucose and insulin levels in the culture medium of pancreatic β-cells and preserved the contraction rhythm of infected heart organoids. These findings highlight a promising drug cocktail for further preclinical studies and clinical trials targeting a broad range of enterovirus-mediated diseases., Competing Interests: Declarations. Ethics approval: The study involved commercially available human cell lines, WT virus strains, which we isolated from patient samples or obtained via collaborations. All experiments with viruses were performed in BSL2 laboratories in compliance with the guidelines of the national authorities, and under appropriate ethical and safety approvals. All experiments conducted with pancreatic cancer organoids have been approved by the Helsinki University Hospital ethical board, HUS/74/2023, HUS/23/2024. The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for study on lung organoids (METC 2012 − 512). Competing interests: The authors have no relevant financial or non-financial interests to disclose., (© 2025. The Author(s).)

Objective: In the Diabetes Virus Detection and Intervention trial, antiviral treatment with pleconaril and ribavirin decreased the decline, compared with placebo, in endogenous C-peptide 1 year after diagnosis of type 1 diabetes (T1D) in children and adolescents. This article reports the results 2 and 3 years after diagnosis., Research Design and Methods: This was a multicenter, randomized, placebo-controlled (1:1) trial of 96 children and adolescents aged 6-15.9 years newly diagnosed with T1D. Antiviral treatment (pleconaril and ribavirin) or placebo was given for 6 months from diagnosis, and participants were followed for 3 years. The primary outcome was residual C-peptide secretion, reported as the area under the curve (AUC), assessed by 2-h mixed-meal tolerance test. Secondary outcomes included insulin doses and HbA1c., Results: At the 3-year follow-up, 75 participants attended. At 2 years, the mean ± SD AUC for C-peptide in the placebo group was 0.27 ± 0.33 compared with 0.34 ± 0.37 in the pleconaril and ribavirin group. After 3 years, the AUC had decreased to 0.17 ± 0.23 and 0.25 ± 0.34, respectively. There was no statistically significant difference between the groups. The groups were also comparable with regard to secondary end points., Conclusions: The decreased reduction in C-peptide levels after antiviral treatment is no longer present after 2 or 3 years. Further investigations are needed to explore options to use antiviral treatment in the prevention and treatment of T1D., (© 2025 by the American Diabetes Association.)

Children who develop diabetes in their first years of life risk being exposed to many decades of hyperglycemia, hence having a high risk of early complications and premature death. An additional age-dependent risk is that dysglycemia, especially hyperglycemia, negatively affects the developing brain. In evaluating the outcome of insulin treatment at an individual and group level, cutoff thresholds for glucose values are needed. Time in tight range (TITR) was defined as a measurement of time spent in a state of normoglycemia. The International Society of Pediatric and Adolescent Diabetes recommended that for preschoolers with type 1 diabetes (T1D), either >70% of time with glucose in range 70-180 mg/dL (3.9-10 mmol/L) or >50% of time in a tighter range 70-140 mg/dL (TITR) can be used as continuous glucose monitoring targets. In Sweden, over the past two decades, pediatric diabetes teams set glycemic targets to 70-140 mg/dL (3.9-7.8 mmol/L). Swedish registry data show that >50% of children <7 years old have >50% TITR. The purpose of this review is to share and discuss international knowledge and experiences of working with TITR as a health-promoting strategy in preschoolers with T1D on a structural and individual level. We conclude that as insulin treatment improves, a reasonable goal is to strive for as much time in a state of normoglycemia as possible, and this can easily be explained to families of children with diabetes. For children with access to an experienced health care team and diabetes technologies a currently realistic target can be at least half of the time in normoglycemic range, i.e., TITR >50%., (© 2024 by the American Diabetes Association.)

Aims/hypothesis: Defensins play a crucial role in the innate immune system's first defense against microbial threats. However, little is known about the defensin system in the pancreas, especially in relation to Type 1 diabetes. We explore the expression of defensins in different disease stages of Type 1 diabetes and correlated obtained findings to the degree of inflammation, providing new insights into the disease and the innate immune system., Material and Methods: Pancreases from non-diabetic human organ donors of different age groups and donors with Type 1 diabetes with different disease duration were examined. Sections from head, body and tail of the pancreas were stained for eight different defensins and for immune cells; CD3+, CD45+, CD68+ and NES+ (granulocytes)., Results: In non-diabetic adult controls the level of expression for defensins Beta-1,Alpha-1, Cathelicidin and REG3A correlated with the level of inflammation. In contrast, individuals with Type  1 diabetes exhibit a reduction or absence of several central defensins regardless of the level of inflammation in their pancreas. The expression of Cathelicidin is present in neutrophils and macrophages but not in T-cells in subjects with Type 1 diabetes., Conclusions: Obtained findings suggest a pancreatic dysfunction in the innate immune system and the bridging to the adaptive system in Type 1 diabetes. Further studies on the role of the local innate immune system in Type 1 diabetes is needed., (© 2024. The Author(s).)

Aims: Sulfatide is a chaperone for insulin manufacturing in beta cells. Here we explore whether the blood glucose values normally could be associated with this sphingolipid and especially two of its building enzymes CERS2 and CERS6. Both T1D and T2D have low blood sulfatide levels, and insulin resistance on beta cells at clinical diagnosis. Furthermore, we examined islet pericytes for sulfatide, and beta-cell receptors for GLP-1, both of which are related to the insulin production., Materials and Methods: We examined mRNA levels in islets from the DiViD and nPOD studies, performed genetic association analyses, and histologically investigated pericytes in the islets for sulfatide., Results: Polymorphisms of the gene encoding the CERS6 enzyme responsible for synthesising dihydroceramide, a precursor to sulfatide, are associated with random blood glucose values in non-diabetic persons. This fits well with our finding of sulfatide in pericytes in the islets, which regulates the capillary blood flow in the islets of Langerhans, which is important for oxygen supply to insulin production. In the islets of newly diagnosed T1D patients, we observed low levels of GLP-1 receptors; this may explain the insulin resistance in their beta cells and their low insulin production. In T2D patients, we identified associated polymorphisms in both CERS2 and CERS6., Conclusions: Here, we describe several polymorphisms in sulfatide enzymes related to blood glucose levels and HbA1c in non-diabetic individuals. Islet pericytes from such persons contain sulfatide. Furthermore, low insulin secretion in newly diagnosed T1D may be explained by beta-cell insulin resistance due to low levels of GLP-1 receptors., (© 2024 The Authors. Diabetes/Metabolism Research and Reviews published by John Wiley & Sons Ltd.)

