Xinyu Xu

Inhibition of increased circulating Tfh cell by anti-CD20 monoclonal antibody in patients with type 1 diabetes.

Objectives Follicular helper T (Tfh) cells exert an important role in autoimmune diseases. Whether it might be involved in type 1 diabetes (T1D) is unknown. Our aim was to investigate the role of Tfh cells in patients with T1D and the effect of anti-CD20 monoclonal antibody (rituximab) on Tfh cells from T1D patients. Patients and Methods Fifty-four patients with T1D and 37 healthy controls were enrolled in the current study. 20 of those patients were treated with rituximab. The frequencies of circulating CD4+CXCR5+ICOS+T cells were analyzed by flow cytometry. The serum autoantibodies were detected by radioligand assay. The levels of IL-21, IL-6 and BCL-6 were assessed using ELISA and/or real-time PCR. Results Increased frequencies of circulating Tfh cells together with enhanced expression of IL-21 were detected in patients. The correlation between the frequencies of circulating Tfh cells and the serum autoantibodies or C-peptide level was comfirmed. After rituximab therapy, follow-up analysis demonstrated that the frequencies of circulating Tfh cell and serum IA2A were decreased. The levels of IL-21, IL-6 and Bcl-6 mRNA were decreased after treatment. Furthermore, beta cell function in 10 of 20 patients was improved. Conclusions These data indicate Tfh cells may participate in the T1D-relatede immune responses and B cells might play a role in the development of Tfh responses in the disease progression.

UCP2 -866G/A, Ala55Val and UCP3 -55C/T Polymorphisms in Association with Obesity Susceptibility — A Meta-Analysis Study

Aims/hypothesis Variants of UCP2 and UCP3 genes have been reported to be associated with obesity, but the available data on the relationship are inconsistent. A meta-analysis was performed to determine whether there are any associations between the UCP2 -866G/A, Ala55Val, and UCP3 -55C/T polymorphisms and obesity susceptibility. Methods The PubMed, Embase, Web of Science and CNKI, CBMdisc databases were searched for all relevant case-control studies. The fixed or random effect pooled measure was determined on the bias of heterogeneity test among studies. Publication bias was examined by the modified Beggs and Eggers test. Results Twenty-two published articles with thirty-two outcomes were included in the meta-analysis: 12 studies with a total of 7,390 cases and 9,860 controls were analyzed for UCP2 -866G/A polymorphism with obesity, 9 studies with 1,483 cases and 2,067 controls for UCP2 Ala55Val and 8 studies with 2,180 cases and 2,514 controls for UCP3 -55C/T polymorphism. Using an additive model, the UCP2 -866G/A polymorphism showed no significant association with obesity risk in Asians (REM OR = 0.81, 95% CI: 0.65–1.01). In contrast, a statistically significant association was observed in subjects of European descent (FEM OR = 1.06, 95% CI: 1.01–1.12). But neither the UCP2 Ala55Val nor the UCP3 -55C/T polymorphism showed any significant association with obesity risk in either subjects of Asian (REM OR = 0.84, 95% CI: 0.67–1.06 for Ala55Val; REM OR = 0.94, 95% CI: 0.55–1.28 for -55C/T) or of European descent (REM OR = 1.04, 95% CI: 0.80-1.36 for Ala55Val; FEM OR = 1.08, 95% CI: 0.97–1.20 for -55C/T). Conclusions and Interpretation Our meta-analysis revealed that the UCP2 -866G/A polymorphism may be a risk factor for susceptibility to obesity in subjects of European descent, but not in individuals of Asian descent. And our results did not support the association between UCP2 Ala55Val, UCP3 -55C/T polymorphisms and obesity in the populations investigated. This conclusion warrants confirmation by further studies.

Association between rs13266634 C/T polymorphisms of solute carrier family 30 member 8 (SLC30A8) and type 2 diabetes, impaired glucose tolerance, type 1 diabetes—A meta-analysis

