Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated GRP78 and reduced spontaneous NETosis of bone marrow-derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate, characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of IFNγ-producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes

The INNODIA Type 1 Diabetes Natural History Study: a European cohort of newly diagnosed children, adolescents and adults

Aims/hypothesis: Type 1 diabetes is an heterogenous condition. Characterising factors explaining differences in an individual’s clinical course and treatment response will have important clinical and research implications. Our aim was to explore type 1 diabetes heterogeneity, as assessed by clinical characteristics, autoantibodies, beta cell function and glycaemic outcomes, during the first 12 months from diagnosis, and how it relates to age at diagnosis. Methods: Data were collected from the large INNODIA cohort of individuals (aged 1.0–45.0 years) newly diagnosed with type 1 diabetes, followed 3 monthly, to assess clinical characteristics, C-peptide, HbA1c and diabetes-associated antibodies, and their changes, during the first 12 months from diagnosis, across three age groups: <10 years; 10–17 years; and ≥18 years. Results: The study population included 649 individuals (57.3% male; age 12.1±8.3 years), 96.9% of whom were positive for one or more diabetes-related antibodies. Baseline (IQR) fasting C-peptide was 242.0 (139.0–382.0) pmol/l (AUC 749.3 [466.2–1106.1] pmol/l × min), with levels increasing with age (p<0.001). Over time, C-peptide remained lower in participants aged <10 years but it declined in all age groups. In parallel, glucose levels progressively increased. Lower baseline fasting C-peptide, BMI SD score and presence of diabetic ketoacidosis at diagnosis were associated with lower stimulated C-peptide over time. HbA1c decreased during the first 3 months (p<0.001), whereas insulin requirement increased from 3 months post diagnosis (p<0.001). Conclusions/interpretation: In this large cohort with newly diagnosed type 1 diabetes, we identified age-related differences in clinical and biochemical variables. Of note, C-peptide was lower in younger children but there were no main age differences in its rate of decline. Graphical Abstract

Aberrant expression of transglutaminase 2 in pancreas and thymus of NOD mice underscores the importance of deamidation in neoantigen generation

Post-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2, the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1β and IFNγ in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD β-cells underscores the role of deamidation in amplifying T-cell reactivity

Study protocol : Minimum effective low dose: anti-human thymocyte globulin (MELD-ATG): phase II, dose ranging, efficacy study of antithymocyte globulin (ATG) within 6 weeks of diagnosis of type 1 diabetes

Introduction Type 1 diabetes (T1D) is a chronic autoimmune disease, characterised by progressive destruction of the insulin-producing beta cells of the pancreas. One immunosuppressive agent that has recently shown promise in the treatment of new-onset T1D subjects aged 12-45 years is antithymocyte globulin (ATG), Thymoglobuline, encouraging further exploration in lower age groups. Methods and analysis Minimal effective low dose (MELD)-ATG is a phase 2, multicentre, randomised, double-blind, placebo-controlled, multiarm parallel-group trial in participants 5-25 years diagnosed with T1D within 3-9 weeks of planned treatment day 1. A total of 114 participants will be recruited sequentially into seven different cohorts with the first cohort of 30 participants being randomised to placebo, 2.5 mg/kg, 1.5 mg/kg, 0.5 mg/kg and 0.1 mg/kg ATG total dose in a 1:1:1:1:1 allocation ratio. The next six cohorts of 12-15 participants will be randomised to placebo, 2.5 mg/kg, and one or two selected middle ATG total doses in a 1:1:1:1 or 1:1:1 allocation ratio, as dependent on the number of middle doses, given intravenously over two consecutive days. The primary objective will be to determine the changes in stimulated C-peptide response over the first 2 hours of a mixed meal tolerance test at 12 months for 2.5 mg/kg ATG arm vs the placebo. Conditional on finding a significant difference at 2.5 mg/kg, a minimally effective dose will be sought. Secondary objectives include the determination of the effects of a particular ATG treatment dose on (1) stimulated C-peptide, (2) glycated haemoglobin, (3) daily insulin dose, (4) time in range by intermittent continuous glucose monitoring measures, (5) fasting and stimulated dry blood spot (DBS) C-peptide measurements. Ethics and dissemination MELD-ATG received first regulatory and ethical approvals in Belgium in September 2020 and from the German and UK regulators as of February 2021. The publication policy is set in the INNODIA (An innovative approach towards understanding and arresting Type 1 diabetes consortium) grant agreement (www.innodia.eu).Peer reviewe

Multi‐omics analysis reveals drivers of loss of β‐cell function after newly diagnosed autoimmune type 1 diabetes: An INNODIA multicenter study

Aims: Heterogeneity in the rate of beta-cell loss in newly diagnosed type 1 diabetes patients is poorly understood and creates a barrier to designing and interpreting disease-modifying clinical trials. Integrative analyses of baseline multi-omics data obtained after the diagnosis of type 1 diabetes may provide mechanistic insight into the diverse rates of disease progression after type 1 diabetes diagnosis. Methods: We collected samples in a pan-European consortium that enabled the concerted analysis of five different omics modalities in data from 97 newly diagnosed patients. In this study, we used Multi-Omics Factor Analysis to identify molecular signatures correlating with post-diagnosis decline in beta-cell mass measured as fasting C-peptide. Results: Two molecular signatures were significantly correlated with fasting C-peptide levels. One signature showed a correlation to neutrophil degranulation, cytokine signalling, lymphoid and non-lymphoid cell interactions and G-protein coupled receptor signalling events that were inversely associated with a rapid decline in beta-cell function. The second signature was related to translation and viral infection was inversely associated with change in beta-cell function. In addition, the immunomics data revealed a Natural Killer cell signature associated with rapid beta-cell decline. Conclusions: Features that differ between individuals with slow and rapid decline in beta-cell mass could be valuable in staging and prediction of the rate of disease progression and thus enable smarter (shorter and smaller) trial designs for disease modifying therapies as well as offering biomarkers of therapeutic effect