Aims/hypothesis: Correctly diagnosing MODY is important, as individuals with this diagnosis can discontinue insulin injections; however, many people are misdiagnosed. We aimed to develop a robust approach for determining the pathogenicity of variants of uncertain significance in hepatocyte nuclear factor-1 alpha (HNF1A)-MODY and to obtain an accurate estimate of the prevalence of HNF1A-MODY in paediatric cases of diabetes., Methods: We extended our previous screening of the Norwegian Childhood Diabetes Registry by 830 additional samples and comprehensively genotyped HNF1A variants in autoantibody-negative participants using next-generation sequencing. Carriers of pathogenic variants were treated by local healthcare providers, and participants with novel likely pathogenic variants and variants of uncertain significance were enrolled in an investigator-initiated, non-randomised, open-label pilot study (ClinicalTrials.gov registration no. NCT04239586). To identify variants associated with HNF1A-MODY, we functionally characterised their pathogenicity and assessed the carriers' phenotype and treatment response to sulfonylurea., Results: In total, 615 autoantibody-negative participants among 4712 cases of paediatric diabetes underwent genetic sequencing, revealing 19 with HNF1A variants. We identified nine carriers with novel variants classified as variants of uncertain significance or likely to be pathogenic, while the remaining ten participants carried five pathogenic variants previously reported. Of the nine carriers with novel variants, six responded favourably to sulfonylurea. Functional investigations revealed their variants to be dysfunctional and demonstrated a correlation with the resulting phenotype, providing evidence for reclassifying these variants as pathogenic., Conclusions/interpretation: Based on this robust classification, we estimate that the prevalence of HNF1A-MODY is 0.3% in paediatric diabetes. Clinical phenotyping is challenging and functional investigations provide a strong complementary line of evidence. We demonstrate here that combining clinical phenotyping with functional protein studies provides a powerful tool to obtain a precise diagnosis of HNF1A-MODY., (© 2023. The Author(s).)

Previous studies showed a low-grade enterovirus infection in the pancreatic islets of patients with newly diagnosed type 1 diabetes (T1D). In the Diabetes Virus Detection (DiViD) Intervention, a phase 2, placebo-controlled, randomized, parallel group, double-blind trial, 96 children and adolescents (aged 6-15 years) with new-onset T1D received antiviral treatment with pleconaril and ribavirin (n = 47) or placebo (n = 49) for 6 months, with the aim of preserving β cell function. The primary endpoint was the mean stimulated C-peptide area under the curve (AUC) 12 months after the initiation of treatment (less than 3 weeks after diagnosis) using a mixed linear model. The model used longitudinal log-transformed serum C-peptide AUCs at baseline, at 3 months, 6 months and 1 year. The primary endpoint was met with the serum C-peptide AUC being higher in the pleconaril and ribavirin treatment group compared to the placebo group at 12 months (average marginal effect = 0.057 in the linear mixed model; 95% confidence interval = 0.004-0.11, P = 0.037). The treatment was well tolerated. The results show that antiviral treatment may preserve residual insulin production in children and adolescent with new-onset T1D. This provides a rationale for further evaluating antiviral strategies in the prevention and treatment of T1D. European Union Drug Regulating Authorities Clinical Trials identifier: 2015-003350-41 ., (© 2023. The Author(s).)

Aims: To investigate if HLA risk haplotypes and HbA1c levels are associated with the expression levels of innate anti-viral immune pathway genes in type 1 diabetes., Materials and Methods: We investigated RNA expression levels of innate anti-viral immune pathway genes in laser-dissected islets from two to five tissue sections per donor from the Diabetes Virus Detection study and the network of Pancreatic Organ Donors in relation to HLA risk haplotypes (non-predisposed and predisposed) and HbA1c levels (normal, elevated, and high)., Results: The expression of innate anti-viral immune genes (TLR7, OAS1, OAS3 etc.) was significantly increased in individuals with predisposing vs non-predisposing HLA haplotypes. Also, the expression of several of the innate anti-viral immune genes from the HLA risk haplotype analysis was significantly increased in the group with high vs normal HbA1c. Furthermore, the gene expression of OAS2 was significantly increased in the group with high HbA1c vs elevated HbA1c., Conclusions: Expression of innate anti-viral immune pathway genes was increased in individuals with predisposing HLA risk haplotypes and those with high HbA1c. This indicates that type 1 diabetes might well begin with alterations in innate anti-viral immunity, and already at this stage be associated with HLA risk haplotypes., (© 2023 The Authors. Diabetes/Metabolism Research and Reviews published by John Wiley & Sons Ltd.)

Continuous glucose monitors (CGM) and insulin pumps have become the preferred treatment option for most young children and adolescents with type 1 diabetes (T1D), by avoiding fingerstick testing and providing real-time glucose measurements. These medical devices and their adhesives contain substances which have been identified as being responsible for allergic contact dermatitis. We describe the case of a toddler who developed severe contact dermatitis from her diabetes devices, leading to secondary infections and hospital admissions. This was followed by the development of a symmetrical exanthema with retroauricular and glutaeal distribution. Patch tests were positive for isobornyl acrylate (IBOA) and 4-tert-butylcatechol (PTBC). Her symmetrical exanthema was interpreted as systemic contact dermatitis due to IBOA and PTBC in her diabetes devices. We suspect that systemic contact dermatitis is an underreported complication in diabetic patients., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. Skin Health and Disease published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

