Evidence for immunological priming and increased frequency of CD4+ CD25+ cord blood T cells in children born to mothers with type 1 diabetes

Maternal transmission of islet autoantibodies to children born to mothers with type 1 diabetes (T1D) has been shown to protect from autoantibodies and diabetes development later in life. However, the factors conferring disease protection are poorly understood. The aim of this study was to evaluate comparatively proinflammatory cytokines, autoantibodies and lymphocyte subsets in cord blood (CB) of children born to mothers with either T1D (n = 13), gestational diabetes (GDM) (n = 32) or healthy mothers (n = 81) in relation to transplacental passage of autoantibodies. The results are consistent with early priming of the fetal immune system only in children born to mothers with T1D. Levels of interleukin (IL)-1beta (P = 0.022), tumour necrosis factor (TNF)-alpha (P = 0.002) and IL-8 (P = 0.0012), as well as the frequency of CD4(+) CD25(+) T cells (P < 0.01) were significantly increased, and the increased levels correlated positively with anti-GAD65 autoantibody (GADA) levels. Moreover, CD4(+) CD25(+) T cells of children born to T1D mothers exhibited a more pronounced memory phenotype with increased CCR4 expression and down-regulation of CD62L. These data suggest that early activation of the fetal immune system as a consequence of maternal autoimmunity and transplacental passage of GADA may influence the generation and expansion of fetal regulatory T cells. This might induce an early antigen-specific immunological tolerance that could protect against T1D later in life.     

Immunology in the clinic review series; focus on type 1 diabetes and viruses: enterovirus, thymus and type 1 diabetes pathogenesis

Thymus dysfunction, especially immune suppression, is frequently associated with various virus infections. Whether viruses may disturb the thymus function and play a role in the pathogenesis of autoimmune diseases is an open issue. Enteroviruses, especially Coxsackievirus B4 (CV-B4), have been largely suggested as potential inducers or aggravating factors of type 1 diabetes (T1D) pathogenesis in genetically predisposed individuals. Several pathogenic mechanisms of enterovirus-induced T1D have been suggested. One of these mechanisms is the impairment of central self-tolerance due to viral infections. Coxsackievirus-B4 is able to infect murine thymus in vitro and in vivo and to infect human thymus in vitro. Thymic epithelial cells and thymocytes are targets of infection with this virus, and several abnormalities, especially disturbance of maturation/differentiation processes, were observed. Altogether, these data suggest that CV-B infection of thymus may be involved in the pathogenesis of T1D. Further investigations are needed to explore this hypothesis.     

Maternal virus infections in pregnancy and type 1 diabetes in their offspring: Systematic review and meta‐analysis of observational studies

Virus infections are implicated in the development of type 1 diabetes based on epidemiological, clinical, in vitro cell‐based and molecular studies, and animal models. We reviewed the association between virus infections in pregnant women and development of islet autoimmunity or type 1 diabetes in their offspring. We performed a systematic review and meta‐analysis, analysed using random effects models, of human studies from Medline and EMBASE without language restriction. Inclusion criteria were as follows: cohort and case‐control studies measuring viral nucleic acid in blood, stool, urine, or tissue, or serological tests for viruses, in pregnant women whose offspring developed islet autoimmunity and/or type 1 diabetes. All studies required sufficient data to calculate odds ratios and 95% confidence intervals. The 10 studies (4 case control, 6 nested‐case control) that met the eligibility criteria included 2992 participants (953 offspring, 2039 mothers), with varying study design. The 2 outcomes examined were islet autoimmunity (n = 466) and type 1 diabetes (n = 2526). Meta‐analysis showed a significant association between virus infection during pregnancy and clinical type 1 diabetes during childhood (odds ratio 2·16, 95% CI 1·22‐3·80; P = 0·008; heterogeneity X² = 1·65, I² = 40%), but no association with islet autoimmunity (1·45, 0·63‐3·31; P = 0·38; X² = 1·34, I² = 25%). The increased risk of type 1 diabetes following maternal virus infection is consistent with viraemia involving the fetus during pregnancy and suggests a potential causative link between antenatal infection and type 1 diabetes. Larger prospective birth studies with more frequent sampling, and pathogenesis studies, are required to more clearly establish an aetiological link.     

