Changes in T‐cell subsets identify responders to FcR‐nonbinding anti‐CD3 mAb (teplizumab) in patients with type 1 diabetes

The mechanisms whereby immune therapies affect progression of type 1 diabetes (T1D) are not well understood. Teplizumab, an FcR nonbinding anti‐CD3 mAb, has shown efficacy in multiple randomized clinical trials. We previously reported an increase in the frequency of circulating CD8⁺ central memory (CD8CM) T cells in clinical responders, but the generalizability of this finding and the molecular effects of teplizumab on these T cells have not been evaluated. We analyzed data from two randomized clinical studies of teplizumab in patients with new‐ and recent‐onset T1D. At the conclusion of therapy, clinical responders showed a significant reduction in circulating CD4⁺ effector memory T cells. Afterward, there was an increase in the frequency and absolute number of CD8CM T cells. In vitro, teplizumab expanded CD8CM T cells by proliferation and conversion of non‐CM T cells. Nanostring analysis of gene expression of CD8CM T cells from responders and nonresponders versus placebo‐treated control subjects identified decreases in expression of genes associated with immune activation and increases in expression of genes associated with T‐cell differentiation and regulation. We conclude that CD8CM T cells with decreased activation and regulatory gene expression are associated with clinical responses to teplizumab in patients with T1D.     

Genetic analysis of the ELOVL6 gene polymorphism associated with type 2 diabetes mellitus

Recent animal studies have indicated that overexpression of the elongation of long-chain fatty acids family member 6 (Elovl6) gene can cause insulin resistance and β-cell dysfunction. These are the major factors involved in the development of type 2 diabetes mellitus (T2DM). To identify the relationship between single nucleotide polymorphisms (SNP) of ELOVL6 and T2DM pathogenesis, we conducted a case-control study of 610 Han Chinese individuals (328 newly diagnosed T2DM and 282 healthy subjects). Insulin resistance and islet first-phase secretion function were evaluated by assessment of insulin resistance in a homeostasis model (HOMA-IR) and an arginine stimulation test. Three SNPs of the ELOVL6 gene were genotyped with polymerase chain reaction-restriction fragment length polymorphism, with DNA sequencing used to confirm the results. Only genotypes TT and CT of the ELOVL6 SNP rs12504538 were detected in the samples. Genotype CC was not observed. The T2DM group had a higher frequency of the C allele and the CT genotype than the control group. Subjects with the CT genotype had higher HOMA-IR values than those with the TT genotype. In addition, no statistical significance was observed between the genotype and allele frequencies of the control and T2DM groups for SNPs rs17041272 and rs6824447. The study indicated that the ELOVL6 gene polymorphism rs12504538 is associated with an increased risk of T2DM, because it causes an increase in insulin resistance.     

Potential antidiabetic activity of benzimidazole derivative albendazole and lansoprazole drugs in different doses in experimental type 2 diabetic rats

Background/aim The aim of this study is to determine the effects of different concentrations of albendazole and lansoprazole, which were benzimidazole derivatives, on endocrinologic and biochemical parameters in experimental type 2 diabetic (T2D) rats. Materials and methods In this study, 46 male Wistar Albino rats were used. Animals were divided as healthy control (0.1 mL/rat/day saline, s.c, n = 6), diabetes control (0.1 mL/rat/day saline, s.c, n = 8), diabetes+low-dose albendazole (5 mg/kg, oral, n = 8), diabetes+high-dose albendazole (10 mg/kg, oral n = 8), diabetes+low-dose lansoprazole (15 mg/kg, subcutaneous, n = 8), and diabetes+high-dose lansoprazole (30 mg/kg, subcutaneous, n = 8). All groups were treated for 8 weeks. The blood samples were analyzed by autoanalyzer and ELISA kits for biochemical and endocrinological parameters, respectively. Results Glucose, HbA1c, triglyceride, low density cholesterol (LDL), leptin, and Homeostatic Model Assessment for insulin resistance (HOMA-IR) levels increased and insulin and HOMA-β levels decreased in the diabetic rats compared to the healthy control group. The glucose, HbA1c, and triglyceride levels were partially decreased; however, insulin and HOMA-β levels were increased by low-dose albendazole therapy. The high dose of lansoprazole treatment increased insulin level. Conclusion The lansoprazole and albendazole treatments can be a potential drug or combined with antidiabetic drugs in T2D treatment by Adenosine 5 ′ -monophosphate activated protein kinase (AMPK), peroxisome proliferator-activated receptor (PPAR), incretin-like effect and other antidiabetic mechanisms. It may be beneficial to create an effective treatment strategy by developing more specific substances with benzimidazole scaffold.     