Background: At diagnosis of Type 1 Diabetes (T1D), 30% of the beta cells are dormant, i.e. alive, but inactive. This could reduce beta cell destruction, as cellular stress contributes to beta cell damage. However, the beta cells, that are still active, must produce more insulin and are therefore more vulnerable. The inactive beta cells represent a potential for restoring the insulin secretion., Methods: We analyzed the expression of selected genes in islets from live, newly diagnosed T1D patients from the DiViD study and organ doners with longer duration of T1D, type 2 diabetes (T2D), or no diabetes from the nPOD study. Additionally, analysis of polymorphisms was performed on all the investigated genes., Findings: Various possibilities were considered for the inactivity of the beta cells: secretion defect, fetal state, hibernation, and insulin resistance. We analyzed genes related to the ceramide and sphingomyelin synthesis and degradation, secretion, circadian rhythm and insulin action, and found changes in T1D islets that resemble fetal dedifferentiation and asynchrony. Furthermore, we found low levels of insulin receptor mRNA in the islets. No polymorphisms were found., Interpretation: Our findings suggest a secretion defect, but also fetal dedifferentiation and desynchronization in the inactive beta cells. Together with previous evidence, that predisposing factors for T2D are also present for T1D development, we raise the idea to treat individuals with ongoing T1D development prophylactically with T2D medicine like GLP-1 receptor agonists, metformin, or others, combined with anti-inflammatory compounds, in order to reactivate the dormant beta cells, and to prevent autoimmune destruction. T2D mechanisms during T1D development should be investigated further., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Josefsen, Krogvold, Gerling, Pociot, Dahl-Jørgensen and Buschard.)

Aims/hypothesis: Enterovirus (EV) infection of pancreatic islet cells is one possible factor contributing to type 1 diabetes development. We have reported the presence of EV genome by PCR and of EV proteins by immunohistochemistry in pancreatic sections. Here we explore multiple human virus species in the Diabetes Virus Detection (DiViD) study cases using innovative methods, including virus passage in cell cultures., Methods: Six recent-onset type 1 diabetes patients (age 24-35) were included in the DiViD study. Minimal pancreatic tail resection was performed under sterile conditions. Eleven live cases (age 43-83) of pancreatic carcinoma without diabetes served as control cases. In the present study, we used EV detection methods that combine virus growth in cell culture, gene amplification and detection of virus-coded proteins by immunofluorescence. Pancreas homogenates in cell culture medium were incubated with EV-susceptible cell lines for 3 days. Two to three blind passages were performed. DNA and RNA were extracted from both pancreas tissue and cell cultures. Real-time PCR was used for detecting 20 different viral agents other than EVs (six herpesviruses, human polyomavirus [BK virus and JC virus], parvovirus B19, hepatitis B virus, hepatitis C virus, hepatitis A virus, mumps, rubella, influenza A/B, parainfluenza 1-4, respiratory syncytial virus, astrovirus, norovirus, rotavirus). EV genomes were detected by endpoint PCR using five primer pairs targeting the partially conserved 5' untranslated region genome region of the A, B, C and D species. Amplicons were sequenced. The expression of EV capsid proteins was evaluated in cultured cells using a panel of EV antibodies., Results: Samples from six of six individuals with type 1 diabetes (cases) and two of 11 individuals without diabetes (control cases) contained EV genomes (p<0.05). In contrast, genomes of 20 human viruses other than EVs could be detected only once in an individual with diabetes (Epstein-Barr virus) and once in an individual without diabetes (parvovirus B19). EV detection was confirmed by immunofluorescence of cultured cells incubated with pancreatic extracts: viral antigens were expressed in the cytoplasm of approximately 1% of cells. Notably, infection could be transmitted from EV-positive cell cultures to uninfected cell cultures using supernatants filtered through 100 nm membranes, indicating that infectious agents of less than 100 nm were present in pancreases. Due to the slow progression of infection in EV-carrying cell cultures, cytopathic effects were not observed by standard microscopy but were recognised by measuring cell viability. Sequences of 5' untranslated region amplicons were compatible with EVs of the B, A and C species. Compared with control cell cultures exposed to EV-negative pancreatic extracts, EV-carrying cell cultures produced significantly higher levels of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP1)., Conclusions/interpretation: Sensitive assays confirm that the pancreases of all DiViD cases contain EVs but no other viruses. Analogous EV strains have been found in pancreases of two of 11 individuals without diabetes. The detected EV strains can be passaged in series from one cell culture to another in the form of poorly replicating live viruses encoding antigenic proteins recognised by multiple EV-specific antibodies. Thus, the early phase of type 1 diabetes is associated with a low-grade infection by EVs, but not by other viral agents., (© 2022. The Author(s).)

Type 1 diabetes (T1D) incidence is increased after COVID-19 infection in children under 18 years of age. Interferon-α-activated oligoadenylate synthetase and downstream RNAseL activation degrade pathogen RNA, but can also damage host RNA when RNAseL activity is poorly regulated. One such regulator is PDE12 which degrades 2'-5' oligoadenylate units, thereby decreasing RNAseL activity. We analyzed PDE12 expression in islets from non-diabetic donors, individuals with newly (median disease duration 35 days) and recently (5 years) diagnosed T1D, and individuals with type 2 diabetes (T2D). We also analyzed PDE12 single-nucleotide polymorphisms (SNPs) relative to T1D incidence. PDE12 expression was decreased in individuals with recently diagnosed T1D, in three of five individuals with newly diagnosed T1D, but not in individuals with T2D. Two rare PDE12 SNPs were found to have odds ratios of 1.80 and 1.74 for T1D development. We discuss whether decreased PDE12 expression after COVID-19 infection might be part of the up to 2.5-fold increase in T1D incidence., (© 2022. The Author(s).)

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 β-cells displayed markedly increased PKR protein levels. These PKR+ β-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 β-cells in both newly diagnosed type 1 diabetes and ND islets, but the expression was increased in β-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 β-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 β-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 β-cells contributes to the pathogenesis of type 1 diabetes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Krogvold, Leete, Mynarek, Russell, Gerling, Lenchik, Mathews, Richardson, Morgan and Dahl-Jørgensen.)

