Early Detection of Peripheral Blood Cell Signature in Children Developing Beta-Cell Autoimmunity at a Young Age

The appearance of Type 1 diabetes (T1D)-associated autoantibodies is the first and only measurable parameter to predict progression toward T1D in genetically susceptible individuals. However, autoantibodies indicate an active autoimmune reaction, wherein the immune tolerance is already broken. Therefore, there is a clear and urgent need for new biomarkers that predict the onset of the autoimmune reaction preceding autoantibody positivity or reflect progressive beta-cell destruction. Here we report the mRNA-sequencing-based analysis of 306 samples including fractionated samples of CD4+ and CD8+ T cells as well as CD4-CD8- cells fractions and unfractionated PBMC samples longitudinally collected from seven children that developed beta-cell autoimmunity (Cases) at a young age and their matched controls. We identified transcripts, including interleukin-32 (IL32) that were upregulated before T1D-associated autoantibodies appeared. Single-cell RNA-seq studies revealed that high IL32 in Case samples were contributed mainly by activated T cells and NK cells. Further, we showed that IL32 expression can be induced by a virus and cytokines in pancreatic islets and beta-cells, respectively. The results provide a basis for early detection of aberrations in the immune system function before T1D and suggest a potential role for IL32 in the pathogenesis of T1D.</p

Tissue Culture of Isolated Human Pancreatic Islets Infected With Different Strains of Coxsackievirus B4: Assessment of Virus Replication and Effects on Islet Morphology and Insulin Release

The aim was to study whether different strains of Coxsackievirus B4 (CBV-4) are able to infect human pancreatic islet cells in vitro and cause morphological and functional damages. Isolated islets maintained in tissue culture were infected with five well- characterised strains of CBV-4. Aliquots of the culture medium were analysed with regard to virus replication and insulin content. Infected and uninfected islets were examined by light microscopy to determine the degree of virus-induced cytopathic effect (CPE). The results showed that the islet cells were susceptible to infection by all the strains of CBV-4 although the outcome of the infection differed. The virus titres obtained at 48 and 72 hours post infection differed significantly between all the CBV-4 strains (p < 0.001), indicating different ability to replicate in islet cells. Pronounced to weak CPE, which was partly due to the origin (donor) of the islets, was induced by four of the five CBV-4 strains. One strain (VD2921) replicated without causing CPE despite high virus titres. One (V89-4557) of the CBV-4 strains always revealed pronounced CPE. Infection by this strain also caused functional impairment that significantly affected insulin response to high glucose at 48 hours post infection (p < 0.001). Replication of another CBV-4 strain (JVB) in the islet cells significantly increased the release of insulin compared to non-infected control cells (p < 0.001) indicating damage of the β-cells leading to leakage of insulin

Enterovirus infection of human islets of Langerhans affects beta-cell function resulting in disintegrated islets, decreased glucose stimulated insulin secretion and loss of Golgi structure

Aims/hypothesis: In type 1 diabetes (T1D), most insulin-producing beta cells are destroyed, but the trigger is unknown. One of the possible triggers is a virus infection and the aim of this study was to test if enterovirus infection affects glucose stimulated insulin secretion and the effect of virus replication on cellular macromolecules and organelles involved in insulin secretion. Methods: Isolated human islets were infected with different strains of coxsackievirus B (CVB) virus and the glucose-stimulated insulin release (GSIS) was measured in a dynamic perifusion system. Classical morphological electron microscopy, large-scale electron microscopy, so-called nanotomy, and immunohistochemistry were used to study to what extent virus-infected beta cells contained insulin, and real-time PCR was used to analyze virus induced changes of islet specific genes. Results: In islets infected with CVB, GSIS was reduced in correlation with the degree of virus-induced islet disintegration. The expression of the gene encoding insulin was decreased in infected islets, whereas the expression of glucagon was not affected. Also, in islets that were somewhat disintegrated, there were uninfected beta cells. Ultrastructural analysis revealed that virus particles and virus replication complexes were only present in beta cells. There was a significant number of insulin granules remaining in the virus-infected beta cells, despite decreased expression of insulin mRNA. In addition, no typical Golgi apparatus was detected in these cells. Exposure of islets to synthetic dsRNA potentiated glucose-stimulated insulin secretion. Conclusions/interpretation: Glucose-stimulated insulin secretion; organelles involved in insulin secretion and gene expression were all affected by CVB replication in beta cells

Echovirus 6 infects human exocrine and endocrine pancreatic cells and induces pro-inflammatory innate immune response

