The use of peptide-major-histocompatibility-complex multimers in type 1 diabetes mellitus

Major histocompatibility complex (MHC) class I and MHC class II molecules present short peptides that are derived from endogenous and exogenous proteins, respectively, to cognate T-cell receptors (TCRs) on the surface of T cells. The exquisite specificity with which T cells recognize particular peptide-major-histocompatibility-complex (pMHC) combinations has permitted development of soluble pMHC multimers that bind exclusively to selected T-cell populations. Because the pathogenesis of type 1 diabetes mellitus (T1DM) is driven largely by islet-reactive T-cell activity that causes β-cell death, these reagents are useful tools for studying and, potentially, for treating this disease. When coupled to fluorophores or paramagnetic nanoparticles, pMHC multimers have been used to visualize the expansion and islet invasion of T-cell effectors during diabetogenesis. Administration of pMHC multimers to mice has been shown to modulate T-cell responses by signaling through the TCR or by delivering a toxic moiety that deletes the targeted T cell. In the nonobese diabetic mouse model of T1DM, a pMHC-I tetramer coupled to a potent ribosome-inactivating toxin caused long-term elimination of a specific diabetogenic cluster of differentiation 8+ T-cell population from the pancreatic islets and delayed the onset of diabetes. This review will provide an overview of the development and use of pMHC multimers, particularly in T1DM, and describe the therapeutic promise these reagents have as an antigen-specific means of ameliorating deleterious T-cell responses in this autoimmune disease.     

Genetic variants in the complement system and their potential link in the aetiology of type 1 diabetes

Type 1 diabetes is an autoimmune disease in which one's own immune system destroys insulin-secreting beta cells in the pancreas. This process results in life-long dependence on exogenous insulin for survival. Both genetic and environmental factors play a role in disease initiation, progression, and ultimate clinical diagnosis of type 1 diabetes. This review will provide background on the natural history of type 1 diabetes and the role of genetic factors involved in the complement system, as several recent studies have identified changes in levels of these proteins as the disease evolves from pre-clinical through to clinically apparent disease.     

Three microsatellites from the T1DGC MHC data set show highly significant association with type 1 diabetes, independent of the HLA-DRB1, -DQA1 and -DQB1 genes

Aim:  The aim of this study was to test the microsatellites in the Type 1 Diabetes Genetics Consortium major histocompatibility complex (MHC) data set for association with type 1 diabetes (T1D) independent of the HLA-DRB1 , -DQA1 and -DQB1 genes.
Methods:  The data set was edited to contain only one affected child per family, and broad ethnic subgroups were defined. Genotypes for HLA-DRB1 , -DQA1 and -DQB1 were replaced by a haplotype code spanning all three loci, with phase inferred based on common haplotypes. The final data set contained 8190 samples in 2301 families, 59 microsatellites and the DRB1–DQA1–DQB1 haplotype code. Statistical analyses consisted of conditional logistic regression and haplotype estimations and linkage disequilibrium calculations.
Results:  The data set was screened using a main effects test approach adjusted for DRB1–DQA1–DQB1 , and significant results tested for validity. After these procedures, four markers remained significant at the Bonferroni-corrected threshold: D6S2773 (p = 0.00014), DG6S185 (p = 0.00015), DG6S398 (p = 0.00043) and D6S2998 (p = 0.00015). These results were supported by allelic tests conditioned on DRB1–DQA1–DQB1 haplotypes, except for DG6S185 , which may contain artefacts.
Conclusions:  We have identified three microsatellites that mark additional risk factors for T1D at highly significant levels in the MHC. Further analyses are needed to establish the relationship with other possible genetic determinants in this region.     

