SummaryThe immunopathology of type 1 diabetes (T1D) has proved difficult to study in man because of the limited availability of appropriate samples, but we now report a detailed study charting the evolution of insulitis in human T1D. Pancreas samples removed post-mortem from 29 patients (mean age 11·7 years) with recent-onset T1D were analysed by immunohistochemistry. The cell types constituting the inflammatory infiltrate within islets (insulitis) were determined in parallel with islet insulin content. CD8+ cytotoxic T cells were the most abundant population during insulitis. Macrophages (CD68 + ) were also present during both early and later insulitis, although in fewer numbers. CD20+ cells were present in only small numbers in early insulitis but were recruited to islets as beta cell death progressed. CD138+ plasma cells were infrequent at all stages of insulitis. CD4+ cells were present in the islet infiltrate in all patients but were less abundant than CD8 + or CD68 + cells. Forkhead box protein P3+ regulatory T cells were detected in the islets of only a single patient. Natural killer cells were detected rarely, even in heavily inflamed islets. The results suggest a defined sequence of immune cell recruitment in human T1D. They imply that both CD8+ cytotoxic cells and macrophages may contribute to beta cell death during early insulitis. CD20+ cells are recruited in greatest numbers during late insulitis, suggesting an increasing role for these cells as insulitis develops. Natural killer cells and forkhead box protein P3 + T cells do not appear to be required for beta cell death.

To the Editor: Recently evidence has emerged that supports a role for islet inflammation in the development of type 2 diabetes in man, suggesting that there may be certain common features underlying the pathology of beta cell loss in both type 1 and type 2 diabetes. In particular, data have recently been presented revealing an increased number of macrophages infiltrating the islets of nine type 2 diabetic patients, as well as in several animal models of type 2 diabetes (including high-fat-fed C57BL6/6J mice, GK rats and the db/db mouse) when compared with relevant controls [1]. Those authors argued that this evidence implies that macrophage infiltration could be involved in mediating beta cell dysfunction and loss in type 2 diabetes. In view of these conclusions, we considered it important to verify whether increased macrophage infiltration is also observed in a different and larger cohort of human patients with type 2 diabetes and to assess the magnitude of this response.Serial sections of paraffin-embedded pancreas recovered at autopsy from 15 type 2 diabetic patients (mean age [±SEM] 69.2±1.8 years) and 16 non-diabetic controls (age 52.9±3.9 years) were processed and stained with antiinsulin and anti-CD68 antibodies (DAKO, Ely, UK) using a standard immunoperoxidase technique. The use of all tissue was undertaken with full ethical permission. A quantitative analysis of up to 50 randomly selected islets per individual was carried out and the number of CD68 + cells (taken to indicate the presence of macrophages) either within the islets or in the peri-islet area was counted. Statistical comparisons were performed by χ 2 analysis. Within the control group, we did not observe any tendency for the number of macrophages present within islets to change with age. Therefore we consider that, although the mean age of the type 2 diabetic patients was slightly lower than the controls, this difference per se is unlikely to account for variations in macrophage infiltration. In order to confirm that the number of CD68 + cells counted per islet was not distorted by a change in islet size or area in the patients vs controls, random images were examined from slides stained for insulin from six cases of type 2 diabetes (mean age 62.7± 2.3 years) and four controls (mean age 64.0 ± 2.6 years). This revealed that the percentage of pancreatic tissue occupied by endocrine cells was similar in the two groups (1.99±0.23% in type 2 diabetes; 2.17±0.32% in controls). In addition, the mean endocrine cell area within the islets was also unchanged in the sections studied, implying that the overall size of the islets was not decreased in the cohort of patients with type 2 diabetes compared with the controls. A total of 545 and 564 islets were analysed Diabetologia

Type 1 diabetes is a chronic autoimmune disease characterised by the selective destruction of pancreatic beta (β) cells. The understanding of the aetiology of this disease has increased dramatically in recent years by the study of tissue recovered from patients, from analysis of the responses of isolated islet and β-cells in tissue culture and via the use of animal models. However, knowledge of the immunopathology of type 1 diabetes in humans is still relatively deficient due largely to the difficulty of accessing appropriate samples. Here we review the state of current knowledge in relation to the histopathological features of the disease in humans. We focus specifically on recent-onset type 1 diabetes cases since in such patients, evidence of the ongoing disease process is still present. We chart the progression of the disease by describing the characteristic features of the pancreas, consider the sequence of immune cell infiltration and discuss the abnormalities of MHC antigen expression. The possibility that these changes might derive from a persistent enteroviral infection of the islet beta cells is examined.

Cell-to-cell interactions play an important role in the development and maintenance of the beta-cell phenotype. Here, we have investigated whether E-cadherin plays a role in regulating the growth of insulin-secreting MIN6 cells configured as three-dimensional islet-like clusters (pseudoislets). Pseudoislets form by cell aggregation rather than by proliferation from individual cells and attain the size of primary mouse islets after approximately 7 days of maintenance in culture. E-cadherin is known to mediate homotypic cell adhesion between beta-cells and has also been implicated in a number of cellular processes, including proliferation, apoptosis, and differentiation. E-cadherin and its associated intracellular elements, alpha- and beta-catenin, were upregulated in MIN6 pseudoislets. Pseudoislet formation was associated with an increased expression of cyclin-dependent kinase inhibitors and a concomitant downregulation of Ki67, suggesting an overall reduction in cellular proliferation. However, measurements of 5-bromo-2'-deoxyuridine incorporation revealed that there were no differences in the rate of MIN6 cell proliferation whether they were configured as monolayers or as pseudoislets, which is likely to be a result of their being a transformed cell line. Cells within pseudoislets were not necrotic, but apoptosis appeared to be upregulated in the islet-like structures. However, no differential expression of Fas and FasL was detected in monolayers and pseudoislets. These results suggest that cell-to-cell interactions within islet-like structures may initiate antiproliferative and proapoptotic signals.

Aims/hypothesis Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes.Methods Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. Results Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1.Electronic supplementary material The online version of this article (doi:10.1007/s00125-013-3094-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.