单纯饮食控制的2型糖尿病患者糖化血红蛋白与全天各点血糖的相关性分析

了解单纯饮食控制的2型糖尿病患者全天8个点血糖值与糖化血红蛋白(HbAlc)的相关性。收集8周观察期中16d的每日8个点血糖谱各点平均值,与8周末的HbAlc进行简单线性相关分析。8个点血糖中,全天各点的平均值与HbAlc的相关性最好,r值为0．84。凌晨3：00点,清晨6：00点及睡前(22：00～23：00)血糖与8周末的HbAlc的相关系数r值分别为0．81,0．79和0．78。结果表明血糖控制稳定的单纯饮食控制的2型糖尿病患者空腹血糖和吸收后状态血糖与HbAlc的相关性较好,而全天血糖的平均值与HbAlc的相关性更好。     

The role of autoimmunity in women with type 1 diabetes and adverse pregnancy outcome: A missing link

The incidence of pregnancy complications in women with type 1 Diabetes Mellitus (T1D) is greater than in healthy pregnant women. This has mostly been attributed to hyperglycemia. However, despite the implementation of stricter guidelines regarding glycemic control, pregnancy complications remain more common in women with T1D. This may suggest that other etiological factors are involved. We suggest that the immune response may play a role, since the immune response has to adapt during pregnancy in order to facilitate implantation, placental and fetal development, and aberrant immunological adaptations to pregnancy are involved in various pregnancy complications. Since T1D is an autoimmune disorder, the question rises whether the immune response of women with T1D is able to adapt properly during pregnancy. Here we review the current proof and views on the role of aberrant immunological adaptations in pregnancy complications and whether such aberrant adaptations could be involved in the pregnancy complications of T1D patients.     

HTLV‐1 and ‐2 in a first‐time blood donor population in Northeastern Brazil: Prevalence,molecular characterization,and evidence of intrafamilial transmission

Independent epidemiology for respective human T‐cell lymphotropic virus (HTLV) types 1 and 2 is little known in blood donors in Brazil, where screening for HTLV‐1/2 is mandatory at blood banks, but no testing to confirm/differentiate these viruses. Therefore, this study aims to assess the prevalence of HTLV‐1 and ‐2 in a first‐time blood donor population in Northeastern Brazil and to carry out molecular characterization of respective isolates. A cross‐sectional study was conducted at the State Blood Bank in Piauí. Samples were screened for anti–HTLV‐1/2 by enzyme immunoassay, and reactive samples were confirmed using a line immunoassay and polymerase chain reaction (PCR). Of 37 306 blood donors, 47 were anti–HTLV‐1/2 reactive by enzyme immunoassay. After confirmed by line immunoassay, 22 were positive for HTLV‐1 (0.59 per 1000; 95% CI: 0.38‐0.87), 14 were positive for HTLV‐2 (0.37 per 1000; 95% CI: 0.21‐0.61), 1 was indeterminate, and the remaining donors were negative. The HTLV‐1 infection was also confirmed by PCR in all anti–HTLV‐1‐positive samples, and sequencing classified these isolates as belonging to the Transcontinental (A) subgroup of the Cosmopolitan (1a) subtype. Of 14 anti–HTLV‐2‐positive samples, 11 were also PCR positive, which belonged to subtype a (HTLV‐2a/c). In addition, 38 family members of 5 HTLV‐1‐ and 3 HTLV‐2‐infected donors were analyzed. Familial transmission of HTLV‐1 and ‐2 was evidenced in 3 families. In conclusion, in Northeastern Brazil, where HTLV‐1 and ‐2 are endemic, counseling blood donor candidates and their families might play a key role in limiting the spread of these viruses.     

