Sarah J. Richardson

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.

Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.

Aims/hypothesisEndoplasmic reticulum (ER) stress may play a role in cytokine-mediated beta cell death in type 1 diabetes, but it remains controversial whether ER stress markers are present in islets from type 1 diabetic individuals. Therefore, we evaluated by immunostaining the expression of markers of the three main branches of the ER stress response in islets from 13 individuals with and 15 controls without type 1 diabetes (eight adults and seven children).MethodsAntibodies against the ER stress markers C/EBP homologous protein (CHOP), immunoglobulin heavy chain (BIP) and X-box binding protein 1 (XBP-1) were validated using HeLa cells treated with the ER stressor thapsigargin. These antibodies were then used to stain serial sections of paraffin-embedded pancreas from type 1 diabetic and non-diabetic individuals; samples were also immunostained for CD45, insulin and glucagon. Immunostaining intensities of the ER stress markers were quantified using a software-based, unbiased quantitative approach.ResultsIslets from individuals with type 1 diabetes showed increased levels of CHOP and, at least for insulitis-positive and beta cell-containing islets, BIP. XBP-1 expression was not, however, increased.Conclusions/interpretationIslet cells from individuals with type 1 diabetes display a partial ER stress response, with evidence of the induction of some, but not all, components of the unfolded protein response.

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

The diagnosis of insulitis in human type 1 diabetes

To the Editor: During a workshop concerning the histopathological characteristics of insulitis in human type 1 diabetes (fifth annual meeting of the JDRF Network for Pancreatic Organ Donors with Diabetes [nPOD], 10 February 2013, Jacksonville, FL, USA), a consensus opinion was reached on the criteria necessary for the diagnosis of insulitis, and a definition of insulitis was agreed, as detailed in the text box. Workshop attendees included many leading researchers in the

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.

Detection of a low-grade enteroviral infection in the islets of Langerhans of living patients newly diagnosed with type 1 diabetes

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 six 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. Nondiabetic organ donors served as controls. VP1 was detected in the islets of all type 1 diabetic patients (two of nine controls). Hyperexpression of class I HLA molecules was found in the islets of all patients (one of nine controls). Enterovirus-specific RNA sequences were detected in four of six patients (zero of six controls). The results were confirmed in various laboratories. Only 1.7% of the islets contained VP1+ cells, and the amount of enterovirus RNA was low. The results provide evidence for the presence of enterovirus in pancreatic islets of type 1 diabetic patients, which is consistent with the possibility that a low-grade enteroviral infection in the pancreatic islets contributes to disease progression in humans.

Single-agent lenalidomide in relapsed/refractory mantle cell lymphoma: results from a UK phase II study suggest activity and possible gender differences

We present data from a phase II study investigating a novel treatment strategy for relapsed/refractory mantle cell lymphoma (MCL). Twenty‐six patients received lenalidomide 25 mg/d (days 1–21 of a 28‐d cycle) for up to 6 cycles followed by low‐dose maintenance lenalidomide (15 mg) in responding patients. Eight patients achieved complete or partial response to give an overall response rate of 31% with median response duration of 22·2 months [95% confidence interval (CI) 0·0–53·6] and median progression‐free survival (PFS) of 3·9 months (95% CI 0·0–11·1). An additional six patients (23%) achieved stable disease. Eleven patients received maintenance with median PFS of 14·6 months (95% CI 7·3–21·9). Correlative studies showed that peripheral T and Natural Killer (NK) cells increased in responding patients by 40–60% over the first 6 cycles with an initial dip in NK cells suggestive of tumour infiltration. Peripheral regulatory T cells were increased in MCL patients (P = 0·001) and expanded further following lenalidomide. Sequential plasma analysis showed increased IL12 p40 and IL7 alongside decreased MMP9, IL10, and adiponectin. Finally, a significant correlation (P = 0·02) between gender and response suggested that female MCL patients were more sensitive to lenalidomide than males. In summary, we confirm the activity, safety and immunomodulatory properties of lenalidomide in MCL and highlight its potential as a low‐dose maintenance agent.

Evidence of increased islet cell proliferation in patients with recent-onset type 1 diabetes

Aims/hypothesisIn adults, the rate of beta cell replication is normally very low, but recent evidence suggests that it may increase during insulitis. We therefore studied tissue from donors with recent-onset type 1 diabetes to establish whether islet cell proliferation is increased during the disease process.MethodsParaffin-embedded pancreatic sections from ten donors with recent-onset type 1 diabetes and a range of relevant controls were stained by immunohistochemical techniques with antibodies against the proliferation markers Ki67 and minichromosome maintenance protein-2 (MCM-2). A combination staining technique involving immunoperoxidase and immunofluorescence methods was developed to quantify the numbers of alpha and beta cells with Ki67-positive nuclei and to investigate the relationship between insulitis and islet cell proliferation.ResultsIn non-diabetic control donors, only 1.1 ± 0.3% (mean ± SEM) of islets contained one or more Ki67+ islet cells, whereas this proportion was increased markedly in recent-onset type 1 diabetes (10.88 ± 2.5%; p < 0.005). An equivalent increase in Ki67+ staining occurred in alpha and beta cells and was correlated positively with the presence of insulitis. A significant increase in the labelling of islet cells from type 1 diabetic donors was also seen when MCM-2 staining was employed. Increased islet cell proliferation was not evident in three donors with longer duration type 1 diabetes or in ten type 2 diabetic donors.Conclusions/interpretationAlpha and beta cells undergo a marked increase in proliferation during the progression of type 1 diabetes in humans. The results imply that islet cell proliferation is re-initiated in response to the autoimmune attack associated with type 1 diabetes.

Expression of the enteroviral capsid protein VP1 in the islet cells of patients with type 1 diabetes is associated with induction of protein kinase R and downregulation of Mcl-1

Aims/hypothesisImmunohistochemical staining reveals that the enteroviral capsid protein VP1 is present at higher frequency in the insulin-containing islets of patients with recent-onset type 1 diabetes than in controls. This is consistent with epidemiological evidence suggesting that enteroviral infection may contribute to the autoimmune response in type 1 diabetes. However, immunostaining of VP1 is not definitive since the antibody widely used to detect the protein (Clone 5D8/1) might also cross-react with additional proteins under some conditions. Therefore, we sought to verify that VP1 immunopositivity correlates with additional markers of viral infection.MethodsAntigen immunoreactivity was examined in formalin-fixed, paraffin-embedded, pancreases from two different collections of type 1 diabetes and control cases: a historical collection from the UK and the nPOD (network of Pancreatic Organ donors with Diabetes) cohort from the USA.ResultsVP1 immunoreactivity was present in ∼20% of insulin-containing islets of both cohorts under stringent conditions but was absent from insulin-deficient islets. The presence of VP1 was restricted to beta cells but only a minority of these contained the antigen. The innate viral sensor, protein kinase R (PKR) was upregulated selectively in beta cells that were immunopositive for VP1. The anti-apoptotic protein myeloid cell leukaemia sequence-1 (Mcl-1) was abundant in beta cells that were immunonegative for VP1 but Mcl-1 was depleted in cells containing VP1.Conclusions/interpretationThe presence of immunoreactive VP1 within beta cells in type 1 diabetes is associated with a cellular phenotype consistent with the activation of antiviral response pathways and enhanced sensitivity to apoptosis. However, definitive studies confirming whether viral infections are causal to beta cell loss in human diabetes are still awaited.