Differential expression of GSK3β and pS9GSK3β in normal human tissues: can pS9GSK3β be an epithelial marker?

Glycogen synthase kinase 3β (GSK3β) and phosphorylated GSK3β at Ser9 (pS9GSK3β) are crucial in cellular proliferation and metabolism. GSK3β and pS9GSK3β are deregulated in many diseases including tumors. Data on altered expression of GSK3β and pS9GSK3β are mainly limited to tumor tissues, thus the expression of GSK3β and pS9GSK3β in normal human tissue has been largely unknown. Thus, we examined the immunohistochemical localization of GSK3β and pS9GSK3β in human fetal and adult tissues, and also compared the expression pattern of GSK3β and pS9GSK3β with that of the CK7 and CK20. We found GSK3β expression in neurons of brain, myenteric plexus in gastrointestinal tract, squamous epithelium of skin, and mammary gland. The expression of pS9GSK3β was restricted to the epithelial cells of breast and pancreaticobiliary duct, distal nephron of kidney, gastrointestinal tract, fallopian tube, epididymis, secretory cell of prostatic gland, and umbrella cell of urinary tract. The staining pattern of pS9GSK3β and CK7 was overlapped in most organs except for gastrointestinal tract where CK7 was negative and CK20 was positive. Our results show that the expression of GSK3β may be associated with differentiation of ectodermal derived tissues and pS9GSK3β with that of epithelial cells of endodermal derived tissues in human. In addition, the expression of pS9GSK3β in the selective epithelial cells may indicate its association with secretory or barrier function of specific cells and may serve as another immunohistochemical marker for epithelial cells.     

Roles of TNF-α, GSK-3β and RANKL in the occurrence and development of diabetic osteoporosis

Objective: To investigate the roles of TNF-α, GSK-3β and RANKL in the occurrence and development of diabetic osteoporosis. Methods: Diabetic rat model was established; tissue section technology was used to observe the situation of osteoporosis in diabetic rats; rat serum levels of OC, RANKL, GSK-3β, P38mapk, TNF-α and INS were detected by Elisa assay; osteoblasts and osteoclasts were primarily cultured and identified by immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining respectively. The effects of GSK-3β inhibitors, lithium chloride, TNF-α antagonists and RANKL antagonists on the proliferation of osteoblasts and osteoclasts were evaluated; quantitative PCR was used to assess the effects of GSK-3β inhibitors, lithium chloride, on TNF-α and RANKL gene expression in osteoblasts and osteoclasts, and the effects of TNF-α and RANKL antagonists on GSK-3β gene expression in osteoblasts and osteoclasts. Results: Diabetic rat model was successfully established; osteoblasts and osteoclasts were successfully isolated and cultured. Elisa experiments showed that in diabetic model group, the levels of RANKL, GSK-3β, P38mapk and TNF-α were significantly increased, while the levels of osteocalcin (OC) and insulin (INS) were significantly reduced; MTT results showed that osteoclast proliferation in GSK-3β inhibitor and lithium chloride groups were weaker than the untreated group, while osteoclast proliferation in TNF-α antagonist group and RANKL antagonist Group was very close to the untreated group. Osteoblast proliferation in GSK-3β inhibitor and lithium chloride groups were weaker than the untreated group, while osteoblast proliferation in TNF-α antagonist group and RANKL antagonist group was higher than the untreated group. In all of the corresponding groups, cell proliferation in the diabetic group was stronger than the untreated group. In GSK-3β inhibitor and lithium oxide groups, TNF-α and RANKL gene expression levels were elevated, but TNF-α and RANKL gene expression levels in the diabetic group were slightly lower than the control group. GSK-3β gene expression level in TNF-α antagonist group and RANKL antagonist group was reduced; GSK-3β gene expression level in diabetic group was lower than the control group. Conclusion: In diabetic rats, TNF-α, GSK-3β and RANKL levels were elevated; GSK-3β could promote the proliferation of osteoblasts and osteoclasts, and inhibit the expression of TNF-α and RANKL; TNF-α and RANKL can suppress the proliferation of osteoblasts while had little effect on osteoclast proliferation; they also can promote the GSK-3β gene expression; interactions between the three broke the balance between osteoblasts and osteoclasts, leading to osteoporosis.     