Aims/hypothesis: The Diabetes Virus Detection (DiViD) study is the first study to laparoscopically collect pancreatic tissue and purified pancreatic islets together with duodenal mucosa, serum, peripheral blood mononuclear cells (PBMCs) and stools from six live adult patients (age 24-35 years) with newly diagnosed type 1 diabetes. The presence of enterovirus (EV) in the pancreatic islets of these patients has previously been reported., Methods: In the present study we used reverse transcription quantitative real-time PCR (RT-qPCR) and sequencing to characterise EV genomes present in different tissues to understand the nature of infection in these individuals., Results: All six patients were found to be EV-positive by RT-qPCR in at least one of the tested sample types. Four patients were EV-positive in purified islet culture medium, three in PBMCs, one in duodenal biopsy and two in stool, while serum was EV-negative in all individuals. Sequencing the 5' untranslated region of these EVs suggested that all but one belonged to enterovirus B species. One patient was EV-positive in all these sample types except for serum. Sequence analysis revealed that the virus strain present in the isolated islets of this patient was different from the strain found in other sample types. None of the islet-resident viruses could be isolated using EV-permissive cell lines., Conclusions/interpretation: EV RNA can be frequently detected in various tissues of patients with type 1 diabetes. At least in some patients, the EV strain in the pancreatic islets may represent a slowly replicating persisting virus., (© 2021. The Author(s).)

Aims/hypothesis: The incidence of type 1 diabetes is increasing more rapidly than can be explained by genetic drift. Viruses may play an important role in the disease, as they seem to activate the 2'-5'-linked oligoadenylate (2'-5'A) pathway of the innate antiviral immune system. Our aim was to investigate this possibility., Methods: Innate antiviral immune pathways were searched for type 1 diabetes-associated polymorphisms using genome-wide association study data. SNPs within ±250kb flanking regions of the transcription start site of 64 genes were examined. These pathways were also investigated for type 1 diabetes-associated RNA expression profiles using laser-dissected islets from two to five tissue sections per donor from the Diabetes Virus Detection (DiViD) study and the network of Pancreatic Organ Donors (nPOD)., Results: We found 27 novel SNPs in genes nominally associated with type 1 diabetes. Three of those SNPs were located upstream of the 2'-5'A pathway, namely SNP rs4767000 (p = 1.03 × 10 -9 , OR 1.123), rs1034687 (p = 2.16 × 10 -7 , OR 0.869) and rs739744 (p = 1.03 × 10 -9 , OR 1.123). We also identified a large group of dysregulated islet genes in relation to type 1 diabetes, of which two were novel. The most aberrant genes were a group of IFN-stimulated genes. Of those, the following distinct pathways were targeted by the dysregulation (compared with the non-diabetic control group): OAS1 increased by 111% (p < 1.00 × 10 -4 , 95% CI -0.43, -0.15); MX1 increased by 142% (p < 1.00 × 10 -4 , 95% CI -0.52, -0.22); and ISG15 increased by 197% (p = 2.00 × 10 -4 , 95% CI -0.68, -0.18)., Conclusions/interpretation: We identified a genetic predisposition in the 2'-5'A pathway that potentially contributes to dysregulation of the innate antiviral immune system in type 1 diabetes. This study describes a potential role for the 2'-5'A pathway and other components of the innate antiviral immune system in beta cell autoimmunity.

Exposure of isolated human islets to proinflammatory cytokines leads to up-regulation of inducible nitric oxide synthase (iNOS), raised NO, and beta cell toxicity. These findings have led to increasing interest in the clinical utility of iNOS blockade to mitigate beta cell destruction in human type 1 diabetes (T1D). However, recent studies show that iNOS-derived NO may also confer beta cell protection. To investigate this dichotomy, we compared islet cell distributions and intensity of iNOS immunostaining in pancreatic sections, co-stained for insulin and glucagon, from new-onset T1D donors (group 1), with non-diabetic autoantibody-negative (group 2), non-diabetic autoantibody-positive (group 3) and long-term diabetic donors (group 4). The cellular origins of iNOS, its frequency and graded intensities in islets and number in peri-islet, intra-islet and exocrine regions were determined. All donors showed iNOS positivity, irrespective of disease and presence of beta cells, had variable labelling intensities, without significant differences in the frequency of iNOS-positive islets among study groups. iNOS was co-localised in selective beta, alpha and other endocrine cells, and in beta cell-negative islets of diabetic donors. The number of peri- and intra-islet iNOS cells was low, being significantly higher in the peri-islet area. Exocrine iNOS cells also remained low, but were much lower in group 1. We demonstrate that iNOS expression in islet cells is variable, heterogeneous and independent of co-existing beta cells. Its distribution and staining intensities in islets and extra-islet areas do not correlate with T1D or its duration. Interventions to inactivate the enzyme to alleviate disease are currently not justified.

In type 1 diabetes (T1D), a lifelong autoimmune disease, T cells infiltrate the islets and the exocrine pancreas in high numbers. CD8 + T cells are the main cell type found in the insulitic lesion, and CD8 + T cells reactive against β-cell antigens have been detected in peripheral blood and in the pancreas of patients with short- or long-term disease. In the Diabetes Virus Detection (DiViD) study, researchers collected pancreatic tissue, by pancreatic tail resection, from living patients with recent-onset T1D. These tissues have been extensively studied by the scientific community, but the autoreactive nature of the T-cell infiltrate has remained unexplored. Our objective was to determine the number and localization of these cells in pancreas samples obtained through the DiViD study. Here, we demonstrate the presence of high frequencies of CD8 + T cells reactive against a highly relevant epitope derived from the preproinsulin signal peptide in pancreatic tissue samples from these donors. We also show the heterogeneity of islet distribution and CD8 + T-cell infiltration. Our findings contribute to the current limited existing knowledge of T-cell reactivity in the pancreas of donors with recent-onset T1D and indicate that antigen-specific therapies directed toward preproinsulin could have high clinical impact., (© 2021 by the American Diabetes Association.)

