| 1. |
- Lin, Jake, et al.
(author)
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Distinct transcriptomic profiles in children prior to the appearance of type 1 diabetes-linked islet autoantibodies and following enterovirus infection
- 2023
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In: Nature Communications. - 2041-1723. ; 14, s. 1-13
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Journal article (peer-reviewed)abstract
- Although the genetic basis and pathogenesis of type 1 diabetes have been studied extensively, how host responses to environmental factors might contribute to autoantibody development remains largely unknown. Here, we use longitudinal blood transcriptome sequencing data to characterize host responses in children within 12 months prior to the appearance of type 1 diabetes-linked islet autoantibodies, as well as matched control children. We report that children who present with insulin-specific autoantibodies first have distinct transcriptional profiles from those who develop GADA autoantibodies first. In particular, gene dosage-driven expression of GSTM1 is associated with GADA autoantibody positivity. Moreover, compared with controls, we observe increased monocyte and decreased B cell proportions 9-12 months prior to autoantibody positivity, especially in children who developed antibodies against insulin first. Lastly, we show that control children present transcriptional signatures consistent with robust immune responses to enterovirus infection, whereas children who later developed islet autoimmunity do not. These findings highlight distinct immune-related transcriptomic differences between case and control children prior to case progression to islet autoimmunity and uncover deficient antiviral response in children who later develop islet autoimmunity.
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| 2. |
- Busse, Niels, et al.
(author)
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Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes
- 2017
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In: Oncotarget. - : IMPACT JOURNALS LLC. - 1949-2553. ; 8:8, s. 12620-12636
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Journal article (peer-reviewed)abstract
- Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed. To detect the presence of very low copies of the virus genome in tissue samples from T1D patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family. With these probes enteroviral RNA was detected with high sensitivity and specificity in infected cells and tissues, including in FFPE pancreas sections from patients with T1D. Detection was not impeded by variations in sample processing and storage thereby overcoming the potential limitations of fragmented RNA. Co-staining of small RNA probes in parallel with classical immunstaining enabled virus detection in a cell-specific manner and more sensitively than by viral protein.
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| 3. |
- Krogvold, Lars, et al.
(author)
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Detection of a low-grade enteroviral infection in the islets of Langerhans of living patients newly diagnosed with type 1 diabetes
- 2015
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In: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 64:5, s. 1682-1687
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Journal article (peer-reviewed)abstract
- The Diabetes Virus Detection study (DiViD) is the first to examine fresh pancreatic tissue at the diagnosis of type 1 diabetes for the presence of viruses. Minimal pancreatic tail resection was performed 3-9 weeks after onset of type 1 diabetes in 6 adult patients (age 24-35 years). The presence of enteroviral capsid protein 1 (VP1) and the expression of class I HLA were investigated by immunohistochemistry. Enterovirus RNA was analyzed from isolated pancreatic islets and from fresh frozen whole pancreatic tissue using PCR and sequencing. Non-diabetic organ donors served as controls. VP1 was detected in the islets of all type 1 diabetes patients (2 of 9 controls). Hyperexpression of class I HLA molecules was found in the islets of all patients (1 of 9 controls). Enterovirus specific RNA sequences were detected in 4 of 6 cases (0 of 6 controls). The results were confirmed in different laboratories. Only 1.7 % of the islets contained VP1 positive cells and the amount of enterovirus RNA was low. The results provides evidence for the presence of enterovirus in pancreatic islets of type 1 diabetic patients, being consistent with the possibility that a low grade enteroviral infection in the pancreatic islets contribute to disease progression in humans.
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| 4. |
- Laiho, Jutta E., et al.
(author)
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Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells
- 2016
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In: Journal of Clinical Virology. - : Elsevier BV. - 1386-6532 .- 1873-5967. ; 77, s. 21-28
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Journal article (peer-reviewed)abstract
- Background: Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. Objectives: This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 ( CVB1), in acutely infected human A549 cells in vitro. Study design: A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10-1 to 10-8. Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. Results: RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10-8). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10-7. The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10(-6), while ISH detected the virus at dilutions of 10(-4). Conclusions: All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.
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| 5. |
- Oikarinen, Sami, et al.
(author)
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Characterisation of enterovirus RNA detected in the pancreas and other specimens of live patients with newly diagnosed type 1 diabetes in the DiViD study
- 2021
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In: Diabetologia. - : Springer Nature. - 0012-186X .- 1432-0428. ; 64:11, s. 2491-2501
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Richardson, Sarah J., et al.
(author)
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Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas
- 2014
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In: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 57:2, s. 392-401
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Journal article (peer-reviewed)abstract
- Aims/hypothesisEnteroviral 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.MethodsEnteroviral 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.ResultsClone 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/interpretationWhen 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.
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