AIMS To investigate the association of solute carrier family 30 member 8 (SLC30A8) rs13266634 C/T polymorphism with type 2 diabetes (T2DM), impaired glucose tolerance (IGT), and type 1 diabetes (T1DM). METHODS We searched all the publications about the association between SLC30A8 and diabetes from PubMed, and evaluated the association between SLC30A8 rs13266634 C/T polymorphism and T2DM, IGT and T1DM, respectively, by meta-analysis of all the validated studies. Allelic and genotypic comparisons between cases and controls were evaluated. RESULTS Thirty six studies were included in the meta-analysis: 31 studies were analysed for rs13266634 C/T polymorphism with T2DM, 3 studies with IGT and 4 studies with T1DM. The pooled odds ratios (ORs) for allelic and genotypic comparisons (including additive model, co-dominant model, dominant model and recessive model) showed that rs13266634 C/T polymorphism was significantly associated with increased T2DM risk: OR=1.15, 95% confidence interval (CI)=1.13-1.17, P<0.001, P(heterogeneity)=0.041, OR=1.34, 95% CI=1.26-1.41, P<0.001, P(heterogeneity)=0.908, OR=1.20, 95% CI=1.16-1.24, P<0.001, P(heterogeneity)=0.699, and OR=1.23, 95% CI=1.17-1.30, P<0.001, P(heterogeneity)=0.801, respectively. In subgroup analyses, we found that rs13266634 C/T polymorphism was associated with T2DM risk both in Asian and European subgroup (P<0.001), but not in African (P>0.05). And the pooled odds ratio (OR) for allelic frequency comparison showed that rs13266634 C/T polymorphism was also significantly associated with IGT: OR=1.15, 95% CI=1.06-1.26, P<0.001, P(heterogeneity)=0.364. Meanwhile, our meta-analysis did not suggest that rs13266634 C/T polymorphism was associated with T1DM risk (P>0.05): OR=1.02, 95% CI=0.98-1.06, P=0.328, P(heterogeneity)=0.488 for allelic frequency comparison. CONCLUSIONS Our meta-analysis results revealed the significant association between rs13266634 C/T polymorphism and T2DM and IGT, but did not support the association between this polymorphism and T1DM.

Fat Mass and Obesity-Associated Gene Enhances Oxidative Stress and Lipogenesis in Nonalcoholic Fatty Liver Disease

Background and AimNonalcoholic fatty liver disease (NAFLD) is strongly associated with obesity, hyperlipidemia, and type 2 diabetes mellitus. Several studies have found that fat mass and the obesity-associated (FTO) gene is linked to obesity. The aim of this work is to investigate the expression and function of FTO in liver with lipid metabolism diseases.MethodsWe investigated the basal FTO expression in an NAFLD rat model and compared it with control subjects. The function of FTO in lipid metabolism was further studied in L02 cells through overexpression experiments.ResultsA significant increase in FTO mRNA and protein levels was found in the NAFLD group. In addition, the FTO levels were positively associated with malondialdehyde and superoxide dismutase concentrations. FTO overexpression in L02 cells enhanced lipogenesis and oxidative stress.ConclusionsThis study demonstrates that increased FTO levels in the liver are involved in oxidative stress and lipid deposition, which characterize NAFLD.

Systematic review and meta-analysis of islet autotransplantation after total pancreatectomy in chronic pancreatitis patients [Review]

Islet autotransplantation (IAT) is a viable treatment for patients with severe chronic pancreatitis, this modality may prevent brittle diabetes mellitus after pancreatectomy. This systematic review and meta-analysis was performed to evaluated the outcomes of IAT after TP and discuss the factors that may affect the efficacy of this procedure. MEDLINE, Embase, Web of Science and the Cochrane Central Register of Controlled Trials (CENTRAL) were searched from 1977 to 30 April 2014. Cohort Studies reported patients with IAT after TP were included. The studies and data were identified and extracted by two reviewers independently. Data were analyzed using STATA 12.0 and Comprehensive Meta AnalysisV2 software. Random effects model, meta-regression analysis, sensitivity analysis and publication bias were conducted to improve the comprehensive analysis. Twelve studies reporting the outcomes of 677 patients were included in this review. The insulin independent rate for IAT after TP at last follow-up was 3.72 per 100 person-years (95% CI: 1.00-6.44). The 30-day mortality was 2.1% (95% CI: 1.2-3.8%). The mortality at last follow-up was 1.09 per 100 person-years (95% CI: 0.21-1.97). Factors associated with incidence density of insulin independence in univariate meta-regression analyses included islet equivalents per kg body weight (IEQ/kgBW) (P=0.026). Our systematic review suggests that IAT is a safe modality for patients with CP need to undergo TP. A significant number of patients will achieve insulin independence for a long time after receiving enough IEQ/kgBW.