Induced regulatory T cells suppress Tc1 cells through TGF-β signaling to ameliorate STZ-induced type 1 diabetes mellitus

Type 1 diabetes mellitus (T1D) is a chronic autoimmune condition in which the immune system destroys insulin-producing pancreatic β cells. In addition to well-established pathogenic effector T cells, regulatory T cells (Tregs) have also been shown to be defective in T1D. Thus, an increasing number of therapeutic approaches are being developed to target Tregs. However, the role and mechanisms of TGF-β-induced Tregs (iTregs) in T1D remain poorly understood. Here, using a streptozotocin (STZ)-induced preclinical T1D mouse model, we found that iTregs could ameliorate the development of T1D and preserve β cell function. The preventive effect was associated with the inhibition of type 1 cytotoxic T (Tc1) cell function and rebalancing the Treg/Tc1 cell ratio in recipients. Furthermore, we showed that the underlying mechanisms were due to the TGF-β-mediated combinatorial actions of mTOR and TCF1. In addition to the preventive role, the therapeutic effects of iTregs on the established STZ-T1D and nonobese diabetic (NOD) mouse models were tested, which revealed improved β cell function. Our findings therefore provide key new insights into the basic mechanisms involved in the therapeutic role of iTregs in T1D.     

Inflammation,diet, and type 2 diabetes: a mini-review

ABSTRACT

Inflammation is a common feature of type 2 diabetes (T2D). Inflammatory cytokines increase in patients with type 2 diabetes, metabolic syndrome, and heart disease. Various types of cells can produce inflammatory cytokines and then release them into the bloodstream, where their complex interactions with target tissues raise a tissue-specific immune response. This review focused on C-reactive protein (CRP), tumor necrosis factor (TNF)-α as an inflammatory cytokine, and adiponectin produced by adipose tissues. Despite the major role of cytokines in the development of T2D, further studies are required to investigate the possible effects of the macronutrient composition of diet on these cytokines.     

Natural killer cells are required for accelerated type 1 diabetes driven by interferon-beta

The destruction of beta cells by the islet infiltrating lymphocytes causes type 1 diabetes. Transgenic mice models expressing interferon (IFN)-beta in beta cells, in the non-obese diabetic (NOD) strain and in a diabetes-free, major histocompatibility complex-matched, homologous strain, the non-obese resistant (NOR) mice, developed accelerated type 1 diabetes after 3 weeks of age. Our aim was to determine if natural killer (NK) cells could affect the acceleration of the disease. We determined the amount of NK cells in the pancreas, spleen and lymph nodes from NOD rat insulin promoter (RIP)-IFN-beta mice. Pancreatic cytokines were assessed by quantitative real-time polymerase chain reaction and protein arrays. To confirm the relevance of NK cells in the acceleration of autoimmune diabetes this subset was depleted with anti-asialo GM1 antibodies. An increase of intrapancreatic NK cells characterized the accelerated onset of diabetes both in NOD and NOR RIP-IFN-beta transgenic models. Cytokines involved in NK function and migration were found to be hyperexpressed in the pancreas from accelerated diabetic mice. Interestingly, the depletion of NK cells in vivo abolished completely the acceleration of diabetes. NK cells connect innate to adaptive immunity and might play a role in autoimmunity. We report here that NK cells are required critically in the pancreas for accelerated diabetes. This model links inflammation to acceleration of beta cell-specific autoimmunity mediated by NK cells.     