Luxury brands consumption: The segment of “Chandlers”

The purpose of this paper is to introduce the segment of “chandlers” to the Russian academic society and to describe the specifics of their contemporary consumer behavior. The term “chandler” for this study was borrowed from American classical literature and applied to marketing. The study was conducted in April 2016 and comprised of two stages. The first stage was a series of in-depth interviews with seven representatives of the target audience from Moscow. It allowed to formulate the hypotheses which were proved/disproved by these hypotheses during the online survey. 117 relevant respondents were chosen for the study (72 — from Moscow, 45 — from regional city Ufa). The results allowed to formulate a preliminary conclusion there are no сhandlers in Ufa now. The most popular luxury brands for the Moscow сhandlers and specifics of their consumption were determined. This research is the first descriptive step to understanding the specifics of contemporary сhandlers — how they manage to consume luxury in the form of material artefacts and services, while being kept on a shoestring budget. The research entails a few limitations. The investigation comprised only a limited numbers of the respondents from Russian cities as Moscow and Ufa. In future, more consumers will be involved in the sample to cover more cities in Russia and respondents from other countries will be included. Upon the research completion a range of the recommendations has been provided to the luxury producers whose brands are already presented in Moscow and also for those who are planning to open their stores there. The results may serve as a guide for marketing tools development in the luxury industry. The originality of the paper lies in the term “chandlers’ segment” which is introduced in marketing theory for the first time

Glucagon-Like Peptide-1 Protects Human Islets against Cytokine-Mediated β-Cell Dysfunction and Death: A Proteomic Study of the Pathways Involved

Glucagon-like peptide-1 (GLP-1) has been shown to protect pancreatic β-cells against cytokine-induced dysfunction and destruction. The mechanisms through which GLP-1 exerts its effects are complex and still poorly understood. The aim of this study was to analyze the protein expression profiles of human islets of Langerhans treated with cytokines (IL-1β and IFN-γ) in the presence or absence of GLP-1 by 2D difference gel electrophoresis and subsequent protein interaction network analysis to understand the molecular pathways involved in GLP-1-mediated β-cell protection. Co-incubation of cytokine-treated human islets with GLP-1 resulted in a marked protection of β-cells against cytokine-induced apoptosis and significantly attenuated cytokine-mediated inhibition of glucose-stimulated insulin secretion. The cytoprotective effects of GLP-1 coincided with substantial alterations in the protein expression profile of cytokine-treated human islets, illustrating a counteracting effect on proteins from different functional classes such as actin cytoskeleton, chaperones, metabolic proteins, and islet regenerating proteins. In summary, GLP-1 alters in an integrated manner protein networks in cytokine-exposed human islets while protecting them against cytokine-mediated cell death and dysfunction. These data illustrate the beneficial effects of GLP-1 on human islets under immune attack, leading to a better understanding of the underlying mechanisms involved, a prerequisite for improving therapies for diabetic patients.status: publishe

A proteome reference map of INS-1E cells

Diabetes is an emerging global epidemic disease that can be traced back to rapid increases in overweight, obesity and physical inactivity. Total deaths are projected to rise by more than 50% in the next 10 years. Two major types can be discriminated. Type 1 diabetes is characterized by a total insulin deficiency, caused by immune-mediated beta-cell destruction and type 2 diabetes results from beta-cell dysfunction and peripheral insulin resistance, leading to a relative insulin deficiency. The aim of this study was to provide a useful tool in facilitating proteomic research in diabetes. We used the rat insulin-producing cell line, INS-1E (provided by Claes Wolheim), a widely used model to study different aspects of diabetes. Total cell lysate was separated in the first dimension on 24 cm pH 4-7 strips and on a 12.5% SDS-polyacrylamide gel in the second dimension. Spots were picked using a an Ettan™ Spot Picker, digested with Trypsin Gold and identified with an AB 4800 MALDI TOF/TOF. Up till now, 424 protein spots out of an approximate 2200 spots have been identified with a MOWSE score higher than the cut-off score indicating identity or extensive homology (p<0.05). This resulted in 271 unique proteins. Proteins were grouped according to their Gene Ontology classification. Major groups identified include: metabolic processes of all kind, protein folding, response to stress and ubiquitin-dependent protein catabolic processes. The full reference map will help us and others to speed up differential analyses in the quest for understanding diabetes.status: accepte

Understanding type 1 diabetes through proteomics

Auto-immunity against pancreatic beta-cells leads to an absolute shortage of the hormone insulin, resulting in hyperglycemia and the onset of type 1 diabetes (T1D). Proteomic approaches have been used to elucidate the mechanisms of beta-cell dysfunction and death. Areas covered: In the present review, we discuss discoveries in the beta-cell proteome that have contributed to better insights in the role of the beta-cell in T1D. Techniques, such as 2D-DIGE and MALDI imaging, together with new approaches for sample preparation, including laser capture microdissection and immunopeptidomics, have resulted in novel mechanistic insights in the pathogenesis of T1D. We describe how proteomic studies in beta-cell lines as well as isolated islets from animal models and humans have discovered intracellular signaling pathways leading to beta-cell destruction, the generation of neo-antigens through post-translational modifications of beta-cell antigens as well as better biomarkers of disease progression. Expert commentary: Proteomics has contributed to the discovery of beta-cell neo-autoantigen generation through post-translational modifications, hybrid insulin peptide formation and the generation of defective ribosomal gene products. These concepts are revolutionizing our insights in the pathogenesis of T1D, acknowledging a central role for the beta-cell in its own destruction.status: publishe