Histological validation of a type 1 diabetes clinical diagnostic model for classification of diabetes.

AIMS: Misclassification of diabetes is common due to an overlap in the clinical features of type 1 and type 2 diabetes. Combined diagnostic models incorporating clinical and biomarker information have recently been developed that can aid classification, but they have not been validated using pancreatic pathology. We evaluated a clinical diagnostic model against histologically defined type 1 diabetes. METHODS: We classified cases from the Network for Pancreatic Organ donors with Diabetes (nPOD) biobank as type 1 (n = 111) or non-type 1 (n = 42) diabetes using histopathology. Type 1 diabetes was defined by lobular loss of insulin-containing islets along with multiple insulin-deficient islets. We assessed the discriminative performance of previously described type 1 diabetes diagnostic models, based on clinical features (age at diagnosis, BMI) and biomarker data [autoantibodies, type 1 diabetes genetic risk score (T1D-GRS)], and singular features for identifying type 1 diabetes by the area under the curve of the receiver operator characteristic (AUC-ROC). RESULTS: Diagnostic models validated well against histologically defined type 1 diabetes. The model combining clinical features, islet autoantibodies and T1D-GRS was strongly discriminative of type 1 diabetes, and performed better than clinical features alone (AUC-ROC 0.97 vs. 0.95; P = 0.03). Histological classification of type 1 diabetes was concordant with serum C-peptide [median < 17 pmol/l (limit of detection) vs. 1037 pmol/l in non-type 1 diabetes; P < 0.0001]. CONCLUSIONS: Our study provides robust histological evidence that a clinical diagnostic model, combining clinical features and biomarkers, could improve diabetes classification. Our study also provides reassurance that a C-peptide-based definition of type 1 diabetes is an appropriate surrogate outcome that can be used in large clinical studies where histological definition is impossible. Parts of this study were presented in abstract form at the Network for Pancreatic Organ Donors Conference, Florida, USA, 19-22 February 2019 and Diabetes UK Professional Conference, Liverpool, UK, 6-8 March 2019.

Untargeted metabolomic analysis in non-fasted diabetic dogs by UHPLC-HRMS.

INTRODUCTION: We recently identified variances in serum metabolomic profiles between fasted diabetic and healthy dogs, some having similarities to those identified in human type 1 diabetes. OBJECTIVES: Compare untargeted metabolomic profiles in the non-fasted state. METHODS: Serum from non-fasted diabetic (n = 6) and healthy control (n = 6) dogs were analyzed by liquid chromatography-high resolution mass spectrometry. RESULTS: Clear clustering of metabolites between groups were observed, with multiple perturbations identified that were similar to those previously observed in fasted diabetic dogs. CONCLUSION: These findings further support the development of targeted assays capable of detecting metabolites that may be useful as biomarkers of canine diabetes.

Validation of a rapid type 1 diabetes autoantibody screening assay for community-based screening of organ donors to identify subjects at increased risk for the disease.

The Network for Pancreatic Organ donors with Diabetes (nPOD) programme was developed in response to an unmet research need for human pancreatic tissue obtained from individuals with type 1 diabetes mellitus and people at increased risk [i.e. autoantibody (AAb)-positive] for the disease. This necessitated the establishment of a type 1 diabetes-specific AAb screening platform for organ procurement organizations (OPOs). Assay protocols for commercially available enzyme-linked immunosorbent assays (elisas) determining AAb against glutamic acid decarboxylase (GADA), insulinoma-associated protein-2 (IA-2A) and zinc transporter-8 (ZnT8A) were modified to identify AAb-positive donors within strict time requirements associated with organ donation programmes. These rapid elisas were evaluated by the international islet AAb standardization programme (IASP) and used by OPO laboratories as an adjunct to routine serological tests evaluating donors for organ transplantation. The rapid elisas performed well in three IASPs (2011, 2013, 2015) with 98-100% specificity for all three assays, including sensitivities of 64-82% (GADA), 60-64% (IA-2A) and 62-68% (ZnT8A). Since 2009, nPOD has screened 4442 organ donors by rapid elisa; 250 (5·6%) were identified as positive for one AAb and 14 (0.3%) for multiple AAb with 20 of these cases received by nPOD for follow-up studies (14 GADA+, two IA-2A(+) , four multiple AAb-positive). Rapid screening for type 1 diabetes-associated AAb in organ donors is feasible, allowing for identification of non-diabetic, high-risk individuals and procurement of valuable tissues for natural history studies of this disease.

The Anatomical Society core regional anatomy syllabus for undergraduate medicine.

