Antibodies against different epitopes of heat-shock protein 60 in children with type 1 diabetes mellitus

The aim of this study was to investigate the amounts and epitope specificity of antibodies against heat shock protein 60 (hsp60) in the sera of type 1 diabetic and healthy children. Antibodies specific for peptide p277 of human hsp60 and of M. bovis as well as for human hsp60, M. bovis hsp65 proteins were measured by ELISA. Other autoantibodies (islet cell antibodies, glutamate decarboxylase antibodies and IA-2 antibodies) were also determined. A total number of 83 serum samples from children with type 1 diabetes mellitus and 81 samples of control children were investigated. Epitope scanning of the hsp60 for linear antibody epitopes was carried out using synthetic peptides attached to pins. The antibody levels specific for peptide p277 of human- and of M. bovis origin were significantly (human: P=0.0002, M. bovis: P=0.0044) higher in the diabetic children group than in the healthy children. We could not find significant difference in the antibody levels to whole, recombinant hsp proteins among the examined groups of children. Antibodies to two epitope regions on hsp60 (AA394–413 and AA435–454) were detected in high titres in sera of children with diabetes mellitus. The first region similar to the sequence found in glutamate decarboxylase, whereas the second one overlaps with p277 epitope to a large extent. Presence of antibodies to certain epitopes of hsp60 (AA394–413—glutamic acid decarboxylase-like epitope; AA435–454—p277-like epitope) in diabetic children may reflect their possible role in the autoimmune diabetogenic process of the early diabetes.     

T cells and autoantibodies to human HSP70 in type 1 diabetes in children

We studied T-cell proliferative responses (stimulation index: SI) and autoantibodies to human HSP60, HSP70 and HSP90 proteins in 25 children (mean age 10.1+/-3.8 years) newly diagnosed with Type 1 diabetes. The control group for T cells included 25 adults and three pediatric donors without Type 1 diabetes. Controls for antibodies included 10 pediatric subjects. The T-cell responses to HSP70 of the test group (mean SI=4.5+/-3.1) were significantly greater than those of the control group (meanSI=1.4+/-0.6; p<0.0001); the incidence of HSP70 responders was (85%) compared to 14% in the control group. All but three of the Type 1 children who responded to HSP70 also responded to HSP60 (85%). The T-cell responses of the Type 1 group to HSP90 (mean SI=1.7+/-1.1) were similar to those of the control group (mean SI=1.5+/-0.7). We mapped HSP70 epitopes recognized by T cells in seven subjects using overlapping peptides of the molecule. Among the Type 1 subjects, IgG seropositivity was 45% to HSP60, 30% to HSP70, and 15% to HSP90. Thus, we conclude that children with newly diagnosed Type 1 diabetes manifest heightened T-cell autoimmunity to HSP70 and HSP60, but not to HSP90.     

Reduced CD4+ T-cell-specific gene expression in human type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) in humans is characterized by the T-cell-dependent destruction of the insulin producing pancreatic beta cells; however, the precise pathogenesis of the disease, especially the initiation of pathologic immune response, is still largely unknown. We hypothesized that the function of human CD4+ T cells is altered in T1DM and analyzed unstimulated human peripheral blood CD4+ T-cell gene expression. We used a novel three-way comparison of DNA microarray data of CD4+ T cells isolated from patients with new onset T1DM, patients with long-term Type 2 diabetes (T2DM), and from healthy control subjects in order to eliminate any possible influence of glucose homeostasis on our findings. We analyzed the T1DM specific gene-expression changes and their functional relevance to T1DM autoimmunity. Our genetic and functional data show that T1DM CD4+ T cells are down-regulated specifically affecting key immune functions and cell cycle. Histone deacetylase gene expression, a key regulator of epigenetic modification is also reduced. The CD4+ T cells showed impaired function, including an abnormal immune response, which may be a key element that leads to the breakdown of self-tolerance.     