DNA methylation and mRNA expression of HLA‐DQA1 alleles in type 1 diabetes mellitus

Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA‐DQA1 gene. Our aim was to investigate DNA methylation of HLA‐DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA‐DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA‐DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA‐DQA1 promoter alleles was analysed. Individual mRNA HLA‐DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA‐DQA1 allele 02:01 expression (PPIA normalization, P_corr = 0·041; DRA normalization, P_corr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA‐DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA‐DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA‐DQA1 allele in patients with T1D have been described for the first time.     

Multi‐parametric flow cytometric and genetic investigation of the peripheral B cell compartment in human type 1 diabetes

The appearance of circulating islet-specific autoantibodies before disease diagnosis is a hallmark of human type 1 diabetes (T1D), and suggests a role for B cells in the pathogenesis of the disease. Alterations in the peripheral B cell compartment have been reported in T1D patients; however, to date, such studies have produced conflicting results and have been limited by sample size. In this study, we have performed a detailed characterization of the B cell compartment in T1D patients (n = 45) and healthy controls (n = 46), and assessed the secretion of the anti-inflammatory cytokine interleukin (IL)-10 in purified B cells from the same donors. Overall, we found no evidence for a profound alteration of the B cell compartment or in the production of IL-10 in peripheral blood of T1D patients. We also investigated age-related changes in peripheral B cell subsets and confirmed the sharp decrease with age of transitional CD19⁺CD27⁻CD24^hiCD38^hi B cells, a subset that has recently been ascribed a putative regulatory function. Genetic analysis of the B cell compartment revealed evidence for association of the IL2–IL21 T1D locus with IL-10 production by both memory B cells (P = 6·4 × 10⁻⁴) and islet-specific CD4⁺ T cells (P = 2·9 × 10⁻³). In contrast to previous reports, we found no evidence for an alteration of the B cell compartment in healthy individuals homozygous for the non-synonymous PTPN22 Trp⁶²⁰ T1D risk allele (rs2476601; Arg⁶²⁰Trp). The IL2–IL21 association we have identified, if confirmed, suggests a novel role for B cells in T1D pathogenesis through the production of IL-10, and reinforces the importance of IL-10 production by autoreactive CD4⁺ T cells.     

Fine‐scale geographic clustering pattern of human T‐cell leukemia virus type 1 infection among blood donors in Kyushu‐Okinawa,Japan

Human T‐cell leukemia virus type I (HTLV‐1) infection is endemic in Japan, particularly clustered in the southwestern district, Kyushu‐Okinawa, which consists of eight prefectures that further consist of 274 municipalities. However, no information is available about the fine‐scale distribution of HTLV‐1 infection within Kyushu‐Okinawa. To assess the municipal‐level distribution of people with HTLV‐1 infection in Kyushu‐Okinawa, we performed a cross‐sectional study using a fine‐scale geographic information system map based on HTLV‐1 screening test results from the Japanese Red Cross database from September 2012 to February 2014. Of the 881 871 (646 914 male, 234 957 female) screened blood donors, 981 were seropositive for HTLV‐1 by confirmatory test. The seroprevalence was 0.11% (95% confidence interval [CI] 0.10%‐0.12%) for all, 0.094% (95% CI, 0.09%‐0.10%) for male, and 0.16% (95% CI, 0.14%‐0.18%) for female individuals. The sex‐ and age‐specific HTLV‐1 seroprevalence varied significantly across municipalities; particularly, the seroprevalence among women aged 50 years was significantly higher than that of men in both the mainland of Kyushu‐Okinawa and the satellite island, in all of which the seroprevalence of HTLV‐1 was more than 1.2%. These results show that, even in the Kyushu‐Okinawa district, there are endemic clusters of HTLV‐1 in small areas. This suggests that public health education programs are needed to eliminate new HTLV‐1 infection in these areas.     