NF-κB is a well-characterized transcription factor, widely known for its roles in inflammation and immune responses, as well as in control of cell division and apoptosis. However, its function in β-cells is still being debated, as it appears to depend on the timing and kinetics of its activation. To elucidate the temporal role of NF-κB in vivo, we have generated two transgenic mouse models, the ToIβ and NOD/ToIβ mice, in which NF-κB activation is specifically and conditionally inhibited in β-cells. In this study, we present a novel function of the canonical NF-κB pathway during murine islet β-cell development. Interestingly, inhibiting the NF-κB pathway in β-cells during embryogenesis, but not after birth, in both ToIβ and NOD/ToIβ mice, increased β-cell turnover, ultimately resulting in a reduced β-cell mass. On the NOD background, this was associated with a marked increase in insulitis and diabetes incidence. While a robust nuclear immunoreactivity of the NF-κB p65-subunit was found in neonatal β-cells, significant activation was not detected in β-cells of either adult NOD/ToIβ mice or in the pancreata of recently diagnosed adult T1D patients. Moreover, in NOD/ToIβ mice, inhibiting NF-κB post-weaning had no effect on the development of diabetes or β-cell dysfunction. In conclusion, our data point to NF-κB as an important component of the physiological regulatory circuit that controls the balance of β-cell proliferation and apoptosis in the early developmental stages of insulin-producing cells, thus modulating β-cell mass and the development of diabetes in the mouse model of T1D.

C-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine specifically recognized by the ligand receptor CXCR3 which is mostly expressed in T-lymphocytes. Although CXCL10 expression and secretion have been widely associated to pancreatic islets both in non-obese diabetic (NOD) mice and in human type 1 diabetic (T1D) donors, the specific expression pattern among pancreatic endocrine cell subtypes has not been clarified yet. Therefore, the purpose of this study was to shed light on the pancreatic islet expression of CXCL10 in NOD, in C57Bl/6J and in NOD-SCID mice as well as in human T1D pancreata from new-onset T1D patients (DiViD study) compared to non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD). CXCL10 was expressed in pancreatic islets of normoglycaemic and new-onset diabetic NOD mice but not in C57Bl/6J and NOD-SCID mice. CXCL10 expression was increased in pancreatic islets of new-onset diabetic NOD mice compared to normoglycaemic NOD mice. In NOD mice, CXCL10 colocalized both with insulin and glucagon. Interestingly, CXCL10-glucagon colocalization rate was significantly increased in diabetic vs. normoglycaemic NOD mouse islets, indicating an increased expression of CXCL10 also in alpha-cells. CXCL10 was expressed in pancreatic islets of T1D patients but not in non-diabetic donors. The analysis of the expression pattern of CXCL10 in human T1D pancreata from DiViD study, revealed an increased colocalization rate with glucagon compared to insulin. Of note, CXCL10 was also expressed in alpha-cells residing in insulin-deficient islets (IDI), suggesting that CXCL10 expression in alpha cells is not driven by residual beta-cells and therefore may represent an independent phenomenon. In conclusion, we show that in T1D CXCL10 is expressed by alpha-cells both in NOD mice and in T1D patients, thus pointing to an additional novel role for alpha-cells in T1D pathogenesis and progression., (Copyright © 2020 Nigi, Brusco, Grieco, Licata, Krogvold, Marselli, Gysemans, Overbergh, Marchetti, Mathieu, Dahl Jørgensen, Sebastiani and Dotta.)

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells.

IL-6 is a pro-inflammatory cytokine upregulated in some autoimmune diseases. The role of IL-6 in the development of type 1 diabetes (T1D) is unclear. Clinical studies are investigating whether tocilizumab (anti-IL-6 receptor) can help preserve beta cell function in patients recently diagnosed with T1D. However, in some rodent models and isolated human islets, IL-6 has been found to have a protective role for beta cells by reducing oxidative stress. Hence, we systematically investigated local tissue expression of IL-6 in human pancreas from non-diabetic, auto-antibody positive donors and donors with T1D and T2D. IL-6 was constitutively expressed by beta and alpha cells regardless of the disease state. However, expression of IL-6 was highly reduced in insulin-deficient islets of donors with T1D, and the expression was then mostly restricted to alpha cells. Our findings suggest that the implication of IL-6 in T1D pathogenesis might be more complex than previously assumed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Insulin deficiency in type 1 diabetes (T1D) is generally considered a consequence of immune-mediated specific beta-cell loss. Since healthy pancreatic islets consist of ~65% beta cells, this would lead to reduced islet size, while the number of islets per pancreas volume (islet density) would not be affected. In this study, we compared the islet density, size, and size distribution in biopsies from subjects with recent-onset or long-standing T1D, with that in matched non-diabetic subjects. The results presented show preserved islet size and islet size distribution, but a marked reduction in islet density in subjects with recent onset T1D compared with non-diabetic subjects. No further reduction in islet density occurred with increased disease duration. Insulin-negative islets in T1D subjects were dominated by glucagon-positive cells that often had lost the alpha-cell transcription factor ARX while instead expressing PDX1, normally only expressed in beta cells within the islets. Based on our findings, we propose that failure to establish a sufficient islet number to reach the beta-cell mass needed to cope with episodes of increased insulin demand contributes to T1D susceptibility. Exhaustion induced by relative lack of beta cells could then potentially drive beta-cell dedifferentiation to alpha-cells, explaining the preserved islet size observed in T1D compared to controls., (© 2019 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Type 1 diabetes studies consistently generate data showing islet β-cell dysfunction and T cell-mediated anti-β-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the β-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single β-cells. Consistent with immunohistological studies, β-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These β-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 - β-cells, specifically found in islets with lymphocytic infiltrates. β-Cell surface expression of HLA Class II was detected on a portion of CD45 - insulin + β-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic β-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. β-Cell expression of Class II molecules suggests that β-cells may interact directly with islet-infiltrating CD4 + T cells and may play an immunopathogenic role., (© 2019 by the American Diabetes Association.)