耐受性树突状细胞过继转移建立1型糖尿病小鼠感染性免疫耐受

目的探讨耐受性树突状细胞(DC)过继转移,在T1DM模型小鼠体内建立感染性免疫耐受的方法和机制。方法在链脲佐菌素(STZ)所致1型糖尿病模型(type 1 diabetes mellitus,T1DM)内采用胰岛素与IFA混合乳剂皮下注射的方式诱导免疫耐受。体外分离和纯化耐受性树突状细胞。另取BALB/c小鼠,通过体内腹腔注射STZ,尾静脉注射T1DM发病小鼠脾脏T淋巴细胞(DTL),诱导T1DM。同时将不同来源DC经腹腔注射入模型小鼠体内。每周测定血糖,第4周时处死动物,取胰腺进行病理组织学检查。MTF法测定小鼠脾淋巴细胞增殖反应,采用流式细胞术检测CIM~ CD25~ 调节性T细胞。结果采用2次小剂量STZ加DTL联合注射的方式可在小鼠体内建立以胰腺β细胞炎性破坏为主要病理特征的T1DM。通过过继转移免疫耐受性DC,可明显降低糖尿病高血糖以及小鼠脾淋巴细胞增殖能力,与模型对照组差异有统计学意义(P<0.01);组织病理检查发现胰岛内炎症细胞浸润减少,组织结构完整;FACS显示CD4~ CD25~ T细胞亚群比例显著升高。结论过继转移耐受性DC可以在次一级T1DM模型内诱导感染性免疫耐受并防止T1DM的发生,其机制与促进体内CD4~ CD25~ T细胞亚群产生相关。     

Immune intervention in type 1 diabetes

Type 1 diabetes (T1D) is a chronic autoimmune disease that results in the specific immune destruction of insulin producing beta cells. Currently there is no cure for T1D and treatment for the disease consists of lifelong administration of insulin. Immunotherapies aimed at preventing beta cell destruction in T1D patients with residual c-peptide or in individuals developing T1D are being evaluated. Networks of researchers such as TrialNet and the Immune Tolerance Network in the U.S. and similar networks in Europe have been established to evaluate such immunotherapies. This review focuses on immune intervention for the prevention and amelioration of human T1D with a focus on potential immune suppressive, antigen specific and environmental therapies.     

MTNR1B基因多态性与2型糖尿病

2型糖尿病是一种与多基因、多因素相关联的具有明显遗传异质性的疾病.全基因组关联分析显示MTNR1B基因变异与胰岛素分泌、葡萄糖水平以及2型糖尿病发病有显著相关性.MTNR1B基因是2型糖尿病重要的易感基因之一,其变异可能通过减少β细胞胰岛素分泌,从而增加2型糖尿病的易感性.     

HMGB1 is activated in type 2 diabetes mellitus patients and in mesangial cells in response to high glucose

Diabetic nephropathy (DN) is one of the most devastating complications of diabetes, leading the cause of end-stage renal disease (ESRD). And investigations into mechanisms underlying renal inflammation may provide new insight into novel therapeutic targets for patients with DN. However, little is known about the promotion of inflammation in DN. In the present study, we examined the promotion by high glucose to High-mobility group box-1 (HMGB1) in patients with type 2 diabetes mellitus or in renal mesangial SV40 MES 13 cells. Results demonstrated that high glucose promoted the pre-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in patients with T2DM or in SV40 MES 13 cells. And the serum HMGB1 was also upregulated in T2DM patients, correlating with serum IL-6 and TNFα. The in vitro results indicated that HMGB1 mediated the D-glucose-induced pro-inflammatory cytokines in mesangial cells. And the NF-κB signaling pathway involved in the promotion of pro-inflammatory cytokines by D-glucose. In summary, the present study indicated that HMGB1 was significantly promoted by the glucose in vivo or in vitro, in an association with an upregulation of pro-inflammatory cytokines, via activating NF-κB signaling pathway. And the strategy of HMGB1 inhibition reduced the upregulation of pro-inflammatory cytokines in response to high glucose, via inhibiting the NF-κB signaling pathway. It implies the regulatory role of HMGB1 in the inflammatory responses in DN.     