Haplotype-based search for SNPs associated with differential type 1 diabetes risk among chromosomes carrying a specific HLA DRB1–DQA1–DQB1 haplotype

Aim:  The aim of this study was to test chromosomes carrying the same DRB1–DQA1–DQB1 haplotype for single nucleotide polymorphisms (SNPs) in the major histocompatibility complex (MHC) that might mark subgroups of the haplotype with different risks for type 1 diabetes (T1D).
Methods:  Chromosomes from T1D children, their parents and non-diabetic siblings in families of the Type 1 Diabetes Genetics Consortium (T1DGC) were analysed by two haplotype-based methods: (i) logistic regression analysis restricted to phased chromosomes carrying the same DRB1–DQA1–DQB1 haplotype but differentiated by the two alleles at MHC SNPs, which were individually tested for association with T1D and (ii) homozygous parent transmission disequilibrium test (TDT) for biased transmission of a SNP allele to diabetic children from parents who are heterozygous at the SNP but homozygous for the specific DRB1–DQA1–DQB1 haplotype being evaluated.
Results:  A number of SNPs gave nominally significant (p < 0.05) evidence of marking two subsets of the 301–501–201 haplotype that might differ with respect to their diabetogenic potency. However, none of the SNPs achieved experiment-wide significance and hence may be false-positive associations.
Conclusions:  We discuss limitations and possible deficiencies of our study suggesting further work that might yield more robust SNP associations marking two subgroups of a DRB1–DQA1–DQB1 haplotype with different T1D risks.     

Identification of T1D susceptibility genes within the MHC region by combining protein interaction networks and SNP genotyping data

Aim:  To develop novel methods for identifying new genes that contribute to the risk of developing type 1 diabetes within the Major Histocompatibility Complex (MHC) region on chromosome 6, independently of the known linkage disequilibrium (LD) between human leucocyte antigen ( HLA ) -DRB1 , - DQA1 , - DQB1 genes.
Methods:  We have developed a novel method that combines single nucleotide polymorphism (SNP) genotyping data with protein–protein interaction (ppi) networks to identify disease-associated network modules enriched for proteins encoded from the MHC region. Approximately 2500 SNPs located in the 4 Mb MHC region were analysed in 1000 affected offspring trios generated by the Type 1 Diabetes Genetics Consortium (T1DGC). The most associated SNP in each gene was chosen and genes were mapped to ppi networks for identification of interaction partners. The association testing and resulting interacting protein modules were statistically evaluated using permutation.
Results:  A total of 151 genes could be mapped to nodes within the protein interaction network and their interaction partners were identified. Five protein interaction modules reached statistical significance using this approach. The identified proteins are well known in the pathogenesis of T1D, but the modules also contain additional candidates that have been implicated in β-cell development and diabetic complications.
Conclusions:  The extensive LD within the MHC region makes it important to develop new methods for analysing genotyping data for identification of additional risk genes for T1D. Combining genetic data with knowledge about functional pathways provides new insight into mechanisms underlying T1D.     

Aberrant expression of Class II major histocompatibility complex molecules by B cells and hyperexpression of Class I major histocompatibility complex molecules by insulin containing islets in Type 1 (insulin-dependent) diabetes mellitus

Summary Twenty-three patients with recent onset Type 1 (insulin-dependent) diabetes in whom residual insulin secreting B cells were present and 12 patients with disease of more prolonged duration (maximum 9 years), 8 of whom had residual B cells, were studied. Aberrant expression of Class II major histocompatibility complex molecules was demonstrated immunohistochemically on insulin secreting B cells in 21 out of 23 patients with recent onset disease and 6 of the patients with more prolonged disease. No such expression was seen on glucagon secreting A cells or somatostatin secreting D cells. Islets where there was marked hyperexpression of Class I major histocompatibility complex molecules on islet endocrine cells were seen in all cases in which residual B cells were present. Ninety-two per cent of insulin containing islets but only 1% of insulin deficient islets exhibited this phenomenon (p<0.001, Chi-squared test). There was evidence to suggest that both these abnormalities of major histocompatibility complex expression preceded insulitis within a given islet. They also appeared to be unique to Type 1 diabetes, being absent in pancreases of patients with Type 2 (non-insulin-dependent) diabetes, chronic pancreatitis, cystic fibrosis, graft-versus-host disease and Coxsackie B viral pancreatitis. The development of autoimmunity to B cells in Type 1 diabetes may be a multistep process in which abnormalities of major histocompatibility complex expression on islet endocrine cells are crucial events.     