Type 1 diabetes (T1D) is an autoimmune disease where pancreatic β-cells are destroyed by islet-infiltrating T cells. Although a role for β-cell defects has been suspected, β-cell abnormalities are difficult to demonstrate. We show a β-cell DNA damage response (DDR), presented by activation of the 53BP1 protein and accumulation of p53, in biopsy and autopsy material from patients with recently diagnosed T1D as well as a rat model of human T1D. The β-cell DDR is more frequent in islets infiltrated by CD45 + immune cells, suggesting a link to islet inflammation. The β-cell toxin streptozotocin (STZ) elicits DDR in islets, both in vivo and ex vivo, and causes elevation of the proinflammatory molecules IL-1β and Cxcl10. β-Cell-specific inactivation of the master DNA repair gene ataxia telangiectasia mutated (ATM) in STZ-treated mice decreases the expression of proinflammatory cytokines in islets and attenuates the development of hyperglycemia. Together, these data suggest that β-cell DDR is an early event in T1D, possibly contributing to autoimmunity., (© 2018 by the American Diabetes Association.)

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-βH1, 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-1β, induced PDL1 expression in vitro in human islet cells and EndoC-βH1 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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Neutrophils and their inflammatory mediators are key pathogenic components in multiple autoimmune diseases, while their role in human type 1 diabetes (T1D), a disease that progresses sequentially through identifiable stages prior to the clinical onset, is not well understood. We previously reported that the number of circulating neutrophils is reduced in patients with T1D and in presymptomatic at-risk subjects. The aim of the present work was to identify possible changes in circulating and pancreas-residing neutrophils throughout the disease course to better elucidate neutrophil involvement in human T1D., Methods: Data collected from 389 subjects at risk of developing T1D, and enrolled in 4 distinct studies performed by TrialNet, were analyzed with comprehensive statistical approaches to determine whether the number of circulating neutrophils correlates with pancreas function. To obtain a broad analysis of pancreas-infiltrating neutrophils throughout all disease stages, pancreas sections collected worldwide from 4 different cohorts (i.e., nPOD, DiViD, Siena, and Exeter) were analyzed by immunohistochemistry and immunofluorescence. Finally, circulating neutrophils were purified from unrelated nondiabetic subjects and donors at various T1D stages and their transcriptomic signature was determined by RNA sequencing., Results: Here, we show that the decline in β cell function is greatest in individuals with the lowest peripheral neutrophil numbers. Neutrophils infiltrate the pancreas prior to the onset of symptoms and they continue to do so as the disease progresses. Of interest, a fraction of these pancreas-infiltrating neutrophils also extrudes neutrophil extracellular traps (NETs), suggesting a tissue-specific pathogenic role. Whole-transcriptome analysis of purified blood neutrophils revealed a unique molecular signature that is distinguished by an overabundance of IFN-associated genes; despite being healthy, said signature is already present in T1D-autoantibody-negative at-risk subjects., Conclusions: These results reveal an unexpected abnormality in neutrophil disposition both in the circulation and in the pancreas of presymptomatic and symptomatic T1D subjects, implying that targeting neutrophils might represent a previously unrecognized therapeutic modality., Funding: Juvenile Diabetes Research Foundation (JDRF), NIH, Diabetes UK.

Indoleamine 2,3 dioxygenase-1 (IDO1) is a powerful immunoregulatory enzyme that is deficient in patients with type 1 diabetes (T1D). In this study, we present the first systematic evaluation of IDO1 expression and localization in human pancreatic tissue. Although IDO1 was constitutively expressed in β-cells from donors without diabetes, less IDO1 was expressed in insulin-containing islets from double autoantibody-positive donors and patients with recent-onset T1D, although it was virtually absent in insulin-deficient islets from donors with T1D. Scatter plot analysis suggested that IDO1 decay occurred in individuals with multiple autoantibodies, prior to β-cell demise. IDO1 impairment might therefore contribute to β-cell demise and could potentially emerge as a promising therapeutic target., (© 2018 by the American Diabetes Association.)

Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize bacteria-infected cells and are thought to play a role in autoimmune diseases. Translocation of duodenal bacteria and viruses to the pancreas through the pancreatic duct has been hypothesized to initiate an innate inflammatory response that could contribute to the development of type 1 diabetes, a process that could involve MAIT cells. In this study, we used immunohistochemistry and quantitative PCR to search for evidence of MAIT cells in the insulitic lesions in the pancreas of human patients recently diagnosed with type 1 diabetes. Only a few scattered MAIT cells were found within the exocrine parenchyma in all pancreatic samples, but no MAIT cells were found in association to the islets. Also, only low gene expression levels of the MAIT T-cell receptor Vα7.2-Jα33 were found in the pancreas of patients with type 1 diabetes, in similar levels as that in nondiabetic organ donors used as control. The absence of MAIT cells shown in insulitic lesions in humans questions the direct cytotoxic role of these cells in β-cell destruction., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aims/hypothesis: Sphingolipids play important roles in beta cell physiology, by regulating proinsulin folding and insulin secretion and in controlling apoptosis, as studied in animal models and cell cultures. Here we investigate whether sphingolipid metabolism may contribute to the pathogenesis of human type 1 diabetes and whether increasing the levels of the sphingolipid sulfatide would prevent models of diabetes in NOD mice., Methods: We examined the amount and distribution of sulfatide in human pancreatic islets by immunohistochemistry, immunofluorescence and electron microscopy. Transcriptional analysis was used to evaluate expression of sphingolipid-related genes in isolated human islets. Genome-wide association studies (GWAS) and a T cell proliferation assay were used to identify type 1 diabetes related polymorphisms and test how these affect cellular islet autoimmunity. Finally, we treated NOD mice with fenofibrate, a known activator of sulfatide biosynthesis, to evaluate the effect on experimental autoimmune diabetes development., Results: We found reduced amounts of sulfatide, 23% of the levels in control participants, in pancreatic islets of individuals with newly diagnosed type 1 diabetes, which were associated with reduced expression of enzymes involved in sphingolipid metabolism. Next, we discovered eight gene polymorphisms (ORMDL3, SPHK2, B4GALNT1, SLC1A5, GALC, PPARD, PPARG and B4GALT1) involved in sphingolipid metabolism that contribute to the genetic predisposition to type 1 diabetes. These gene polymorphisms correlated with the degree of cellular islet autoimmunity in a cohort of individuals with type 1 diabetes. Finally, using fenofibrate, which activates sulfatide biosynthesis, we completely prevented diabetes in NOD mice and even reversed the disease in half of otherwise diabetic animals., Conclusions/interpretation: These results indicate that islet sphingolipid metabolism is abnormal in type 1 diabetes and suggest that modulation may represent a novel therapeutic approach., Data Availability: The RNA expression data is available online at https://www.dropbox.com/s/93mk5tzl5fdyo6b/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%2C%20RNA%20expression.xlsx?dl=0 . A list of SNPs identified is available at https://www.dropbox.com/s/yfojma9xanpp2ju/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%20SNP.xlsx?dl=0 .