128例Ⅱ型糖尿病患者线粒体ND1基因突变位点的研究

目的通过对128例Ⅱ型糖尿病(Type 2 diabetes mellitus,T2DM)患者线粒体DNA ND1基因进行突变位点筛查,探索ND1基因点突变与山西人群T2DM的相关性。方法 PCR扩增患者ND1基因所在区段,PCR产物直接测序分析。结果128例患者共有38例患者检测到基因点突变,突变检出率为29.7%。38例患者共筛出22个突变位点,2种突变类型。其中31例存在单个位点突变,5例存在2个位点突变,2例存在3个位点突变。22个突变位点中,3552(T→A)突变频率最高,为40.9%(9/22);3394(T→C)、3435(C→T)、3497(C→T)、3316(G→A)、3571(C→T)、3537(A→G)的突变频率分别为22.7%(5/22)、22.7%(5/22)、18.2%(4/22)、13.6%(3/22)、13.6%(3/22)和10.1%(2/22);其余突变位点的突变率均为4.5%(1/22)。在所有突变位点中,除3688(G→C)为异质性突变外,其余突变均为同质性。此外,22个突变位点中存在一个新的突变位点3499(A→T),属首次报道。结论 3552(T→A)和3394(T→C)突变频率最高,可能与山西地区T2DM发病相关;新发现的突变位点3499A→T(Thr→Ser),其致病性需要进一步研究。     

Investigation of KIR gene frequencies in type 1 diabetes mellitus

The frequency of killer immunoglobulinlike receptors (KIR) genes was examined in type 1 diabetes mellitus patients and controls from Finland. The KIR gene 2DS5 was significantly decreased in patients versus controls, but this was no longer significant after correction for the number of comparisons made.     

脂联素受体1基因-1927T/C多态位点与2型糖尿病的关系

目的探讨脂联素受体基因1927 T/C多态位点与2型糖尿病易感性的关系,为糖尿病的有效防治提供科学依据。方法采用PCR及测序技术对141例2型糖尿病患者和141例健康者的AdipoR1基因1927突变位点进行检测。同时测定两组人群的临床相关指标。结果糖尿病组中AdipoR1等位基因频率及基因型频率与正常对照组比较差异无显著性（P〉0.05）。糖尿病组中,CC基因型FBG、FINS、C肽、HOMA-IR与TX基因型比较有统计学差异。结论本研究提示AdipoR1 1927 T/C多态可能与宁波地区汉族人群2型糖尿病发病无关,与糖代谢及胰岛素抵抗有关。     

Effects of nifedipine on renal responses to human atrial natriuretic peptide in healthy subjects and normoglycemic patients with type 1 diabetes mellitus