High T-helper-1 cytokines but low T-helper-3 cytokines, inflammatory cytokines and chemokines in children with high risk of developing type 1 diabetes

BACKGROUND: Type 1 diabetes (T1D) is suggested to be of T-helper (Th)1-like origin. However, recent reports indicate a diminished interferon (IFN)-gamma secretion at the onset of the disease. We hypothesize that there is a discrepancy in subsets of Th-cells between children with a high risk of developing T1D, children newly diagnosed with T1D and healthy children. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from children at high risk for T1D (islet cells antibodies [ICA] >/= 20 IJDF-U), those newly diagnosed and healthy children carrying the HLA-risk gene DQB1*0302 or DQB1*0201 and DQA1*0501. Th1- (IFN-gamma, tumour necrosis factor [TNF]-beta, interleukin [IL]-2), Th2- (IL-4,-5,-13), Th3- (transforming growth factor [TGF-beta], IL-10) and inflammatory associated cytokines (TNF-alpha, IL-1alpha,-6) and chemokines (monocyte chemoattractant protein [MCP]-1,-2,-3, Monokine unregulated by IFN-gamma [MIG], Regulated on Activation, Normal T-cell Expressed and Secreted [RANTES], IL-7,-8,-15) were detected in cell-culture supernatants of PBMC, stimulated with glutamic acid decarboxylase 65 (GAD(65)) and phytohaemagglutinin (PHA), by protein micro array and enzyme linked immunospot (ELISPOT) technique. RESULTS: The Th1 cytokines IFN-gamma and TNF-beta, secreted both spontaneously and by GAD(65)- and mitogen stimulation, were seen to a higher extent in high-risk children than in children newly diagnosed with T1D. In contrast, TNF-alpha and IL-6, classified as inflammatory cytokines, the chemokines RANTES, MCP-1 and IL-7 as well as the Th3 cytokines TGF-beta and IL-10 were elevated in T1D children compared to high-risk children. CONCLUSION: High Th-1 cytokines were observed in children with high risk of developing TID, whereas in children newly diagnosed with T1D Th3 cytokines, inflammatory cytokines and chemokines were increased. Thus, an inverse relation between Th1-like cells and markers of inflammation was shown between children with high risk and those newly diagnosed with T1D.     

Sodium-glucose co-transporter inhibitors,their role in type 1 diabetes treatment and a risk mitigation strategy for preventing diabetic ketoacidosis: The STOP DKA Protocol

Recent phase 3 clinical trials have evaluated the impact of adding sodium-glucose co-transporter (SGLT) inhibitors to the type 1 diabetes armamentarium. These trials studied SGLT2 inhibitors (dapagliflozin and empagliflozin) and a dual SGLT1 and SGLT2 inhibitor (sotagliflozin), and demonstrated that these oral non-insulin antihyperglycaemic medications are able not only to improve glycaemic control, but also to reduce body weight and extend time in range without increasing rates of hypoglycaemia in type 1 diabetes. Diabetic ketoacidosis (DKA) is a feature of type 1 diabetes and the risk is increased when SGLT inhibitors are used in type 1 diabetes. To minimize the risk of DKA and still gain the multiple benefits, we developed the “STOP DKA Protocol “, an easily accessible and practical tool, that provides a risk mitigation strategy for reducing DKA in patients with type 1 diabetes being treated with SGLT inhibitors.     

Change in DASH diet score and cardiovascular risk factors in youth with type 1 and type 2 diabetes mellitus: The SEARCH for Diabetes in Youth Study

Youth with diabetes are at an increased risk of cardiovascular disease (CVD). Adherence to the Dietary Approaches to Stop Hypertension (DASH) diet has been shown to improve CVD risk. In this study, we evaluated whether changes in diet quality as characterized by DASH are associated with changes in CVD risk factors in youth with diabetes over time. Longitudinal mixed models were applied to data from 797 participants in the SEARCH for Diabetes in Youth Study representing three time points: baseline, 12- and 60-month follow-up. Data were restricted to youth whose diabetes was first diagnosed in 2002–2005. DASH-related adherence was poor and changed very little over time. However, an increase in DASH diet score was significantly associated with a decrease in HbA_1c levels in youth with type 1 diabetes (β=−0.20, P-value=0.0063) and a decrease in systolic blood pressure among youth with type 2 diabetes (β=−2.02, P-value=0.0406). Improvements in dietary quality may be beneficial in youth with type 1 or type 2 diabetes. However, further work in larger groups of youth with type 1 and 2 diabetes is desirable.     