The Anatomical Society's core syllabus for anatomy (2003 and later refined in 2007) set out a series of learning outcomes that an individual medical student should achieve on graduation. The core syllabus, with 182 learning outcomes grouped in body regions, referenced in the General Medical Council's Teaching Tomorrow's Doctors, was open to criticism on the grounds that the learning outcomes were generated by a relatively small group of anatomists, albeit some of whom were clinically qualified. We have therefore used a modified Delphi technique to seek a wider consensus. A Delphi panel was constructed involving 'experts' (n = 39). The revised core syllabus of 156 learning outcomes presented here is applicable to all medical programmes and may be used by curriculum planners, teachers and students alike in addressing the perennial question: 'What do I need to know ?'

Factors impeding the discovery of an intervention-based treatment for type 1 diabetes.

Type 1 diabetes (T1D) is one of the most common and severe chronic diseases affecting both children and adults. The aetiology of the disease remains unknown, and thus far no 'true' cure for those affected is available. Indeed, exogenous insulin replacement therapy to manage glucose metabolism to the best degree possible remains the current standard of care. However, despite a recent array of truly impressive improvements designed to enhance disease management (e.g. insulin analogues, continuous glucose monitoring, insulin pumps), it is still difficult for the vast majority of patients to reach recommended target HbA1C levels (< 7.0%). As a result of suboptimal disease management, far too many patients with T1D have an increased risk for disease-associated complications such as nephropathy, neuropathy and retinopathy, as well as hypoglycaemia. New treatment modalities are therefore needed urgently to bring a 'true' cure (disease prevention/disease reversal) to patients with T1D. Here we consider issues that collectively pose a major stumbling block in T1D research with respect to identifying a means to prevent and/or cure the disease. We begin this Perspective by discussing new insights emanating from studies of the pancreas in human T1D; findings which may, at least in part, explain why previous interventions seeking disease prevention/reversal have yielded insufficient benefit. We then turn to suggestions that could optimise the outcome of future clinical trials. Finally, we direct attention to recommendations for the global T1D research community; messages we deem to have the potential to improve our chances of finding the elusive T1D 'cure'.

Mobilization without immune depletion fails to restore immunological tolerance or preserve beta cell function in recent onset type 1 diabetes.

Granulocyte colony-stimulating factor (G-CSF) has been used to restore immune competence following chemoablative cancer therapy and to promote immunological tolerance in certain settings of autoimmunity. Therefore, we tested the potential of G-CSF to impact type 1 diabetes (T1D) progression in patients with recent-onset disease [n = 14; n = 7 (placebo)] and assessed safety, efficacy and mechanistic effects on the immune system. We hypothesized that pegylated G-CSF (6 mg administered subcutaneously every 2 weeks for 12 weeks) would promote regulatory T cell (Treg) mobilization to a degree capable of restoring immunological tolerance, thus preventing further decline in C-peptide production. Although treatment was well tolerated, G-CSF monotherapy did not affect C-peptide production, glycated haemoglobin (HbA1c) or insulin dose. Mechanistically, G-CSF treatment increased circulating neutrophils during the 12-week course of therapy (P < 0·01) but did not alter Treg frequencies. No effects were observed for CD4(+) : CD8(+) T cell ratio or the ratio of naive : memory (CD45RA(+)/CD45RO(+)) CD4(+) T cells. As expected, manageable bone pain was common in subjects receiving G-CSF, but notably, no severe adverse events such as splenomegaly occurred. This study supports the continued exploration of G-CSF and other mobilizing agents in subjects with T1D, but only when combined with immunodepleting agents where synergistic mechanisms of action have previously demonstrated efficacy towards the preservation of C-peptide.

The intestinal microbiome in type 1 diabetes.

Few concepts in recent years have garnered more disease research attention than that of the intestinal (i.e. 'gut') microbiome. This emerging interest has included investigations of the microbiome's role in the pathogenesis of a variety of autoimmune disorders, including type 1 diabetes (T1D). Indeed, a growing number of recent studies of patients with T1D or at varying levels of risk for this disease, as well as in animal models of the disorder, lend increasing support to the notion that alterations in the microbiome precede T1D onset. Herein, we review these investigations, examining the mechanisms by which the microbiome may influence T1D development and explore how multi-disciplinary analysis of the microbiome and the host immune response may provide novel biomarkers and therapeutic options for prevention of T1D.

Does the gut microbiota have a role in type 1 diabetes? Early evidence from humans and animal models of the disease.