Summary We examined renal responses to a pharmacological dosage of human atrial natriuretic peptide (hANP) and the potential interference of nifedipine administration with the effects of hANP on kidney function in healthy subjects and normoglycemic patients with type 1 diabetes mellitus. Ten healthy volunteers (age, 28±1 years) and ten patients (age, 33±2 years; diabetes duration; 14±3 years; HbAI 7.2%±0.2%) were studied. According to a double-blind, randomized, placebo-controlled trial design, three experiments were performed in each subject using the doubledummy technique: placebo only, hANP only, and nifedipine+hANP. As i.v. bolus injection 100 µg hANP was given; nifedipine was applied buccally, at a dose of 10 mg 90 min before and at a dose of 5 mg together with hANP injection. At base-line and in the placebo only experiment, patients did not differ from controls. In the hANP only experiment, in both groups hANP resulted in increased urinary volume and both sodium and chloride excretion (P<0.05 vs placebo only experiment). In patients, hANP-induced increase in electrolyte excretion was greater than in controls (P<0.05). In the nifedipine + hANP experiment, hANP-induced changes in renal indexes were enhanced in controls (P<0.05 vs hANP only experiment) but not in patients. Thus, diuretic response to nifedipine + hANP in patients was decreased in comparison with controls (P<0.05). In patients, however, nifedipine administration decreased the hANP-induced increase in urinary albumin excretion (P<0.05 vs hANP only experiment). Creatinine clearance was uninfluenced throughout the experiments.There were similar decreases in blood pressure in patients and controls after nifedipine administration (P<0.05 vs placebo only experiment). The increase in heart rate after nifedipine was more pronounced in patients than in controls (P<0.05). Conversely, plasma renin activity was stimulated by nifedipine only in controls (P<0.05 vs placebo only experiment). In this study hANP had no effect on heart rate, blood pressure, or plasma renin activity. There was a short-term increase in hANP levels in plasma after nifedipine administration in controls (P<0.05 vs placebo only experiment) but not in patients. In contrast to a previous study, where renal responses to the same pharmacological dosage of hANP were decreased in patients with type 1 diabetes mellitus with HbAI exceeding the normal range, there is no impairment of renal responsiveness to an i.v. bolus of hANP in patients with HbAI within the normal range. Nifedipine and hANP have synergistic effects on kidney function in healthy subjects. It remains to be studied, however, by which mechanism(s) this synergism could be obscured in diabetes patients. Moreover, the increase in hANP levels after nifedipine administration exclusively in controls merits further investigation.Abbreviations hANP Human atrial natriuretic peptide - HbAI Glycosylated hemoglobin AI - HbAI c Glycosylated haemoglobin AI c     

Fulminant type 1 diabetes mellitus observed in insulin receptor substrate 2 deficient mice

The objective of this study was to characterise the fulminant type 1 diabetes mellitus (DM) accompanying abrupt hyperglycaemia and ketonuria observed in insulin receptor substrate 2 (IRS2)-deficient mice. IRS2-deficient mice backcrossed onto the original C57BL/6J:Jc1 background (B6J-IRS2(-/-) mice) for more than 10 generations were used. Eight male IRS2-deficient mice with ketonuria and abrupt increase in plasma glucose concentrations over 25 mmol/l were used as the fulminant type 1 diabetic mice (diabetic mice) and 8 male IRS2-deficient mice (8 weeks old) without glycosuria were used as the control mice. Plasma metabolite, immunoreactive insulin (IRI) and C-peptide concentrations, hepatic energy metabolism related enzyme activities and histopathological change in pancreatic islets were investigated. The diabetic mice showed significantly higher plasma glucose and cholesterol concentrations and lower plasma IRI and C-peptide concentrations than the control mice. In livers of the diabetic mice, glycolytic and malate-aspartate shuttle enzyme activities decreased significantly and gluconeogenic, lipogenic and ketone body synthesis enzyme activities increased significantly compared to those in the control mice. The pancreatic islets of the diabetic mice decreased significantly in size and number of beta cells. The diabetic IRS2-deficient mice did not show the islet-related antibodies observed in the diabetic NOD mice in their sera. The characteristics of the diabetic IRS2-deficient mice resembled those of the human nonautoimmune fulminant type 1 DM. IRS2-deficient mice may be a useful animal model for studying the degradation mechanism of pancreatic beta cells in the process of development of fulminant type 1 DM.     

The influence of type-1 diabetes mellitus on dentition and oral health in children and adolescents