Effects of long‐term diabetes on the structure and cell proliferation of the myometrium in the early pregnancy of mice

It is known that the development of diabetic complications in human pregnancy is directly related to the severity and the duration of this pathology. In this study, we developed a model of long‐term type 1 diabetes to investigate its effects on the cytoarchitecture, extracellular matrix and cell proliferation during the first adaptation phase of the myometrium for pregnancy. A single dose of alloxan was used to induce diabetes in mice prior to pregnancy. To identify the temporal effects of diabetes the mice were divided into two groups: Group D1 (females that became pregnant 90–100 days after alloxan); Group D2 (females that became pregnant 100–110 days after alloxan). Uterine samples were collected after 168 h of pregnancy and processed for light and electron microscopy. In both groups the histomorphometric evaluation showed that diabetes promoted narrowing of the myometrial muscle layers which was correlated with decreased cell proliferation demonstrated by PCNA immunodetection. In D1, diabetes increased the distance between muscle layers and promoted oedema. Contrarily, in D2 the distance between muscle layers decreased and, instead of oedema, there was a markedly deposition of collagen in the myometrium. Ultrastructural analysis showed that diabetes affects the organization of the smooth muscle cells and their myofilaments. Consistently, the immunoreaction for smooth muscle α‐actin revealed clear disorganization of the contractile apparatus in both diabetic groups. In conclusion, the present model demonstrated that long‐term diabetes promotes significant alterations in the myometrium in a time‐sensitive manner. Together, these alterations indicate that diabetes impairs the first phenotypic adaptation phase of the pregnant myometrium.     

Relationship between the incidence of type 1 diabetes and enterovirus infections in different European populations: results from the EPIVIR project

The incidence of type 1 diabetes varies markedly between countries. As enterovirus infections have been linked to type 1 diabetes, we determined whether this variation correlates with the frequency of enterovirus infections in different Caucasian populations in Europe. Enterovirus antibodies were examined in the background population (1-year-old and 10-14-year-old children) in seven countries with either exceptionally high (Finland and Sweden) or low/intermediate incidence of diabetes (Estonia, Germany, Hungary, Lithuania, Russia) using EIA and neutralisation assays. Enterovirus antibodies were less frequent in countries with high diabetes incidence compared to countries with low diabetes incidence (P<0.001). This suggests that enterovirus infections are not particularly common in countries with high diabetes incidence. In contrast, there seems to be an inverse correlation between the incidence of type 1 diabetes and enterovirus infections in the background population, which is in line with the previously proposed polio hypothesis according to which the complications of enterovirus infections become more common in an environment with a decreased rate of infections.     

Mycobacterial antigen‐induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non‐diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects

Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non‐diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell‐mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non‐diabetic TB patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen‐induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐β], and Th2 (IL‐4, IL‐5, IL‐10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1_pool stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1_pool in healthy subjects. In response to complex mycobacterial antigens, both IFN‐γ and TNF‐β were secreted by PBMC of all groups whereas diabetic TB patients secreted IL‐10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN‐γ and IL‐10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions.     

Enteroviruses and the pathogenesis of type 1 diabetes revisited: cross‐reactivity of enterovirus capsid protein (VP1) antibodies with human mitochondrial proteins

Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5‐D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross‐reactivity of clone 5‐D8/1 with normal islet proteins. Western blotting, two‐dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5‐D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B‐type and ATP synthase beta subunit. Immunohistochemistry using the clone 5‐D8/1 revealed a granular cytoplasmic staining pattern in mitochondria‐rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent‐onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5‐D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5‐D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent‐onset type 1 diabetes patients could reflect cross‐reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunology in the clinic review series; focus on type 1 diabetes and viruses: role of antibodies enhancing the infection with Coxsackievirus-B in the pathogenesis of type 1 diabetes