Calorie restriction decreases platelet-derived growth factor (PDGF)-A and thrombin receptor mRNA expression in autoimmune murine lupus nephritis

Calorie restriction (CR) and supplementation with fish oil (FO) are known to increase the life span and diminish histological evidence of glomerulonephritis in lupus prone (NZB×NZW)F_l (B/W) mice. Cellular proliferation is an important pathological element in the development of lupus nephritis, and we have examined the expression of thrombin receptor (TR) and the mitogenic agents PDGF-A and -B. Weanling B/W mice were fed either ad libitum or a calorie restricted (CR; 40% less calories than ad libitum) diet supplemented with either 5% (w/w) corn oil (CO) or FO. CR animals consumed 2.7–3.0 g of wet food per day versus 4.5–5.0 g for the ad libitum animals. Renal RNA was extracted from young (3.5–4.0 months of age) and old (8–10 months of age) mice. Densitometric analysis (reference gene GAPDH) of blots from Northern (PDGF-A and -B) and ribonuclease protection assays (TR) produced the following data: (i) in young mice no signal was detected for PDGF-A, -B and TR in all four groups, while the signals were readily detectable in old mice; (ii) in old mice low and similar levels of PDGF-B were detected, and neither CR nor the source of lipid altered its expression; (iii) CR significantly inhibited PDGF-A and TR expression in both CO (ad libitumversus CR; PDGF-A, 3.25-fold, P<0.025; TR, 3.7-fold, P<0.01) and FO (ad libitumversus CR; PDGF-A, 4.56-fold, P<0.01; TR, 3.6-fold, P<0.025) groups; (iv) although FO (versus CO) produced a trend towards decreased expression, results were not statistically significant. We conclude that suppression of renal disease in lupus-prone mice by CR is accompanied by decreased expression of PDGF-A and the thrombin receptor.     

Preventive advice given by patients with type 2 diabetes to their offspring

Background

Patients'' advice-giving behaviour could be a useful preventive strategy for type 2 diabetes.

Aim

To investigate the conditions under which patients offer advice to their offspring and to assess the factors that facilitate advice giving.

Design of study

Cross-sectional observational study.

Setting

A general hospital with a diabetes clinic in a metropolitan suburb in Japan.

Method

Parents with type 2 diabetes (n = 221) who had offspring aged 20–49 years inclusive without diabetes completed a self-administered questionnaire containing items relating to advice-giving behaviour, demographic characteristics, risk perception, and their disease status.

Results

A total of 184 (83.3%) patients responded that parental advice-giving behaviour is needed for their offspring, while 138 (62.4%) actually advised their offspring. Multiple logistic regression analysis showed that patients who were female (odds ratio [OR] = 1.94, 95% confidence interval [CI] = 1.03 to 3.65, P = 0.041), living with their offspring (OR =1.92, 95% CI = 1.04 to 3.57, P = 0.038), had complications (OR = 2.74, 95% CI = 1.25 to 6.00, P = 0.029), or perceived that their offspring had a high risk of developing diabetes (OR =1.45, 95% CI = 1.09 to 1.93, P = 0.011) were most likely to advise their offspring.

Conclusion

Patients with type 2 diabetes recognised the need to give advice about preventive behaviour to their offspring but were not necessarily engaging in advice-giving behaviour. Advice-giving behaviour was affected by the parents'' own disease status, their perception of their offspring''s risk of developing diabetes, and the relationship between the patients and their offspring.     

Phenotypic and genotypic detection of extended spectrum β-lactamases among Escherichia coli and Klebsiella pneumoniae isolates from type 2 diabetic patients with urinary tract infections