PURPOSE: The present study is to investigate the effects of type 1 diabetes mellitus on dentition and oral health for children and adolescents. MATERIALS AND METHODS: The investigation was carried out on 100 subjects. The first group consisted of 50 subjects with type 1 diabetes mellitus (21 females, 29 males), age 9+/-0.14 years; In the second group, there were 50 healthy subjects who did not suffer from any systemic disease (25 females, 25 males), age 9+/-0.11 years. The subjects were evaluated and divided into two groups of 5-9 years old, and 10-14 years old. The dentition of all participants was examined. Besides, the DFS/dfs index, oral hygiene conditions were evaluated, as well as the plaque index (PI), gingival index (GI) and calculus index (CI). The data obtained from each group were compared statistically. RESULTS: When compared to the non-diabetic group, we observed that dental development was accelerated until the age of 10 in the diabetic group, and there was a delay after the age of 10. The edentulous interval was longer in the group with type 1 diabetes mellitus. This was accompanied by a high ratio of gingival inflammation. Gingival inflammation was 69.7% in the group of 5-9 year-old, and 83.7% in the group of 10-14 year-old with type 1 diabetes mellitus. Though there was a greater loss of teeth in the group with type 1 diabetes mellitus, there were more caries in the control group. The PI, GI and CI values showed an increase with aging in favor of the group with type 1 diabetes mellitus. There was statistically significant difference in PI, GI and CI between the control and type 1 diabetes mellitus groups for 10-14 year-old patients (p<0.001). CONCLUSION: The findings we obtained showed that type 1 diabetes mellitus plays an important part in the dentition and oral health of children and adolescents.     

Crystallization of oral fluid components in patients with type 1 diabetes mellitus

We systematized and described morphological criteria characterizing crystalline aggregates in mixed saliva from patients with type 1 diabetes mellitus.__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 22–24, January, 2005     

抗原处理相关运载体等位基因与I型糖尿病的关联研究

目的 :探讨抗原处理相关运载体 (transporterassociatedwithantigenprocessing ,TAP)等位基因与I型糖尿病 (DM1)的关联性。方法 :用聚合酶链反应 序列特异性寡核苷酸探针杂交技术 ,对 5 2例DM1患者及 6 1例正常对照人群进行TAP1、TAP2等位基因变异位点氨基酸表型频率分析。结果 :DM1患者TAP1333位Ile Ile、6 37位Asp Asp、TAP2 379位Val Val纯合子表型频率显著低于对照 (P <0 0 0 5 ) ,DM1患者TAP1333位Ile Val、6 37位Asp Gly、TAP2 379位Ile Val杂合子表型频率明显高于对照 (P <0 0 0 5 )。TAP基因的其它变异位点氨基酸表型频率在DM1患者与正常对照组之间的差异无显著性意义。结论 :TAP1333位Ile Ile、6 37位Asp Asp、TAP2 379位Val Val纯合子表型可能是DM1的保护基因。TAP1333位Ile Val、6 37位Asp Gly、TAP2 379位Ile Val杂合子表型可能是DM1的易感基因     

1,25-二羟维生素D3对1型糖尿病预防效果观察

目的:1,25-二羟维生素D3[1,25-(OH)2D3]对连续多次小剂量链脲菌素(the mu ltip le low dose streptozotoc in,MLDS)诱导的自身免疫性糖尿病小鼠的预防作用及其机制初探。方法:小鼠分为三组。正常对照组:连续5天腹腔注射与糖尿病组等容量柠檬酸盐缓冲液;糖尿病组:连续5天腹腔注射STZ(40mg.kg-1),以血糖水平持续高于16.7mmol/L为成模标准;预防组:先隔日腹腔注射1,25-(OH)2D3(5μg.kg-1),共15次,然后再连续5天腹腔注射STZ(40mg.kg-1)。实验结束后各组动物处死收集血清并采集胰腺检测诱导型一氧化氮合酶(iNOS)活性及血清胰岛素水平。结果:MLDS诱发的自身免疫性糖尿病模型在第四周基本建成。MLDS使小鼠血糖、血清及胰腺iNOS活性升高,血清胰岛素水平下调;预防组小鼠注射STZ前给予1,25-(OH)2D3有明显降血糖和上调血清胰岛素水平作用,同时抑制血清及胰腺iNOS活性,与糖尿病组比较有显著性差异(P<0.05)。结论:1,25-(OH)2D3可有效预防MLDS诱导的自身免疫性糖尿病的发生。该效应可能与1,25-(OH)2D3抑制iNOS活性有关。     

