Interleukin-21 Is Required for the Development of Type 1 Diabetes in NOD Mice

OBJECTIVE: Interleukin (IL)-21 is a type 1 cytokine that has been implicated in the pathogenesis of type 1 diabetes via the unique biology of the nonobese diabetic (NOD) mouse strain. The aim of this study was to investigate a causal role for IL-21 in type 1 diabetes. RESEARCH DESIGN AND METHODS: We generated IL-21R–deficient NOD mice and C57Bl/6 mice expressing IL-21 in pancreatic β-cells, allowing the determination of the role of insufficient and excessive IL-21 signaling in type 1 diabetes. RESULTS: Deficiency in IL-21R expression renders NOD mice resistant to insulitis, production of insulin autoantibodies, and onset of type 1 diabetes. The lymphoid compartment in IL-21R−/− NOD is normal and does not contain an increased regulatory T-cell fraction or diminished effector cytokine responses. However, we observed a clear defect in autoreactive effector T-cells in IL-21R−/− NOD by transfer experiments. Conversely, overexpression of IL-21 in pancreatic β-cells induced inflammatory cytokine and chemokines, including IL-17A, IL17F, IFN-γ, monocyte chemoattractant protein (MCP)-1, MCP-2, and interferon-inducible protein-10 in the pancreas. The ensuing leukocytic infiltration in the islets resulted in destruction of β-cells and spontaneous type 1 diabetes in the normally diabetes-resistant C57Bl/6 and NOD × C57Bl/6 backgrounds. CONCLUSIONS: This work provides demonstration of the essential prodiabetogenic activities of IL-21 on diverse genetic backgrounds (NOD and C57BL/6) and indicates that IL-21 blockade could be a promising strategy for interventions in human type 1 diabetes

dietary supplementation with high doses of regular vitamin d3 safely reduces diabetes incidence in nod mice when given early and long term

High doses of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) prevent diabetes in the non-obese diabetic (NOD) mouse but also elicit unwanted calcemic side-effects. Because immune cells themselves can convert vitamin D3 into 1,25(OH)2D3 locally, we hypothesized that dietary vitamin D3 can also prevent disease. Thus, we evaluated whether dietary administration of high doses of regular vitamin D3 (800 IU per day) during different periods of life (pregnancy and lactation, early-life (3-14 weeks of age), or lifelong (3-35 weeks of age)) safely prevents diabetes in NOD mice. We found that only lifelong treatment raised serum 25-hydroxyvitamin D3 from 173 nmol/L in controls to 290 nmol/L, without inducing signs of calcemic or bone toxicity, and significantly reduced diabetes development in both male and female NOD mice. This diabetes protection by vitamin D3 correlated with preserved pancreatic insulin content and improved insulitis scores. Moreover, vitamin D3 treatment decreased interferon-γ-positive CD8+ T-cells and increased CD4+(CD25+)FoxP3+ T-cells in pancreatic draining lymph nodes. In conclusion, this study shows for the first time that high doses of regular dietary vitamin D3 can safely prevent diabetes in NOD mice when administered lifelong, although caution is warranted with regards to administering equivalently high doses in humans

Access to primary care for socio-economically disadvantaged older people in rural areas: exploring realist theory using structural equation modelling in a linked dataset

Background: Realist approaches seek to answer questions such as ‘how?’, ‘why?’, ‘for whom?’, ‘in what circumstances?’ and ‘to what extent?’ interventions ‘work’ using context-mechanism-outcome (CMO) configurations. Quantitative methods are not well-established in realist approaches, but structural equation modelling (SEM) may be useful to explore CMO configurations. Our aim was to assess the feasibility and appropriateness of SEM to explore CMO configurations and, if appropriate, make recommendations based on our access to primary care research. Our specific objectives were to map variables from two large population datasets to CMO configurations from our realist review looking at access to primary care, generate latent variables where needed, and use SEM to quantitatively test the CMO configurations. Methods: A linked dataset was created by merging individual patient data from the English Longitudinal Study of Ageing and practice data from the GP Patient Survey. Patients registered in rural practices and who were in the highest deprivation tertile were included. Three latent variables were defined using confirmatory factor analysis. SEM was used to explore the nine full CMOs. All models were estimated using robust maximum likelihoods and accounted for clustering at practice level. Ordinal variables were treated as continuous to ensure convergence. Results: We successfully explored our CMO configurations, but analysis was limited because of data availability. Two hundred seventy-six participants were included. We found a statistically significant direct (context to outcome) or indirect effect (context to outcome via mechanism) for two of nine CMOs. The strongest association was between ‘ease of getting through to the surgery’ and ‘being able to get an appointment’ with an indirect mediated effect through convenience (proportion of the indirect effect of the total was 21%). Healthcare experience was not directly associated with getting an appointment, but there was a statistically significant indirect effect through convenience (53% mediated effect). Model fit indices showed adequate fit. Conclusions: SEM allowed quantification of CMO configurations and could complement other qualitative and quantitative techniques in realist evaluations to support inferences about strengths of relationships. Future research exploring CMO configurations with SEM should aim to collect, preferably continuous, primary data