Despite years of appreciating the potential role of environment to influence the pathogenesis of type 1 diabetes, specific agents or mechanisms serving in such a capacity remain ill defined. This is exceedingly disappointing as the identification of factors capable of modulating the disease, either as triggers or regulators of the autoimmune response underlying type 1 diabetes, would not only provide clues as to why the disorder develops but, in addition, afford opportunities for improved biomarkers of disease activity and the potential to design novel therapeutics capable of disease abatement. Recent improvements in sequencing technologies, combined with increasing appreciation of the role of innate and mucosal immunity in human disease, have stirred strong interest in what is commonly referred to as the 'gut microbiota'. The gut (or intestinal) microbiota is an exceedingly complex microenvironment that is intimately linked with the immune system, including the regulation of immune responses. After evaluating evidence supporting a role for environment in type 1 diabetes, this review will convey current notions for contributions of the gut microbiota to human health and disease, including information gleaned from studies of humans and animal models for this autoimmune disorder.

Clinically relevant fatigue in recurrence-free prostate cancer survivors.

BACKGROUND: Little is known about the prevalence and associations of clinically relevant fatigue (CRF) in recurrence-free prostate cancer survivors. PATIENTS AND METHODS: Four hundred and sixteen recurrence-free prostate cancer survivors who were >1 year post-radiotherapy or radical prostatectomy were surveyed. The prevalence of CRF (defined as Brief Fatigue Inventory >3) was determined and compared with a noncancer control group. Other measures included the Hospital Anxiety and Depression Scale, International Prostate Symptom Score, European Organization for Research and Treatment of Cancer Quality of Life Questionnaire. Relationships between these factors and CRF were explored in univariate and multivariate analyses. RESULTS: Analyzable data were obtained from 91% (377/416) of patients. The prevalence of CRF was 29% (108/377) versus 16% (10/63) in the controls (P=0.031). CRF was more common in post-radiotherapy than in post-prostatectomy 33% (79/240) versus 22% (29/133), P=0.024. However, when other factors (current depression, anxiety, urinary symptoms, medical comorbidities, pain and insomnia) were controlled for, previous treatment did not predict CRF. Current depression [Hospital Anxiety and Depression Scale≥8 was by far the strongest association [odds ratio 9.9, 95% confidence interval 4.2-23.5)]. CONCLUSIONS: Almost one-third of recurrence-free prostate cancer survivors report CRF. Depression, anxiety, urinary symptoms, pain and insomnia measured at outcome are more strongly associated than type of cancer treatment previously received.

Clinically relevant fatigue in men with hormone-sensitive prostate cancer on long-term androgen deprivation therapy.

BACKGROUND: The purpose of the study was to determine the prevalence and associations of clinically relevant fatigue (CRF) in men with biochemically controlled prostate cancer on long-term androgen deprivation therapy (ADT). PATIENTS AND METHODS: One hundred and ninety-eight men were surveyed and the prevalence of CRF (Brief Fatigue Inventory score >3) determined. Associations with other measures (Hospital Anxiety and Depression Scale; International Prostate Symptom Score; European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire; Brief Pain Inventory worst pain; clinical and demographic information) were explored in univariate and multivariate analyses. RESULTS: Eight-one per cent (160 of 198) of questionnaires were analysable. CRF prevalence was 43% (68 of 160). CRF associations included moderate/severe urinary symptoms, anxiety and medical co-morbidities; the strongest associations were depression [odds ratio (OR) 9.8, 95% confidence interval (CI) 4.3-22.8] and pain (OR 9.2, 95% CI 4.0-21.5). After controlling for other factors, the independent associations were depression (OR 4.7, 95% CI 1.6-14.0) and pain (OR 3.1, 95% CI 1.0-8.9). There was no association with age, disease burden or treatment duration. CONCLUSIONS: Two-fifths of men with biochemically controlled prostate cancer on long-term ADT report CRF that interferes with function. Management aimed at improving CRF should address depression and pain.

Combinatorial treatment of bone marrow stem cells and stromal cell-derived factor 1 improves glycemia and insulin production in diabetic mice.

Transdifferentiation of stem cells into insulin-producing cells for the treatment of diabetes have shown promising but inconsistent results. We examined the potential for attracting bone marrow stem cells (BMSCs) to the pancreas using a chemokine, stromal cell-derived factor 1 (SDF-1). SDF-1 treatment markedly increased the number of GFP labeled BMSCs in the pancreas, but surprisingly, the majority was observed in liver. The liver cells had typical pancreatic endocrine cell gene expression including insulin I, insulin II, PDX-1, somatostatin, and glucagon. Combined treatment with SDF-1 and BMSC transplant reduced hyperglycemia and prolonged the long-term survival of diabetic mice, and a sub group had complete normoglycemia (<150 mg/dl), restored blood insulin levels, and normal glucose tolerance. Our results suggest that SDF-1 could potentially be used to improve the homing of stem cells and ß-cell regeneration. The mechanism appears to involve an increase in insulin producing cells mainly in the liver.