Remodeling rodent models to mimic human type 1 diabetes

Type 1 diabetes (T1D) occurs when the immune system attacks the insulin‐producing beta cells located in the islets of Langerhans within the pancreas. Animal models have played a prominent role in developing an understanding of this disease process, through studies of genetic susceptibility, progression of hyperglycemia, and novel approaches to therapy. Here, we critically evaluate the currently available diabetic animal models and their propensity to match and predict disease outcomes in man as well as propose new in vitro and in vivo systems that may facilitate progress in the T1D field.     

Characterization of monocyte-derived dendritic cells in recent-onset diabetes mellitus type 1

Dendritic cells (DC) may play an important role in the immunopathogenesis of type 1 diabetes mellitus (DM-1). In this study, we have analyzed phenotypical changes during cytokine-driven maturation from CD14+ monocytes to mature DC and DC-dependent T-cell stimulation in recent-onset pediatric DM-1 patients and healthy controls. DC maturation was monitored by flow cytometric analyses for the expression of surface markers (HLA-DR, CD1a, CD40, CD80, CD86, CD83, CD14, CD32, mannose-receptor, and CD11c). Flow cytometric analysis of isolated peripheral blood monocytes did not reveal apparent differences between patients and controls. During DC maturation no obvious differences in the expression patterns of surface markers over time or evidence for maturation impairments in DM-1 patients could be appreciated. Solely, a marginal, but significant, transient down-regulation of CD1a on Day 3 (mean MDFI 3.82 vs 7.25; P = 0.021), which was accompanied by an increase of IL-6, could be observed. The comparison of mature DCs (Day 10) between patients and controls indicated no significant differences, except for CD83 (mean MDFI 1.7 vs 1.5; P = 0.042) and CD80 (mean MDFI 15.92 vs 12.73; P = 0.042). Moreover, no difference in T-cell stimulatory capacity was seen. In conclusion, our analysis of a cohort of recent-onset DM-1 patients and controls does not support a role for disease-related alterations in cytokine-driven maturation of monocyte-derived DC.     

血管紧张素原基因M235T分子变异与2型糖尿病肾病的关系

目的　探讨血管紧张素原（ａｎｇｉｏｔｅｎｓｉｎｏｇｅｎ , Ａ Ｇ Ｔ） 基因 Ｍ２３５ Ｔ 分子变异与中国人无肾病并发症的２ 型糖尿病（ｄｉａｂｅｔｅｓ ｍ ｅｌｌｉｔｕｓ , Ｄ Ｍ） 、２ 型糖尿病肾病（ｄｉａｂｅｔｉｃ ｎｅｐｈｒｏｐａｔｈｙ , Ｄ Ｎ） 的关系。方法　用 Ｐ Ｃ Ｒ及 Ｒ Ｆ Ｌ Ｐ 方法对８４ 例 Ｄ Ｍ、９６ 例 Ｄ Ｎ 及９８ 名正常对照进行了 Ａ Ｇ Ｔ 基因 Ｍ２３５ Ｔ 多态性的检测。结果　 Ｄ Ｎ 组 Ｔ 等位基因频率０８２ , Ｔ Ｔ 基因型频率０７０ ,与对照组（０６３ ,０４３） 比较有显著差异（ Ｐ＝ ０００３ , Ｐ＝００００４） ;校正了 Ｄ Ｎ 的几种危险因素后, Ｔ Ｔ 基因型对 Ｄ Ｎ 的 Ｏ Ｒ 为３４７（９５ ％ Ｃ Ｉ 为１５１ ～７９４ , Ｐ＝０００３３） 。 Ｄ Ｍ 组基因型频率分布与对照组比较无显著差异（ Ｐ＞ ００５） 。结论　 Ａ Ｇ Ｔ 基因 Ｔ Ｔ 型可能是中国人群２ 型糖尿病肾病的独立危险因素之一。