Abby Willcox

Analysis of islet inflammation in human type 1 diabetes

The 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.

The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes

Aims/hypothesisEvidence that the beta cells of human patients with type 1 diabetes can be infected with enterovirus is accumulating, but it remains unclear whether such infections occur at high frequency and are important in the disease process. We have now assessed the prevalence of enteroviral capsid protein vp1 (vp1) staining in a large cohort of autopsy pancreases of recent-onset type 1 diabetic patients and a range of controls.MethodsSerial sections of paraffin-embedded pancreatic autopsy samples from 72 recent-onset type 1 diabetes patients and up to 161 controls were immunostained for insulin, glucagon, vp1, double-stranded RNA activated protein kinase R (PKR) and MHC class I.Resultsvp1-immunopositive cells were detected in multiple islets of 44 out of 72 young recent-onset type 1 diabetic patients, compared with a total of only three islets in three out of 50 neonatal and paediatric normal controls. vp1 staining was restricted to insulin-containing beta cells. Among the control pancreases, vp1 immunopositivity was also observed in some islets from ten out of 25 type 2 diabetic patients. A strong correlation was established between islet cell vp1 positivity and PKR production in insulin-containing islets of both type 1 and type 2 diabetic patients, consistent with a persistent viral infection of the islets.Conclusions/interpretationImmunoreactive vp1 is commonly found in the islets of recent-onset type 1 diabetes patients, but only rarely in normal paediatric controls. vp1 immunostaining was also observed in some islets of type 2 diabetes patients, suggesting that the phenomenon is not restricted to type 1 diabetes patients.

Islet-associated macrophages in type 2 diabetes

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 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 (2009) 52:1686–1688 DOI 10.1007/s00125-009-1410-z

Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes

Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of β-cell loss, responsiveness to immunotherapies, and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multiparameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly diagnosed diabetes. Multidimensional cluster analysis showed two equal-sized patient agglomerations characterized by proinflammatory (interferon-γ–positive, multiautoantibody-positive) and partially regulated (interleukin-10–positive, pauci-autoantibody–positive) responses. Multiautoantibody-positive nondiabetic siblings at high risk of disease progression showed similar clustering. Additionally, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesions distinguishable by the degree of cellular infiltrate and presence of B cells that we termed “hyper-immune CD20Hi” and “pauci-immune CD20Lo.” Of note, subjects had only one infiltration phenotype and were partitioned by this into two equal-sized groups that differed significantly by age at diagnosis, with hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment and prevention strategies.

Immunopathology of the human pancreas in type-I diabetes

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.

Differential Insulitic Profiles Determine the Extent of β-Cell Destruction and the Age at Onset of Type 1 Diabetes

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.

Immunohistochemical analysis of the relationship between islet cell proliferation and the production of the enteroviral capsid protein, VP1, in the islets of patients with recent-onset type 1 diabetes

Aims/hypothesisThe enteroviral capsid protein, VP1, was recently shown to be present in some beta cells in more than 60% of patients with recent-onset type 1 diabetes but in very few age-matched controls. The rate of proliferation of islet cells was also markedly increased in the type 1 diabetic patients. As it has been suggested that enteroviruses replicate most efficiently in proliferating cells, we have investigated whether VP1 is preferentially present in proliferating beta cells in type 1 diabetes.MethodsCombined immunoperoxidase and immunofluorescence staining was used to record the presence of enteroviral VP1, insulin and Ki67 in the islets of recent-onset type 1 diabetic patients.ResultsFrom a total of 1,175 islets, 359 (30.5%) contained insulin. VP1-producing endocrine cells were found in 72 islets (6.1% of total), all of which retained insulin. Ki67+ endocrine cells were present in 52 (4.4%) islets, with 44 (84.6%) of these being insulin-positive. Overall, 28 of 1,175 (2.4%) islets contained both Ki67+ cells and VP1+ cells. Dual positivity of these markers accounted for 38.9% of the total VP1+ islets and 53.8% of the total Ki67+ islets. No individual islet cells were dual-positive for Ki67 and VP1.Conclusions/interpretationKi67+ cells were frequently observed in islets that also contained VP1+ cells, suggesting that the factors facilitating viral replication may also drive islet cell proliferation. However, in an individual cell, VP1 production does not require concurrent beta cell proliferation.

Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue.

BACKGROUND The detection of viral infection in paraffin-embedded, formalin-fixed tissue is notoriously difficult and often requires inherent knowledge about the specific virus being sought. For this reason, there is an ongoing need for reagents and methods which can identify a range of different virus types in paraffin embedded tissue. OBJECTIVES The aim of this study was to optimise and validate the use of antisera directed against dsRNA (>50 bp in length) in paraffin-embedded formalin-fixed tissue samples. STUDY DESIGN dsRNA antisera were optimised for use in a range of virally-infected tissue culture cells, Coxsackie-infected mice and human tissues. The specificity of labelling was confirmed by pre-adsorption of antisera with poly-IC and by digestion of dsRNA with RNaseIII. RESULTS Two different polyclonal dsRNA antisera (J2 and K1) were capable of recognising dsRNA encoded by all the multiple different viral types (including (+) ssRNA viruses, dsRNA viruses and DNA viruses) tested in paraffin-embedded formalin fixed infected cells and tissues. In contrast, the enteroviral vp1 antisera detected only a subset of the (+) ssRNA viruses tested. Staining was not seen in uninfected cells or in uninfected control tissues. Positive staining was ablated following incubation of antisera with poly-IC or by pre-treating sections with RNaseIII prior to staining. CONCLUSIONS The dsRNA antisera J2 and K1 are useful for the detection of viral infection in formalin-fixed, paraffin-embedded, human tissue samples.

Germinal centre frequency is decreased in pancreatic lymph nodes from individuals with recent-onset type 1 diabetes

Aims/hypothesisPancreatic lymph nodes (PLNs) are critical sites for the initial interaction between islet autoantigens and autoreactive lymphocytes, but the histology of PLNs in tissue from individuals with type 1 diabetes has not been analysed in detail. The aim of this study was to examine PLN tissue sections from healthy donors compared with those at risk of, or with recent-onset and longer-duration type 1 diabetes.MethodsImmunofluorescence staining was used to examine PLN sections from the following donor groups: non-diabetic (n=15), non-diabetic islet autoantibody-positive (n=5), recent-onset (≤1.5 years duration) type 1 diabetes (n=13), and longer-duration type 1 diabetes (n=15). Staining for CD3, CD20 and Ki67 was used to detect primary and secondary (germinal centre-containing) follicles and CD21 and CD35 to detect follicular dendritic cell networks.ResultsThe frequency of secondary follicles was lower in the recent-onset type 1 diabetes group compared with the non-diabetic control group. The presence of insulitis (as evidence of ongoing beta cell destruction) and diagnosis of type 1 diabetes at a younger age, however, did not appear to be associated with a lower frequency of secondary follicles. A higher proportion of primary B cell follicles were observed to lack follicular dendritic cell networks in the recent-onset type 1 diabetes group.Conclusions/interpretationHistological analysis of rare PLNs from individuals with type 1 diabetes suggests a previously unrecognised phenotype comprising decreased primary B cell follicle frequency and fewer follicular dendritic cell networks in recent-onset type 1 diabetes.

Erratum. Blood and Islet Phenotypes Indicate Immunological Heterogeneity in Type 1 Diabetes. Diabetes 2014;63:3835-3845

In the article listed above, there are two errors in the research design and methods section. In the section with the heading “Studies on Islet-Infiltrating Leukocytes,” the antibody listed as #M0701 should be attributed to Dako and not to Abcam and the Abcam rabbit anti-CD8 catalogue number should read #ab4055 and not #GR404-4. The online version reflects these changes.