Increased Th22 cells are independently associated with Th17 cells in type 1 diabetes

Type 1 diabetes (T1D) is perceived as an autoimmune disease caused by T cell-mediated destruction of the insulin-producing pancreatic β cells. However, the number of inflammatory T cells in blood, as well as the relative importance of each cell type is unclear. Forty-two patients with T1D and 30 controls were enrolled. Circulating primary CD4+ or CD8+ T cells were quantified with 5-color flow cytometry. Serum IL-22 and IL-17 levels were examined by ELISA. Serum autoantibodies were measured by radio-binding assays, using 35S-labeled glutamic acid decarboxylase-65 (GAD65), protein tyrosine phosphatase-2 (IA-2), and zinc transporter 8 (ZnT8). Th17–Th22 and Tc1–Tc17 were significantly elevated in patients with T1D compared to control subjects, while there were no significant differences in Th1 cells. The levels of these T cells in different stages of T1D were investigated. Th22 cells showed a positive correlation with Th17 cells in T1D patients. However, we did not find any correlation between IL-17 and IL-22 in sera. Autoantibodies were not significantly different between patients with early T1D and those who have had it for a longer duration. This study indicates that Th22 may contribute to the pathogenesis of T1D. Blockade of Th22 cells might be of clinical profit in T1D patients.

Effects of islet neogenesis-associated protein pentadecapeptide on cell mass and insulin secretion of pancreatic β-cells

Objective: To explore the effects of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) on proliferation and secretion function of β-cells. Methods: Islets of adult Sprague Dawley rats were isolated by colla-genase digestion and treated with 10 µg/ml INGAP-PP, after 12, 24, 48 h, glucose-stimulated insulin secretion (GSIS) and acridine orange/propidium iodide (AO/PI) staining were used to detect the secretion function and cell viability. The INS-1 cells were treated with 0, 1, 10, 25, 50, 100, 250, and 500 µg/ml INGAP-PP for 24 or 48 h, MTT cell proliferation assay was adopted to survey the dose-response relationship between INGAP-PP and cell proliferation. The mRNA expression of roliferating cell nuclear antigen (PCNA), Cyclin D1, Cdk4, P27, p38MAPK, and JNK in INS-1 cells were examined by RT-PCR, and the protein expression of PCNA was examined by Western blot. The statistical significance was determined by Student’s t-test or one-way analysis of variance. Results: The insulin secreted by islets and the cell viability were increased by INGAP-PP. MTT indicated a dose-response relationship between INGAP-PP and quantity of INS-1 cells, and treatment for 48 h had a stronger effect on cell proliferation than the 24 h. INGAP-PP up-regulated the mRNA expression of PCNA, Cyclin D1, Cdk4 and down-regulated P27, p38MAPK, and JNK. Moreover, the protein expression of PCNA was up-regulated by 45% after INGAP-PP exposure for 48 h. Conclusions: INGAP-PP increased the insulin secretion, enhanced the proliferation and might reduce apoptosis of β-cells. The mechanism may contribute to the changed expression of some genes related to cell cycle.

Characterization of immune response to novel HLA-A2-restricted epitopes from zinc transporter 8 in type 1 diabetes.

OBJECTIVE ZnT8-specific CD8+ T cells in human type 1 diabetes (T1D) have been reported recently, although the results from different laboratories are inconsistent. We aimed to characterize these ZnT8 specific CD8+ T cells and validate assays to screen peptide libraries. METHODS We screened HLA-A2-restricted T cell candidate peptides of ZnT8 with different methods including computer algorithms, MHC-peptide binding and dissociation assays in T2 cell line, identification in HLA-A2 transgenic (Tg) mice and in vivo CTL assays. Then ELISpot assay was used to measure peptide-reactive T cell responses in 49 HLA-A2-restricted T1D patients. RESULTS We demonstrated that ZnT8(107-116)(115), ZnT8(110-118), and ZnT8(177-186) were novel HLA-A*0201-restricted CTL epitopes in T1D patients. ZnT8(107-116)(115), ZnT8(115-123), ZnT8(153-161), ZnT8(177-186) and ZnT8(291-300) represent potentially major biomarkers for T1D. T cell responses against these epitopes showed different distributions between recently diagnosed and long-standing patients. Furthermore, they displayed discriminating performance among different ethnicities. We also compared the performance of the epitope identification strategies used herein. The epitopes which exhibited strong immunogenicity in HLA-A2 Tg mice were also well recognized by T1D patients. CONCLUSIONS The differences in autoimmune T cell responses among T1D individuals may open new avenues toward T1D prediction and prevention. It also provides efficient strategies for immune intervention.

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays.

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8153–161 had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8153–161 is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = − 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.

Discordant association of islet autoantibodies with high‐risk HLA genes in Chinese type 1 diabetes

To reveal the aetiology of diabetes, the relationships between the islet autoantibodies, human leukocyte antigen (HLA)‐A and DRB1 genotypes in the Chinese patients with type l diabetes (T1D) were investigated in our study.