BackgroundT2DM patients are more likely to have UTIs caused by resistant organisms such as ESBLs producing bacteria. Challenging reliable identification and prompt characterization of in-vitro susceptibilities of these bacteria are the first steps of deciding the appropriate antimicrobial therapy for UTIs caused by them.ObjectivesTo isolate and identify E. coli and K. pneumoniae from urine of T2DM patients with UTIs, to determine antibiotic resistance pattern among isolates, and to identify ESBLs production phenotypically and genotypically.Material and methodAll samples were cultured on Cystine-Lactose-Electrolyte-Deficient Agar medium (CLED) by using calibrated loop. Growth of 100 colonies or more, i.e. 105 colony forming units (CFU)/mL urine was considered as significant bacteriuria. Isolation and identification were done according to standard method. All isolates were tested for antibiotic susceptibility testing by the disc diffusion method according to CLSI guidelines. Phenotypic detection of ESBLs was done by double-disk synergy test. Genotypic detection of blaTEM, blaSHV and blaCTX-M genes by using PCR.ResultsResults of this study showed that E. coli and K. pneumoniae were the dominant bacterial isolates, they constituted 103 (91.2%) out of 113 urine isolates. E. coli (58. 4%) K. pneumoniae (32.7%), Enterococcus spp. (4.4%), Proteus spp. (2.7%) and Pseudomonas spp. (1.8%). About 25 (24.3%) out of 103 E. coli and K. pneumoniae isolates were ESBLs positive by DDST, and 22 (88.0%) out of them had ESBLs encoding genes by conventional PCR. The most common gene detected was blaTEM (59.1%), followed by blaSHV (27.3%). CTX-M had not been detected in any of testes isolates.ConclusionblaTEM and blaSHV genes were detected in 22 out of 25 ESBLs producing E. coli and K. pneumoniae isolates phenotypically detected by DDST. blaTEM was found to be the predominant gene (59.1%), while blaCTX-Mene was not detected in any of tested isolates.     

α,β-Amyrin prevents steatosis and insulin resistance in a high-fat diet-induced mouse model of NAFLD via the AMPK-mTORC1-SREBP1 signaling mechanism

Nonalcoholic fatty liver disease (NAFLD), characterized by hepatosteatosis and steatohepatitis, is intrinsically related to obesity. Our previous study reported on the anti-obese activity of α,β-amyrin (AMY), a pentacyclic triterpene isolated from Protium heptaphyllum. This study investigated its ability to prevent fatty liver and the underlying mechanism using the mouse model of NAFLD. NAFLD was induced in male Swiss mice fed a high fat diet (HFD) for 15 weeks. The controls were fed a normal chow diet (ND). The mice were simultaneously treated with AMY at 10 and 20 mg/kg or fenofibrate at 50 mg/kg. Lipid levels along with metabolic and inflammatory parameters were assessed in liver and serum. The liver sections were histologically examined using H&E staining. RT-qPCR and western blotting assays were performed to analyze signaling mechanisms. Mice fed HFD developed severe hepatic steatosis with elevated triglycerides and lipid droplets compared with ND controls. This was associated with a decrease in AMP-activated protein kinase (AMPK) activity, an increase of mechanistic target of rapamycin complex 1 (mTORC1) signaling, and enhanced sterol regulatory element binding protein 1 (SREBP1) expression, which have roles in lipogenesis, inhibition of lipolysis, and inflammatory response. AMY treatment reversed these signaling activities and decreased the severity of hepatic steatosis and inflammatory response, evidenced by serum and liver parameters as well as histological findings. AMY-induced reduction in hepatic steatosis seemed to involve AMPK-mTORC1-SREBP1 signaling pathways, which supported its beneficial role in the prevention and treatment of NAFLD.     

Coincident diabetes mellitus modulates Th1‐, Th2‐, and Th17‐cell responses in latent tuberculosis in an IL‐10‐ and TGF‐β‐dependent manner

Type 2 diabetes mellitus (DM) is a risk factor for the development of active tuberculosis (TB), although its role in the TB‐induced responses in latent TB (LTB) is not well understood. Since Th1, Th2, and Th17 responses are important in immunity to LTB, we postulated that coincident DM could alter the function of these CD4⁺ T‐cell subsets. To this end, we examined mycobacteria‐induced immune responses in the whole blood of individuals with LTB‐DM and compared them with responses of individuals without DM (LTB‐NDM). T‐cell responses from LTB‐DM are characterized by diminished frequencies of mono‐ and dual‐functional CD4⁺ Th1, Th2, and Th17 cells at baseline and following stimulation with mycobacterial antigens‐purified protein derivative, early secreted antigen‐6, and culture filtrate protein‐10. This modulation was at least partially dependent on IL‐10 and TGF‐β, since neutralization of either cytokine resulted in significantly increased frequencies of Th1 and Th2 cells but not Th17 cells in LTB‐DM but not LTB individuals. LTB‐DM is therefore characterized by diminished frequencies of Th1, Th2, and Th17 cells, indicating that DM alters the immune response in latent TB leading to a suboptimal induction of protective CD4⁺ T‐cell responses, thereby providing a potential mechanism for increased susceptibility to active disease.     