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Type 1 diabetes: etiology, immunology, and therapeutic strategies

Type 1 diabetes (T1D) is a chronic autoimmune disease in which destruction or damaging of the beta-cells in the islets of Langerhans results in insulin deficiency and hyperglycemia. We only know for sure that autoimmunity is the predominant effector mechanism of T1D, but may not be its primary cause. T1D precipitates in genetically susceptible individuals, very likely as a result of an environmental trigger. Current genetic data point towards the following genes as susceptibility genes: HLA, insulin, PTPN22, IL2Ra, and CTLA4. Epidemiological and other studies suggest a triggering role for enteroviruses, while other microorganisms might provide protection. Efficacious prevention of T1D will require detection of the earliest events in the process. So far, autoantibodies are most widely used as serum biomarker, but T-cell readouts and metabolome studies might strengthen and bring forward diagnosis. Current preventive clinical trials mostly focus on environmental triggers. Therapeutic trials test the efficacy of antigen-specific and antigen-nonspecific immune interventions, but also include restoration of the affected beta-cell mass by islet transplantation, neogenesis and regeneration, and combinations thereof. In this comprehensive review, we explain the genetic, environmental, and immunological data underlying the prevention and intervention strategies to constrain T1D

TCR-triggered IL-15-expanded CD4⁺ T cells remain dependent on IL-15 for expansion and survival.

<p>CD4⁺ T cells were stimulated with 0.1 µg/ml anti-CD3 and irradiated TdACs without or with 3 ng/ml IL-15. After 3 days, 50 ng/ml soluble IL-15Rα (sIL-15Rα, T1, 50 ng/ml) or control mutated sIL-15Rα (M4), lacking IL-15 binding capacity, were added. (<b>A</b>) Proliferation of CD4⁺ T cells, as determined by ³H-thymidine incorporation of triplicate cultures on day 5. (<b>B</b>) Viable CD4⁺ cell recovery on day 5, determined by flow cytometry. Bar graphs represent the mean±SEM. Statistical significance was calculated using student t-test: ns = not significant, ** <i>P</i><0.01, *** <i>P</i><0.001. Data are representative of three independent experiments.</p

Exogenous IL-15 decreases the fraction but not the absolute number of CD25^High CD4⁺ T cells.

<p>(<b>A</b>) IL-15 alters TCR-induced gene expression of IL-2 and IL-15 receptor subunits. Purified CD4+ T cells were stimulated with 0.1 µg/ml anti-CD3 mAb and irradiated TdACs in the absence (white circles) or presence (black circles) of 3 ng/ml IL-15. At the indicated time points, CD4⁺ T cells were purified for PCR analysis of indicated gene transcripts and expressed as 2^−ΔCt where ΔCt = Ct_{target gene} - mean Ct_{normalization genes}. Statistical significance was calculated using student t-test: ns = not significant, ** <i>P</i><0.01, *** <i>P</i><0.001. (<b>B</b>) Spleen CD4⁺ T cells were left unstimulated or stimulated with anti-CD3 in the presence or absence of IL-15 for two days, as indicated. Shown are histogram overlays of CD25 expression, gated on viable CD4⁺ T cells (d: day). (<b>C</b>) CFSE-labeled CD4⁺ T cells were stimulated with 0.1 µg/ml anti-CD3 and irradiated TdAC, without or with 3 ng/ml IL-15 for four or five days. Shown are flow cytometry dot plots of CFSE dye dilution versus CD25 expression in the viable CD4⁺ gate (left, shown percentages were calculated on viable CD4⁺ T cells) and summarizing bar graph of the CD25 high fraction (left). Statistical significance was calculated using student t-test: * <i>P</i><0.05, ** <i>P</i><0.01. (<b>D</b>) Absolute numbers of viable CD25^High, CD25^Int, CD25^Negative and total CD4⁺ T cells, as determined by flow cytometry using micro-beads as reference. White and black bars represent stimulation with anti-CD3 in the absence or presence of 3 ng/ml rhIL-15, respectively. Statistical analysis was calculated using 2-way ANOVA and Bonferroni posttest: ns = not significant, *** <i>P</i><0.001. Data shown are representative of three independent experiments. (<b>E</b>) CFSE-labeled CD4⁺ T cells were stimulated as in <i>B</i>. Shown are histogram overlays (top) of CD25 expression of the last generation of viable CD4⁺ T cells (by CFSE dilution) on day 5 after stimulation in the absence (dashed line) or presence (shaded) of 3 ng/ml rmIL-15. Bar graph (bottom) represents the mean fluorescenceIntensity of CD25 expression in the last generation, as shown in the overlays. Statistical significance was calculated using student t-test: * <i>P</i><0.05, ** <i>P</i><0.01.</p