GAD65-Specific Cytotoxic T Lymphocytes Mediate Beta-Cell Death and Loss of Function

Autoimmunity to islet cell antigens like glutamic acid decarboxylase 65kD (GAD65) is associated with the destruction of insulin-producing β-cells and progression to type 1 diabetes (T1D) in NOD mice and humans. T cell responses to GAD65 are detectable in the spleen of prediabetic NOD mice and in the peripheral blood of humans prior to the onset of overt hyperglycemia. Previous findings from our lab revealed that GAD65_546-554-specific cytotoxic T lymphocytes (CTL) are present in naïve NOD mice and are able to induce islet inflammation upon adoptive transfer into NOD.scid recipients. Additionally, we found that professional antigen-presenting cells (APC) generate the p546-554 epitope from a soluble GAD65 fragment, p530-554, and from GAD65 released by injured β-cells in vivo. Here, we report that the GAD65 fragment p546-554 is a dominant CTL-inducing epitope which is naturally processed and presented by a GAD65-expressing β-cell line. Further, co-culture of GAD65_546-554-specific CTL with the β-cells leads to a reduction in insulin production and the induction of perforin-mediated cell death. Collectively, these findings support a role for the cross-presentation of GAD65 antigen in the priming and enhancement of dominant GAD65-specific CTL responses, which can directly target β-cells that display GAD65 epitopes.     

Efficacy and safety of alirocumab in insulin‐treated individuals with type 1 or type 2 diabetes and high cardiovascular risk: The ODYSSEY DM‐INSULIN randomized trial

Aims

To investigate the efficacy and safety of alirocumab in participants with type 2 (T2D) or type 1 diabetes (T1D) treated with insulin who have elevated LDL cholesterol levels despite maximally tolerated statin therapy.

Methods

Participants at high cardiovascular risk with T2D (n = 441) or T1D (n = 76) and LDL cholesterol levels ≥1.8 mmol/L (≥70 mg/dL) were randomized 2:1 to alirocumab:placebo administered subcutaneously every 2 weeks, for 24 weeks' double‐blind treatment. Alirocumab‐treated participants received 75 mg every 2 weeks, with blinded dose increase to 150 mg every 2 weeks at week 12 if week 8 LDL cholesterol levels were ≥1.8 mmol/L. Primary endpoints were percentage change in calculated LDL cholesterol from baseline to week 24, and safety assessments.

Results

Alirocumab reduced LDL cholesterol from baseline to week 24 by a mean ± standard error of 49.0% ± 2.7% and 47.8% ± 6.5% vs placebo (both P < .0001) in participants with T2D and T1D, respectively. Significant reductions were observed in non‐HDL cholesterol (P < .0001), apolipoprotein B (P < .0001) and lipoprotein (a) (P ≤ .0039). At week 24, 76.4% and 70.2% of the alirocumab group achieved LDL cholesterol <1.8 mmol/L in the T2D and T1D populations (P < .0001), respectively. Glycated haemoglobin and fasting plasma glucose levels remained stable for the study duration. Treatment‐emergent adverse events were observed in 64.5% of alirocumab‐ vs 64.1% of placebo‐treated individuals (overall population).

Conclusions

Alirocumab produced significant LDL cholesterol reductions in participants with insulin‐treated diabetes regardless of diabetes type, and was generally well tolerated. Concomitant administration of alirocumab and insulin did not raise any safety concerns (NCT02585778).     