Aims/hypothesis: Although IL-1β is considered a key mediator of beta cell destruction, its cellular expression in islets during early type 1 diabetes remains unclear. We compared its expression in rare pancreatic biopsies from new-onset living volunteers with its expression in cadaveric pancreas sections from non-diabetic autoantibody-positive and -negative individuals and those with long-standing disease., Methods: Pancreatic biopsy sections from six new-onset living volunteers (group 1) and cadaveric sections from 13 non-diabetic autoantibody-negative donors (group 2), four non-diabetic autoantibody-positive donors (group 3) and nine donors with diabetes of longer duration (0.25-12 years of disease; group 4) were triple-immunostained for IL-1β, insulin and glucagon. Intra- and peri-islet IL-1β-positive cells in insulin-positive and -negative islets and in random exocrine fields were enumerated., Results: The mean number of IL-1β-positive cells per islet from each donor in peri- and intra-islet regions was <1.25 and <0.5, respectively. In all study groups, the percentage of islets with IL-1β cells in peri- and/or intra-islet regions was highly variable and ranged from 4.48% to 17.59% in group 1, 1.42% to 44.26% in group 2, 7.93% to 17.53% in group 3 and 3.85% to 42.86% in group 4, except in a single case where the value was 75%. In 25/32 donors, a higher percentage of islets showed IL-1β-positive cells in peri-islet than in intra-islet regions. In sections from diabetic donors (groups 1 and 4), a higher mean number of IL-1β-positive cells occurred in insulin-positive islets than in insulin-negative islets. In group 2, 70-90% of islets in 3/13 sections had weak-to-moderate IL-1β staining in alpha cells but staining was virtually absent or substantially reduced in the remaining groups. The mean number of exocrine IL-1β-positive cells in group 1 was lower than in the other groups., Conclusions/interpretation: At onset of type 1 diabetes, the low number of islet-associated IL-1β-positive cells may be insufficient to elicit beta cell destruction. The variable expression in alpha cells in groups 2-4 suggests their cellular heterogeneity and probable physiological role. The significance of a higher but variable number of exocrine IL-1β-positive cells seen in non-diabetic individuals and those with long-term type 1 diabetes remains unclear.

To elucidate the etiology of type 1 diabetes, the affected pancreas needs to be thoroughly characterized. Pancreatic innervation has been suggested to be involved in the pathology of the disease and a reduction of sympathetic innervation of the islets was recently reported. In the present study, we hypothesized that parasympathetic innervation would be altered in the type 1 diabetes pancreas. Human pancreatic specimens were obtained from a unique cohort of individuals with recent onset or long standing type 1 diabetes. Density of parasympathetic axons was assessed by immunofluorescence and morphometry. Our main finding was a reduced density of parasympathetic axons in the exocrine, but not endocrine compartment of the pancreas in individuals with recent onset type 1 diabetes. The reduced density of parasympathetic axons in the exocrine compartment could have functional implications, e.g. be related to the exocrine insufficiency reported in type 1 diabetes patients. Further studies are needed to understand whether reduced parasympathetic innervation is a cause or consequence of type 1 diabetes.

Peroxynitrite-induced nitration of cellular proteins has been shown to associate with various human pathologies. The expression of pancreatic nitrotyrosine and its cellular source relative to insulitis were analysed in cases with increasing duration of type 1 diabetes and compared with non-diabetic autoantibody-negative and -positive cases. Pancreatic tail sections from non-diabetic autoantibody-negative cases (Group 1; n = 7), non-diabetic autoantibody-positive cases (Group 2; n = 6), recently diagnosed cases (Group 3; n = 6), 0.25-5 years of diabetes (Group 4; n = 8) and 7-12 years of diabetes (Group 5; n = 6) were immunostained sequentially for nitrotyrosine, insulin and leucocytes. Nitrotyrosine expression was observed in selective beta cells only. In group 1, the percentage of insulin-positive islets with nitrotyrosine ranged from 7.6 to 58.8%. In group 2, it was minimally expressed in 2 cases and was present in 4.7-19.3% of insulin-positive islets in 3 cases and in all islets in 1 case. In group 3, it was absent in 1 case and in the remaining 5 cases, the values were 17.4-85.7%. In group 4, nitrotyrosine was absent in 6 cases and positive in 1.8 and 22.2% of insulin-positive islets in 2 cases. In group 5, the values were 60% (1 case) and 100% (2 cases), being absent in 3 cases, consistent with insulin-negativity. This case analysis shows that nitrotyrosine immunostaining is independent of the presence and severity of insulitis. Variable nitrotyrosine expression is present in some non-diabetic cases. Its increased expression in beta cells of recent-onset and long-standing disease requires further studies to determine whether beta cell nitration plays a pathogenic role during T1D.