Self-activation of Vγ9Vδ2 T cells by exogenous phosphoantigens involves TCR and butyrophilins

The high cytotoxic activity of Vγ9Vδ2 T lymphocytes against tumor cells makes them useful candidates in anticancer therapies. However, the molecular mechanism of their activation by phosphoantigens (PAgs) is not completely known. Many studies have depicted the mechanism of Vγ9Vδ2 T-cell activation by PAg-sensed accessory cells, such as immune presenting cells or tumor cells. In this study, we demonstrated that pure resting Vγ9Vδ2 T lymphocytes can self-activate through exogenous PAgs, involving their TCR and the butyrophilins BTN3A1 and BTN2A1. This is the first time that these three molecules, concurrently expressed at the plasma membrane of Vγ9Vδ2 T cells, have been shown to be involved together on the same and unique T cell during PAg activation. Moreover, the use of probucol to stimulate the inhibition of this self-activation prompted us to propose that ABCA-1 could be implicated in the transfer of exogenous PAgs inside Vγ9Vδ2 T cells before activating them through membrane clusters formed by γ9TCR, BTN3A1 and BTN2A1. The self-activation of Vγ9Vδ2 T cells, which leads to self-killing, can therefore participate in the failure of γδ T cell-based therapies with exogenous PAgs and should be taken into account.     

Attenuation of islet‐specific T cell responses is associated with C‐peptide improvement in autoimmune type 2 diabetes patients

The clinical efficacy of peroxisome proliferator‐activated receptor gamma (PPAR‐γ) agonists in cell‐mediated autoimmune diseases results from down‐regulation of inflammatory cytokines and autoimmune effector cells. T cell islet autoimmunity has been demonstrated to be common in patients with phenotypic type 2 diabetes mellitus (T2DM) and islet‐specific T cells (T⁺) to be correlated positively with more severe beta cell dysfunction. We hypothesized that the beneficial effects of the PPAR‐γ agonist, rosiglitazone, therapy in autoimmune T2DM patients is due, in part, to the immunosuppressive properties on the islet‐specific T cell responses. Twenty‐six phenotypic T2DM patients positive for T cell islet autoimmunity (T⁺) were identified and randomized to rosiglitazone (n = 12) or glyburide (n = 14). Beta cell function, islet‐specific T cell responses, interleukin (IL)‐12 and interferon (IFN)‐γ responses and islet autoantibodies were followed for 36 months. Patients treated with rosiglitazone demonstrated significant (P < 0·03) down‐regulation of islet‐specific T cell responses, although no change in response to tetanus, a significant decrease (P < 0·05) in IFN‐γ production and significantly (P < 0·001) increased levels of adiponectin compared to glyburide‐treated patients. Glucagon‐stimulated beta cell function was observed to improve significantly (P < 0·05) in the rosiglitazone‐treated T2DM patients coinciding with the down‐regulation of the islet‐specific T cell responses. In contrast, beta cell function in the glyburide‐treated T2DM patients was observed to drop progressively throughout the study. Our results suggest that down‐regulation of islet‐specific T cell autoimmunity through anti‐inflammatory therapy may help to improve beta cell function in autoimmune phenotypic T2DM patients.     

Mouse superkiller‐2‐like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses

IFN‐α/β allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2‐like DExH‐box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN‐α/β by retinoic acid inducible gene 1‐like receptors (RLRs) that detect the presence of RNA viruses in a cell‐intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN‐α/β induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60‐deficient mice revealed no impairment in IFN‐α/β production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60‐deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus‐1. These results put in question the reported role of DDX60 as a broad‐acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses.