Peter Seiron

Introduction: Despite a reduced function and volume of the exocrine pancreas in type 1 diabetes, the acinar cells remain understudied in type 1 diabetes research. The hypothesis of this study is that the acinar tissue is altered in subjects with type 1 diabetes compared with subjects without diabetes. Research design and methods: The cell density, expression of digestive enzymes, and transcriptome of acinar tissue at varying distances from islets were analyzed using histology, immunostaining, and AmpliSeq RNA sequencing of laser capture microdissected tissue. Pancreases examined were from organ donors with or without type 1 diabetes. Results: We demonstrate preserved acinar nuclei density and find no support of acinar atrophy in type 1 diabetes. Staining for digestive enzymes (amylase, lipase, and trypsin) demonstrated an evenly distributed expression in the exocrine parenchyma; although occasional amylase-negative regions appeared in tissue that had been formalin-fixed and paraffin-embedded, this phenomenon was not evident in frozen tissue. Gene set enrichment analysis of whole transcriptome data identified transcriptional alterations in type 1 diabetes that were present in the acinar tissue independent of the distance from islets. Among these, the two most enriched gene sets were Myc Targets V2 and Estrogen Response Early. Conclusion: Taken together, these new data emphasize the involvement of the entire pancreas in type 1 diabetes pathology. The alteration of the gene sets Myc Targets V2 and Estrogen Response Early is a possible link to the increased incidence of pancreatic cancer in type 1 diabetes.

Insulin secretion is impaired with increasing age. In this study, we aimed to determine whether aging induces specific transcriptional changes in human islets. Laser capture microdissection was used to extract pancreatic islet tissue from 37 deceased organ donors aged 1–81 years. The transcriptomes of the extracted islets were analysed using Ion AmpliSeq sequencing. 346 genes that co-vary significantly with age were found. There was an increased transcription of genes linked to senescence, and several aspects of the cell cycle machinery were downregulated with increasing age. We detected numerous genes not linked to aging in previous studies likely because earlier studies analysed islet cells isolated by enzymatic digestion which might affect the islet transcriptome. Among the novel genes demonstrated to correlate with age, we found an upregulation of SPP1 encoding osteopontin. In beta cells, osteopontin has been seen to be protective against both cytotoxicity and hyperglycaemia. In summary, we present a transcriptional profile of aging in human islets and identify genes that could affect disease course in diabetes.

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.

Insulin deficiency in type 1 diabetes (T1D) is generally considered a consequence of specific beta-cell loss. Since healthy pancreatic islets consist of ~65% beta cells, this would lead to reduced islet size if the beta cells are not replaced by other cells or tissue. 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 subjects with recent-onset or long-standing T1D, with that in matched non-diabetic subjects. Results show that subjects with T1D, regardless of disease duration, had a dramatically reduced islet number per mm ² , while the islet size was similar in all groups. Insulin-negative islets in T1D subjects were dominated by glucagon-positive cells that frequently had lost the alpha-cell transcription factor ARX while instead expressing PDX1, normally expressed in beta cells. Based on our findings, we propose that failure during childhood 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.