The threshold hypothesis: solving the equation of nurture vs nature in type 1 diabetes.

For more than 40 years, the contributions of nurture (i.e. the environment) and nature (i.e. genetics) have been touted for their aetiological importance in type 1 diabetes. Disappointingly, knowledge gains in these areas, while individually successful, have to a large extent occurred in isolation from each other. One reason underlying this divide is the lack of a testable model that simultaneously considers the contributions of genetic and environmental determinants in the formation of this and potentially other disorders that are subject to these variables. To address this void, we have designed a model based on the hypothesis that the aetiological influences of genetics and environment, when evaluated as intersecting and reciprocal trend lines based on odds ratios, result in a method of concurrently evaluating both facets and defining the attributable risk of clinical onset of type 1 diabetes. The model, which we have elected to term the 'threshold hypothesis', also provides a novel means of conceptualising the complex interactions of nurture with nature in type 1 diabetes across various geographical populations.

Influence of serum and soluble CD25 (sCD25) on regulatory and effector T-cell function in hepatocellular carcinoma.

Our previous studies showed that high levels of soluble CD25 (sCD25) in the serum of patients with hepatocellular carcinoma (HCC) correlated with blunted effector T-cells (Teff) responses, tumour burden and poor survival. Understanding the interactions between Teff, CD4+CD25+ regulatory T cells (Treg) and soluble factors can identify novel therapeutic targets. In this study, we characterize the mechanisms by which HCC serum and sCD25 mediate suppression of Teff and evaluate the effect of sCD25 on the suppression assays with normal healthy control cells (NHC) at a 1:1 Treg to Teff cell ratio to determine whether sCD25 has any impact on Treg suppression. HCC serum and sCD25 suppressed Teff proliferation and downregulated CD25 expression on HCC Teff in a dose-dependent fashion with sCD25 doses above 3000 pg/ml. Treg from HCC and cirrhosis patients suppressed proliferation of target CD4+CD25- Teff in serum-free medium (SFM). HCC Treg showed a higher degree of suppression than cirrhosis-derived Treg. In contrast, Treg from NHC did not suppress target Teff in SFM. However, isolated Treg from all three study subjects (HCC, cirrhosis and NHC) suppressed CD4+CD25- Teff in serum conditions or in the presence of sCD25 in the range 6000-12,000 pg/ml. In conclusion, downregulation of CD25 cell surface expression on Teff is part of the overall suppressive mechanism of sCD25 and HCC serum on Teff responses. The observed sCD25 and HCC serum-mediated suppression is further influenced via novel immune-inhibitory interaction between CD4+CD25+ Treg and sCD25.

Dimorphic histopathology of long-standing childhood-onset diabetes.

AIMS/HYPOTHESIS: Childhood diabetes is thought to usually result from autoimmune beta cell destruction (type 1A) with eventual total loss of beta cells. Analysis of C-peptide in children characterised at diabetes onset for autoantibodies shows heterogeneous preservation of insulin secretion in long-standing diabetes. The aim of this study was to characterise the pancreases of childhood-onset diabetes in order to define the pathological basis of this heterogeneity. METHODS: We evaluated 20 cadaveric organ donor pancreases of childhood-onset long-term patients for disease heterogeneity and obtained corresponding C-peptide measurements. RESULTS: Pancreases from the majority of cadaveric donors contained only insulin-deficient islets (14 of 20). The remaining six patients (30%) had numerous insulin-positive cells within at least some islets, with two different histological patterns. Pattern A (which we would associate with type 1A diabetes) had lobular retention of areas with 'abnormal' beta cells producing the apoptosis inhibitor survivin and HLA class I. In pattern B, 100% of all islets contained normal-appearing but quantitatively reduced beta cells without survivin or HLA class I. CONCLUSIONS/INTERPRETATION: Our data demonstrate that C-peptide secretion in long-standing diabetic patients can be explained by two different patterns of beta cell survival,possibly reflecting different subsets of type 1 diabetes.

Prophylactic immune tolerance induced by changing the ratio of antigen-specific effector to regulatory T cells.

BACKGROUND: Gene and protein replacement therapies for inherited protein deficiencies such as hemophilia or lysosomal storage disorders are limited by deleterious immune responses directed against their respective therapeutic proteins. Therefore, the development of protocols preventing such responses is key to providing successful long-term therapy. OBJECTIVES: We sought to develop a protocol, utilizing a drug/peptide cocktail, that would effectively shift the antigen-specific CD4+ T-cell population, tipping the balance from effector T cells (Teffs) towards regulatory T cells (Tregs). METHODS: Treg-deficient (DO11.10-tg Rag2(-/-)) BALB/c mice were used to screen for an optimal protocol addressing the aforementioned goal and to study the mechanisms underlying in vivo changes in T-cell populations. Muscle-directed gene transfer to hemophilia B mice was also performed in order to test the optimal protocol in a therapeutically relevant setting. RESULTS: Specific antigen administration (4-week repeated dosing) combined with rapamycin and interleukin-10 led to substantial reductions in Teffs, via activation-induced cell death, and induced CD4+CD25+FoxP3+ Tregs to a large extent in multiple organs. The proportion of apoptotic T cells also increased over time, whereas Teffs and Tregs were differentially affected. When applied to a model of protein deficiency (gene therapy for hemophilia B), the protocol successfully prevented inhibitor formation, whereas non-specific immunosuppression was only marginally effective. CONCLUSIONS: It is feasible to provide a short-term, prophylactic protocol allowing for the induction of immune tolerance. This protocol may provide a marked advance in efforts seeking to improve clinical outcomes in disorders involving therapeutic protein replacement.

Pancreatic adenocarcinoma patients with localised chronic severe pancreatitis show an increased number of single beta cells, without alterations in fractional insulin area.

AIMS/HYPOTHESIS: Recent histological analysis of pancreases obtained from patients with long-standing type 1 diabetes identified chronic islet inflammation and limited evidence suggestive of beta cell replication. Studies in rodent models also suggest that beta cell replication can be induced by certain inflammatory cytokines and by gastrin. We therefore tested the hypothesis that beta cell replication is observed in non-autoimmune human pancreatic disorders in which localised inflammation or elevated gastrin levels are present. METHODS: Resected operative pancreatic specimens were obtained from patients diagnosed with primary adenocarcinoma (with or without chronic severe pancreatitis) or gastrinoma. Additional pancreatic tissue was obtained from autopsy control patients. Immunohistochemistry was used to assess fractional insulin area, beta cell number and replication rate and differentiation factors relevant to beta cell development. RESULTS: Fractional insulin area was similar among groups. Patients with pancreatic adenocarcinoma and localised chronic severe pancreatitis displayed significant increases in the number of single beta cells, as well as increased beta cell replication rate and levels of neurogenic differentiation 1 in islets. Patients with gastrinoma demonstrated significant increases in the number of single beta cells, but the beta cell replication rate and islet differentiation factor levels were similar to those in the control group. CONCLUSIONS/INTERPRETATION: These findings indicate that chronic severe pancreatic inflammation can be associated with significant effects on beta cell number or replication rate, depending on the distribution of the cells. This information may prove useful for attempts seeking to design therapies aimed at inducing beta cell replication as a means of reversing diabetes.

Prolonged islet allograft survival by alpha-1 antitrypsin: the role of humoral immunity.

Immunomodulatory properties have been recognized for human alpha-1 antitrypsin (hAAT). However, production of anti-hAAT antibodies in mice may inactivate the protein. In this study, we evaluated the effects of chronic hAAT administration on allogeneic islet graft survival. Chemically diabetic mice lacking an efficient humoral response due to the targeted disruption of the Ig mu-chain (muMT mice) or wild-type (WT) C57BL/6 mice received DBA/2 mouse islets under the kidney capsule. hAAT (Prolastin or Aralast) was given intraperitoneally on day 0 and every 3 days thereafter. Control animals received no treatment. hAAT administration in WT mice resulted in prolongation of islet allograft survival in a dose-dependent fashion in both hAAT-treated groups. Lack of Ig response (muMT mice) per se conferred a beneficial effect on graft survival that worsened in the Prolastin-treated groups but improved in the Aralast-treated group. Our data indicate that systemic administration of hAAT results in prolongation of islet allograft survival. Absence of mature B cells and Ig mu-chain resulted in improved graft survival, pointing to a role for B cells in the rejection process in this model. Treatment with Prolastin worsened graft survival in muMT mice, whereas Aralast did improve it, suggesting a different efficacy and possible actions of the two drug formulations.