Type 1 diabetes results from an interaction between genetic and environmental factors. Coxsackieviruses B (CV-B) are major environmental candidates, as suggested by epidemiological and experimental studies. The mechanisms leading to the disease involve interactions between the virus, host target tissue (pancreas) and the immune system. The infection of target cells with viruses can be prevented by antibodies. Conversely, the infection can be enhanced by antibodies. The antibody-dependent enhancement (ADE) of infection has been described with various viruses, especially Picornaviruses. In mice infected with CV-B3 this phenomenon resulted in an extended inflammatory reaction and myocarditis. In the human system non-neutralizing antibodies can increase the infection of monocytes with CV-B4 and stimulate the production of interferon (IFN)-α by these cells in vitro. CV-B4/immunoglobulin (Ig)G immune complexes interacted with a specific viral receptor [Coxsackievirus and adenovirus receptor (CAR)] and with IgG Fc fraction receptors (FcγRII and FcγRIII) at the surface of monocytes. The virus-antibody complexes are internalized (CAR) and receptors for the Fc of IgG (FcγRII and FcγRIII). Such antibodies have been detected in patients with type 1 diabetes and they could be responsible for the presence of enteroviral RNA and IFN-α in peripheral blood mononuclear cells (PBMC) of these individuals. The target of enhancing antibodies has been identified as the VP4 protein, which allowed the detection of these antibodies by enzyme-linked immunosorbent assay (ELISA). It cannot be excluded that antibodies enhancing the infection with CV-B may play a role in the pathogenesis of type 1 diabetes, induced or aggravated by these viruses. They can cause a viral escape from the immune response and may participate in the spreading of viruses to β cells. Whether enhancing antibodies raised against VP4 can play a role in iterative homologous and/or heterologous CV-B infections and in the persistence of viruses within the host deserves further study.     

Plasma levels of MASP‐1, MASP‐3 and MAp44 in patients with type 2 diabetes: influence of glycaemic control,body composition and polymorphisms in the MASP1 gene

Mounting evidence indicates that adverse activation of the complement system plays a role in the development of diabetic vascular complications. Plasma levels of the complement proteins mannan‐binding lectin (MBL) and its associated serine proteases (MASP‐1 and MASP‐2) are elevated in diabetes. We hypothesized that single nucleotide polymorphisms (SNPs) in the MASP1 gene may contribute to altered plasma levels of the belonging gene products; MASP‐1, MASP‐3 and mannan‐binding lectin‐associated protein of 44 kDa (MAp44) in patients with type 2 diabetes. To investigate this, we compared plasma levels of MASP‐1, MASP‐3 and MAp44 in 100 patients with type 2 diabetes and 100 sex‐ and age‐matched controls. Ten carefully selected SNPs were analysed using TaqMan^® genotyping assay. Additionally, we included a streptozotocin‐induced diabetes mouse model to directly examine the effect of inducing diabetes on MASP‐1 levels. MASP‐1 levels were significantly higher among patients with type 2 diabetes compared with healthy controls (P = 0·017). Five SNPs (rs874603, rs72549254, rs3774275, rs67143992, rs850312) in the MASP1 gene were associated with plasma levels of MASP‐1, MASP‐3 and MAp44. In the diabetes mouse model, diabetic mice had significantly higher MASP‐1 levels than control mice (P = 0·003). In conclusion, MASP‐1 levels were higher among patients with type 2 diabetes and diabetic mice. The mechanism behind this increase remains elusive.     

Association between G-308A polymorphism of the tumor necrosis factor-alpha gene and 24-hour ambulatory blood pressure values in type 1 diabetic adolescents

Tumor necrosis factor-alpha (TNF-alpha) is an inflammatory cytokine, which also influences blood pressure (BP). The G-308A polymorphism of the TNF-alpha gene is associated with altered TNF-alpha production. The prevalence of the TNF-alpha-308A allele is reportedly higher among patients with type 1 diabetes mellitus (T1DM) than in the healthy population. In this study we investigated whether this genetic polymorphism might correlate with BP values in diabetic adolescents. Ambulatory BP monitoring (ABPM) was performed in 126 adolescents with T1DM (mean age: 14 +/- 2.4 years). The TNF-alpha G-308A genotype was determined by using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methodologies. ABPM results were related to healthy reference values and are given as standard deviation score (SDS). The prevalence of the -308A allele was higher in diabetic adolescents than the Hungarian reference population (0.26 vs 0.14, p < 0.01). TNF-alpha genotype was associated both with systolic and diastolic BP values (p < 0.01 and p < 0.01, respectively). In patients with TNF-alpha-308GG and -308GA/AA genotypes, the 24-h systolic BP average values were 0.37 +/- 1.33 and -0.38 +/- 1.28 SDS, while 24-h diastolic BP average values were 0.09 +/- 1.30 and -0.67 +/- 1.31 SDS. Hence, the TNF-alpha-308A allele carrier state appears to be associated with lower systolic and diastolic BP values.     