Type 1 diabetes is characterized by the loss of insulin production caused by β-cell dysfunction and/or destruction. The hypothesis that β-cell loss occurs early during the prediabetic phase has recently been challenged. Here we show, for the first time in situ, that in pancreas sections from autoantibody-positive (Ab+) donors, insulin area and β-cell mass are maintained before disease onset and that production of proinsulin increases. This suggests that β-cell destruction occurs more precipitously than previously assumed. Indeed, the pancreatic proinsulin-to-insulin area ratio was also increased in these donors with prediabetes. Using high-resolution confocal microscopy, we found a high accumulation of vesicles containing proinsulin in β-cells from Ab+ donors, suggesting a defect in proinsulin conversion or an accumulation of immature vesicles caused by an increase in insulin demand and/or a dysfunction in vesicular trafficking. In addition, islets from Ab+ donors were larger and contained a higher number of β-cells per islet. Our data indicate that β-cell mass (and function) is maintained until shortly before diagnosis and declines rapidly at the time of clinical onset of disease. This suggests that secondary prevention before onset, when β-cell mass is still intact, could be a successful therapeutic strategy., (© 2017 by the American Diabetes Association.)

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. As CAKUT is a genetically heterogeneous disorder and most cases are genetically unexplained, we aimed to identify new CAKUT causing genes. Using whole-exome sequencing and trio-based de novo analysis, we identified a novel heterozygous de novo frameshift variant in the leukemia inhibitory factor receptor (LIFR) gene causing instability of the mRNA in a patient presenting with bilateral CAKUT and requiring kidney transplantation at one year of age. LIFR encodes a transmembrane receptor utilized by IL-6 family cytokines, mainly by the leukemia inhibitory factor (LIF). Mutational analysis of 121 further patients with severe CAKUT yielded two rare heterozygous LIFR missense variants predicted to be pathogenic in three unrelated patients. LIFR mutants showed decreased half-life and cell membrane localization resulting in reduced LIF-stimulated STAT3 phosphorylation. LIFR showed high expression in human fetal kidney and the human ureter, and was also expressed in the developing murine urogenital system. Lifr knockout mice displayed urinary tract malformations including hydronephrosis, hydroureter, ureter ectopia, and, consistently, reduced ureteral lumen and muscular hypertrophy, similar to the phenotypes observed in patients carrying LIFR variants. Additionally, a form of cryptorchidism was detected in all Lifr-/- mice and the patient carrying the LIFR frameshift mutation. Altogether, we demonstrate heterozygous novel or rare LIFR mutations in 3.3% of CAKUT patients, and provide evidence that Lifr deficiency and deactivating LIFR mutations cause highly similar anomalies of the urogenital tract in mice and humans., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Subtypes of CD8 + T cells in insulitic lesions in biopsy specimens from six subjects with recent-onset type 1 diabetes (T1D) and six nondiabetic matched controls were analyzed using simultaneous multicolor immunofluorescence. Also, insulitic islets based on accumulation of CD3 + T cells were microdissected with laser-capture microscopy, and gene transcripts associated with inflammation and autoimmunity were analyzed. We found a substantial proportion, 43%, of the CD8 + T cells in the insulitic lesions to display a tissue resident memory T cell (T RM ) (CD8 + CD69 + CD103 + ) phenotype in T1D subjects. Most T RM cells were located in the insulitic lesion in the endocrine-exocrine interface. T RM cells were also sporadically found in islets of control subjects. Moreover, gene expression analysis showed a lack of active transcription of genes associated with acute inflammatory or cytotoxic T-cell responses. We present evidence that a substantial proportion of T cells in insulitic lesions of recent-onset T1D patients are T RM cells and not classic cytotoxic CD8 + T cells. Our findings highlight the need for further analysis of the T cells involved in insulitis to elucidate their role in the etiology of T1D., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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 β2-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.

A primary insult to the pancreatic islets of Langerhans, leading to the activation of innate immunity, has been suggested as an important step in the inflammatory process in type 1 diabetes (T1D). The aim of this study was to examine whether interferon (IFN)-stimulated genes (ISGs) are overexpressed in human T1D islets affected with insulitis. By using laser capture microdissection and a quantitative PCR array, 23 of 84 examined ISGs were found to be overexpressed by at least fivefold in insulitic islets from living patients with recent-onset T1D, participating in the Diabetes Virus Detection (DiViD) study, compared with islets from organ donors without diabetes. Most of the overexpressed ISGs, including GBP1, TLR3, OAS1, EIF2AK2, HLA-E, IFI6, and STAT1, showed higher expression in the islet core compared with the peri-islet area containing the surrounding immune cells. In contrast, the T-cell attractant chemokine CXCL10 showed an almost 10-fold higher expression in the peri-islet area than in the islet, possibly partly explaining the localization of T cells mainly to this region. In conclusion, insulitic islets from recent-onset T1D subjects show overexpression of ISGs, with an expression pattern similar to that seen in islets infected with virus or exposed to IFN-γ/interleukin-1β or IFN-α., (© 2016 by the American Diabetes Association.)

Type 1 diabetes (T1D) results from a T cell-mediated destruction of pancreatic β-cells following the infiltration of leukocytes (including CD8(+), CD4(+), and CD20(+) cells) into and around pancreatic islets (insulitis). Recently, we reported that two distinct patterns of insulitis occur in patients with recent-onset T1D from the U.K. and that these differ principally in the proportion of infiltrating CD20(+) B cells (designated CD20Hi and CD20Lo, respectively). We have now extended this analysis to include patients from the Network for Pancreatic Organ Donors with Diabetes (U.S.) and Diabetes Virus Detection (DiViD) study (Norway) cohorts and confirm that the two profiles of insulitis occur more widely. Moreover, we show that patients can be directly stratified according to their insulitic profile and that those receiving a diagnosis before the age of 7 years always display the CD20Hi profile. By contrast, individuals who received a diagnosis beyond the age of 13 years are uniformly defined as CD20Lo. This implies that the two forms of insulitis are differentially aggressive and that patients with a CD20Hi profile lose their β-cells at a more rapid rate. In support of this, we also find that the proportion of residual insulin-containing islets (ICIs) increases in parallel with age at the onset of T1D. Importantly, those receiving a diagnosis in, or beyond, their teenage years retain ∼40% ICIs at diagnosis, implying that a functional deficit rather than an absolute β-cell loss may be causal for disease onset in these patients. We conclude that appropriate patient stratification will be critical for correct interpretation of the outcomes of intervention therapies targeted to islet-infiltrating immune cells in T1D., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)