Human enteroviruses (HEV), especially coxsackievirus serotype B (CVB) and echovirus (E), have been associated with diseases of both the exocrine and endocrine pancreas, but so far evidence on HEV infection in human pancreas has been reported only in islets and ductal cells. This study aimed to investigate the capability of echovirus strains to infect human exocrine and endocrine pancreatic cells. Infection of explanted human islets and exocrine cells with seven field strains of E6 caused cytopathic effect, virus titer increase and production of HEV protein VP1 in both cell types. Virus particles were found in islets and acinar cells infected with E6. No cytopathic effect or infectious progeny production was observed in exocrine cells exposed to the beta cell-tropic strains of E16 and E30. Endocrine cells responded to E6, E16 and E30 by upregulating the transcription of interferon-induced with helicase C domain 1 (IF1H1), 2ʹ-5ʹ-oligoadenylate synthetase 1 (OAS1), interferon-β (IFN-β), chemokine (C-X-C motif) ligand 10 (CXCL10) and chemokine (C-C motif) ligand 5 (CCL5). Echovirus 6, but not E16 or E30, led to increased transcription of these genes in exocrine cells. These data demonstrate for the first time that human exocrine cells represent a target for E6 infection and suggest that certain HEV serotypes can replicate in human pancreatic exocrine cells, while the pancreatic endocrine cells are permissive to a wider range of HEV

Dendrograms showing phylogenetic relationships between sequenced E30 isolated during the Cuban epidemic of aseptic meningitis in 2001 and the E30 isolate of the GenBank sequence database.

<p>The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Strains of E30 known to be highly destructive of primary human insulin producing beta cells are shown in the tree by small black triangles.</p

Innate immunity gene expression in primary human islets cultured after three days of infection with clinical strains of E4, E16 and E30.

<p>Gene expression levels are presented as mRNA expression relative to expression of the housekeeping gene 18(2^_dct). Data are presented as means ± SD and were based on observations from at least three donors. ^†<i>P</i><0.05, between all groups (Islets infected with E4, E16, E30 and uninfected controls). *<i>P</i><0.05, Islets infected with E16 and E30 were compared to E4-inoculated islets and the uninfected control.</p

Virus-induced cytopathic effect in primary human pancreatic islets cells.

<p>A. Uninfected islet. B. Islets infected with the E4 isolate 5 days post infection. C. Islets infected with E16 isolates 3 days post infection. D. Islets infected with E30 isolates 3 days post infection. The figure is representative of seven islet donors.</p

Viral titers of the clinical strains of E16, E30 strains and E4 in the culture medium of infected primary human islets during 3 days post-infection.

<p>Aliquots of the culture medium were withdrawn day 0 and day 3. Virus titers were obtained using the cell culture infectious dose 50 (CCID50) titration methods. The results are shown as the means ± SD from experiments performed in triplicate.</p

Human enterovirus species B in ileocecal Crohn's disease

OBJECTIVES: Advanced ileocecal Crohn's disease (ICD) is characterized by strictures, inflammation in the enteric nervous system (myenteric plexitis), and a high frequency of NOD2 mutations. Recent findings implicate a role of NOD2 and another CD susceptibility gene, ATG16L1, in the host response against single-stranded RNA (ssRNA) viruses. However, the role of viruses in CD is unknown. We hypothesized that human enterovirus species B (HEV-B), which are ssRNA viruses with dual tropism both for the intestinal epithelium and the nervous system, could play a role in ICD. METHODS: We used immunohistochemistry and in situ hybridization to study the general presence of HEV-B and the presence of the two HEV-B subspecies, Coxsackie B virus (CBV) and Echovirus, in ileocecal resections from 9 children with advanced, stricturing ICD and 6 patients with volvulus, and in intestinal biopsies from 15 CD patients at the time of diagnosis. RESULTS: All patients with ICD had disease-associated polymorphisms in NOD2 or ATG16L1. Positive staining for HEV-B was detected both in the mucosa and in myenteric nerve ganglia in all ICD patients, but in none of the volvulus patients. Expression of the cellular receptor for CBV, CAR, was detected in nerve cell ganglia. CONCLUSIONS: The common presence of HEV-B in the mucosa and enteric nervous system of ICD patients in this small cohort is a novel finding that warrants further investigation to analyze whether HEV-B has a role in disease onset or progress. The presence of CAR in myenteric nerve cell ganglia provides a possible route of entry for CBV into the enteric nervous system.De två sista författarna delar sistaförfattarskapet.</p

Characterisation of enterovirus RNA detected in the pancreas and other specimens of live patients with newly diagnosed type 1 diabetes in the DiViD study

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 EVnegative 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.Funding Agencies|South-Eastern Norway Regional Health Authority; Novo Nordisk FoundationNovo Nordisk Foundation; Persistent Virus Infection in Diabetes Network (PEVNET) Study Group, the European Unions Seventh Framework Programme [FP7/2007-2013] [261441 PEVNET]; JDRF nPOD program; Sigrid Juselius FoundationSigrid Juselius Foundation; Diabetes Research Foundation in Finland; Academy of FinlandAcademy of FinlandEuropean Commission [288671]</p