Effects of C‐peptide on forearm blood flow and brachial artery dilatation in patients with type 1 diabetes mellitus

Recent studies suggest that C‐peptide increases blood flow in both exercising and resting forearm in patients with type 1 diabetes. Now we have studied the effect of C‐peptide administration on endothelial‐mediated and non‐endothelial‐mediated arterial responses as well as central haemodynamics in 10 patients with type 1 diabetes in a placebo‐controlled double‐blind study. Euglycaemia was maintained with an i.v. insulin infusion before and during the study. A high‐resolution ultrasound technique and Doppler echocardiography were used to assess haemodynamic functions. Brachial artery blood flow and brachial artery diameter were measured in the basal state, 1 and 10 min after reactive hyperaemia and 4 min after sublingual glyceryl trinitrate administration (GTN; endothelial‐independent vasodilatation), both before and after the end of 60‐min C‐peptide (6 pmol kg^–1 min^–1) or saline infusion periods. Echocardiographic measurements were also performed before and at the end of the infusion periods. Seven healthy age‐matched males served as controls for vascular studies. The patients showed a blunted brachial dilatation after reactive hyperaemia in comparison with the healthy controls (2.1 ± 0.5% vs. 9.3 ± 0.3%, P < 0.001), indicating a disturbed endothelial function. C‐peptide infusion compared with saline resulted in increased basal blood flow (33 ± 6%, P < 0.001) and brachial arterial dilatation (4 ± 1%, P < 0.05). Left ventricular ejection fraction seemed to be improved (5 ± 2%, P < 0.05) at the end of C‐peptide infusion compared with placebo. The vascular response to reactive hyperaemia and GTN was not affected by C‐peptide infusion. Our results demonstrate that physiological concentrations of C‐peptide increase resting forearm blood flow, brachial artery diameter and left ventricular systolic function in patients with type 1 diabetes.     

Effects of combination therapy with dipeptidyl peptidase‐IV and histone deacetylase inhibitors in the non‐obese diabetic mouse model of type 1 diabetes

Type 1 diabetes (T1D) results from T helper type 1 (Th1)‐mediated autoimmune destruction of insulin‐producing β cells. Novel experimental therapies for T1D target immunomodulation, β cell survival and inflammation. We examined combination therapy with the dipeptidyl peptidase‐IV inhibitor MK‐626 and the histone deacetylase inhibitor vorinostat in the non‐obese diabetic (NOD) mouse model of T1D. We hypothesized that combination therapy would ameliorate T1D by providing protection from β cell inflammatory destruction while simultaneously shifting the immune response towards immune‐tolerizing regulatory T cells (T_regs). Although neither mono‐ nor combination therapies with MK‐626 and vorinostat caused disease remission in diabetic NOD mice, the combination of MK‐626 and vorinostat increased β cell area and reduced the mean insulitis score compared to diabetic control mice. In prediabetic NOD mice, MK‐626 monotherapy resulted in improved glucose tolerance, a reduction in mean insulitis score and an increase in pancreatic lymph node T_reg percentage, and combination therapy with MK‐626 and vorinostat increased pancreatic lymph node T_reg percentage. We conclude that neither single nor combination therapies using MK‐626 and vorinostat induce diabetes remission in NOD mice, but combination therapy appears to have beneficial effects on β cell area, insulitis and T_reg populations. Combinations of vorinostat and MK‐626 may serve as beneficial adjunctive therapy in clinical trials for T1D prevention or remission.