Insulin detemir used in basal-bolus therapy in people with type 1 diabetes is associated with a lower risk of nocturnal hypoglycaemia and less weight gain over 12 months in comparison to NPH insulin

AIM: The aim of this study was to compare the long-term safety and efficacy of twice-daily insulin detemir or NPH insulin as the basal component of basal-bolus therapy in people with type 1 diabetes. METHODS: A multicentre, open-label, parallel-group study was conducted over 12 months and completed by 308 people (from an original randomized cohort of 428). Patients were randomized in a 2:1 ratio to receive insulin detemir or NPH insulin before breakfast and dinner, with insulin aspart at mealtimes. RESULTS: Glycaemic control improved in both groups with HbA(1c) decreasing by 0.64 and 0.56% point in the insulin detemir and NPH insulin groups, reaching baseline-adjusted final values of 7.53 +/- 0.10% and 7.59 +/- 0.13%, respectively. No significant difference was apparent between treatments in terms of HbA(1c), fasting plasma glucose or 9-point blood glucose profiles. Fewer hypoglycaemic events (major and minor) occurred in association with insulin detemir compared with NPH insulin, but the overall hypoglycaemic risk did not differ statistically significantly (RR for detemir, 0.78 [0.56-1.08]). However, the risk of nocturnal hypoglycaemia during the maintenance phase (month 2-12) was 32% lower in the detemir group (p = 0.02) and lower in every month. This risk reduction remained statistically significant after correction for HbA(1c). After 12 months, baseline-adjusted mean body weight was significantly lower in the insulin detemir group than in the NPH insulin group (p < 0.001). CONCLUSIONS: In long-term basal-bolus therapy, insulin detemir with insulin aspart as mealtime insulin is well tolerated and reduces the risks of nocturnal hypoglycaemia and weight gain compared to NPH insulin.     

Two distinct HLA-DR3 haplotypes are associated with age related heterogeneity in Type 1 (insulin-dependent) diabetes

Summary Heterogeneity between two haplotypes in linkage disequilibrium with DR3: B8, C4AQOB1,BfS,DR3 and B18,C4A3BQO,BfF1,DR3, with regard to age at onset of Type 1 (insulin-dependent) diabetes mellitus, was investigated in 325 unrelated French patients (146 males and 179 females, age at onset 1 month to 29 years) who were genotyped for HLA-A, B, C, DR and Bf and 225 of whom were typed for the C4A, B complement components. A subgroup of 82 patients and 75 control subjects were tested for DR and DQ DNA restriction fragment length polymorphism. The distribution according to age at onset and the mean ages at onset were compared between patients bearing B8, DR3 (n=58), B18,DR3 (n=62) or other DR3 haplotypes (Bx, DR3, n=70), the haplotype segments C4AQOB1.DR3 (n=41) or C4A3BQO,DR3 (n = 52) and the C4 null alleles C4AQO (N=48) or C4BQO (n=112) alone. The B8,DR3 haplotype, its smaller segment C4AQOB1,DR3 or C4AQO alone were associated with age at onset after 6 years (p<0.01, <0.08 and <0.02 respectively); on the other hand, the B18,DR3 haplotype, its segment C4A3BQO,DR3 or C4BQO alone were significantly more frequent in patients aged less than 6 years at onset (p<0.02, <0.01 and <0.01 respectively). Accordingly, the mean age of onset was significantly lower in the latter compared with the former patiens (p<0.02, <0.02 and <0.01 respectively). No age-related variation was observed in BX,DR3 patients and their mean age of onset was intermediate. The observed age distributions were sex dependent: that of C4AQO was mainly observed in males, that of C4BQO in females. Restriction fragment length polymorphism analysis in 37 patients and 32 control subjects positive for DR3 showed distinct patterns which correlated with DR3 and/or DQW2 borne by the B8 (n=11) and the B18 (n=18) haplotypes, respectively. BX,DR3 subjects exhibited either one or the other of these patterns. In the patients, the B18 associated fragments were found in most cases; whereas the B8 associated pattern was more frequent in the control subjects. These results provide evidence that a heterogeneous genetic background associated with two subsets of DR3 and/or silent alleles of the fourth component of complement could account for differences in the clinical expression of Type 1 diabetes.