Aims/hypothesis According to the consensus criteria developed for type 1 diabetes, an individual can be diagnosed with insulitis when ≥ 15 CD45⁺ cells are found within the parenchyma or in the islet–exocrine interface in ≥ 3 islets. The aim of this study was to determine the frequency of individuals with type 2 diabetes fulfilling these criteria with reference to non-diabetic and type 1 diabetic individuals. Methods Insulitis was determined by examining CD45⁺ cells in the pancreases of 50, 13 and 44 organ donors with type 2 diabetes, type 1 diabetes and no diabetes, respectively. CD3⁺ cells (T cells) infiltrating the islets were evaluated in insulitic donors. In insulitic donors with type 2 diabetes, the pancreases were characterised according to the presence of CD68 (macrophages), myeloperoxidase (MPO; neutrophils), CD3, CD20 (B cells) and HLA class I hyperstained islets. In all type 2 diabetic donors, potential correlations of insulitis with dynamic glucose-stimulated insulin secretion in vitro or age, BMI, HbA_1c or autoantibody positivity were examined. Results Overall, 28% of the type 2 diabetic donors fulfilled the consensus criteria for insulitis developed for type 1 diabetes. Of the type 1 diabetic donors, 31% fulfilled the criteria. None of the non-diabetic donors met the criteria. Only type 1 diabetic donors had ≥ 15 CD3⁺ cells in ≥ 3 islets. Type 2 diabetic donors with insulitis also had a substantial number of CD45⁺ cells in the exocrine parenchyma. Macrophages constituted the largest fraction of CD45⁺ cells, followed by neutrophils and T cells. Of type 2 diabetic pancreases with insulitis, 36% contained islets that hyperstained for HLA class I. Isolated islets from type 2 diabetic donors secreted less insulin than controls, although with preserved dynamics. Insulitis in the type 2 diabetic donors did not correlate with glucose-stimulated insulin secretion, the presence of autoantibodies, BMI or HbA_1c. Conclusions/interpretation The current definition of insulitis cannot be used to distinguish pancreases retrieved from individuals with type 1 diabetes from those with type 2 diabetes. On the basis of our findings, we propose a revised definition of insulitis, with a positive diagnosis when ≥ 15 CD3⁺ cells, not CD45⁺ cells, are found in ≥ 3 islets.

Purpose: To investigate the cytokine composition and anti‐inflammatory effects of allogeneic serum preparations for improved use as serum eye drops.Methods: Serum of 15 healthy blood donors was extensively screened for cytokines, including IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐9, IL‐10, IL‐12p70, IL‐13, IL‐15, 1L‐17A, E and F, IL‐21, IL‐22, IL‐23, IL‐27, IL‐28A, IL‐31, IL‐33, granulocyte macrophage colony‐stimulating factor (GM‐CSF), chemokine ligand 20 (CCL20), tumour necrosis factor (TNF)‐α and TNF‐β, interferon (IFN)‐γ and transforming growth factor (TGF)‐β. The levels of cytokines were assessed before and after heat‐induced inactivation. Individual serum preparations were tested for their anti‐inflammatory effect using an in vitro test to differentiate effector T lymphocytes into anti‐inflammatory regulatory T cells.Results: The anti‐inflammatory cytokine TGF‐β was readily detected in the serum of all blood donors and was only modestly affected by heat‐induced inactivation. Serum containing high amounts of TGF‐β was more effective at inducing anti‐inflammatory regulatory T cells. The serum of one healthy blood donor displayed high levels of inflammatory cytokines.Conclusion: We propose that serum used as eye drops is screened for its cytokine content, making it possible to correlate the composition to the clinical outcome. Based on the findings in this study, tailored serum eye drops produced from allogeneic donors may provide increased anti‐inflammatory effects. This may be superior to autologous serum eye drops, which in many cases are retrieved from patients with inflammatory diseases.

With the increasing interest in clinical trials with regulatory T cells (Tregs), immunological profiling of prospective target groups and standardized procedures for Treg isolation are needed. In this study, flow cytometry was used to assess peripheral blood lymphocyte profiles of young healthy individuals and patients undergoing haemodialysis treatment. Tregs obtained from the former may be used in haematopoietic stem cell transplantation and Tregs from the latter in the prevention of kidney transplant rejection. FOXP3 mRNA expression with accompanying isoform distribution was also assessed by the quantitative reverse transcriptase polymerase chain reaction. Flow‐cytometric gating strategies were systematically analysed to optimize the isolation of Tregs. Our findings showed an overall similar immunological profile of both cohorts in spite of great differences in both age and health. Analysis of flow‐cytometric gating techniques highlighted the importance of gating for both CD25high and CD127low expression in the isolation of FOXP3‐positive cells. This study provides additional insight into the immunological profile of young healthy individuals and uraemic patients as well as in‐depth analysis of flow‐cytometric gating strategies for Treg isolation, supporting the development of Treg therapy using cells from healthy donors and uraemic patients.