NOD小鼠自然状态下CD4~+CD25~+T细胞的动态变化及意义

研究自发的1型糖尿病雌鼠模型(NOD)在自然状态下发生1型糖尿病过程中CD4+CD25+T细胞的动态变化,旨在初步探讨调节性T细胞参与1型糖尿病发病的可能机制。采用雌性NOD小鼠作动物模型,每2周尾静脉采血1次,采用三色流式细胞术测定NOD小鼠外周血中CD4+CD25+T细胞(CD3+CD4+CD25+)的百分率。在32周时,对比发生糖尿病和未发生糖尿病NOD小鼠不同脏器中的CD4+CD25+T细胞阳性率。HE法检测胰岛炎。结果显示:(1)自第6周起NOD小鼠CD4+CD25+T细胞百分率逐渐降低。发生糖尿病NOD小鼠CD4+CD25+T细胞比率低于未发病NOD小鼠对照组(外周血分别为0.94%±0.21%、1.62%±0.23%,P=0.01;脾脏2.09%±0.14%、2.77%±0.36%,P=0.019),提示糖尿病NOD小鼠外周血中存在异常比例的CD4+CD25+T细胞;(2)32周龄糖尿病NOD小鼠与未发病NOD小鼠的CD4+CD25+T细胞抑制功能减低,与阳性对照组有显著性差异;(3)HE染色结果示糖尿病NOD小鼠胰岛结构完全破坏,胰岛炎程度较未发病NOD小鼠严重。该结果提示NOD小鼠发生糖尿病时免疫功能紊乱与CD4+CD25+T细胞参与调节及T细胞亚群变化相关,糖尿病的发生受致病性T细胞和调节性T细胞的调节。     

自身免疫性1型糖尿病相关的CD8~+ T细胞表位

自身免疫性1型糖尿病是遗传易感个体中T细胞介导的选择性破坏胰腺β细胞引起的一种自身免疫性疾病。自身反应性CD4+T辅助细胞和CD8+细胞毒性T淋巴细胞(CTLs)在MHCⅡ和Ⅰ类分子的背景下分别识别β细胞表面肽段,作用于一个最终导致β细胞死亡的过程中。而CTLs被认为是导致β细胞破坏更为主要的原因。自身反应性CTLs在识别β细胞上的表位后随着T细胞受体的激活从而引发β细胞凋亡,因此近来涌现的研究均致力于发现并验证这些特异性CD8+T细胞表位中。     

小檗碱对非肥胖性糖尿病小鼠1型糖尿病的预防作用

目的探讨小檗碱对非肥胖性糖尿病(NOD)小鼠1型糖尿病的影响及其可能的分子机制。方法40只4周龄NOD雌性小鼠随机分为小檗碱干预组(Ber,20只)和生理盐水对照组(NS,20),监测血糖,记录糖尿病发病率,40周后处死小鼠,分别应用Western blot方法与real time PCR方法检测两组小鼠胰腺内Fas、iNOS、bcl-2、SOD蛋白与mRNA的表达水平。结果小檗碱干预组NOD小鼠1型糖尿病发生率较对照组明显降低(4/20,20%;18/20,90%),平均发病时间也明显延缓。与对照组相比,小檗碱干预组NOD小鼠胰腺组织Fas、iNOS的蛋白与mRNA表达水平明显下调,Bcl-2、SOD的蛋白与mRNA表达水平明显上调,P0.05。结论小檗碱预防NOD鼠糖尿病的发生可能与上调胰腺组织Fas、iNOS的蛋白表达,下调Bcl-2、SOD的蛋白表达相关。     

Tyk2信号传导在表达OVA的重组卡介菌(rBCG-OVA)所致慢性胞内细菌感染小鼠模型的CD8+Tc1反应中的作用

目的 探讨酪氨酸激酶2(tyrosine kinase 2,Tyk2)在IL-12和IFN-γ激发的信号传导中所起的作用.方法 为阐明Tyk2在CD8+Tc1反应中的作用,我们跟踪了Tyk2基因敲除(Tyk2-/-)小鼠和Tyk2野生型(Tyk2+/+)小鼠感染表达卵清蛋白的重组卡介菌(rBCG-OVA)后功能性CD8+T细胞的增殖分化过程.结果 与Tyk2+/+小鼠相比,在rBCG-OVA感染后,Tyk2-/-小鼠的OVA257-264抗原特异的CD8+T细胞能够随着感染的发生和发展开始增生和收缩,但是CD8+T细胞总数明显减少,OVA257-264/Kb-四聚物阳性的CD8+T细胞和IFN-γ产生阳性的CD8+Tc1细胞数量明显不足,动力曲线与增加的体内细菌的增长相对应.结论 Tyk2的信号缺失使rBCG-OVA致慢性细胞内病原菌感染导致的功能型CD8+Tc1反应减弱.

Abstract：

Objective To elucidate potential roles of tyrosine kinase 2 (Tyk2) in the generation and maintenance of Ag-specific CD8+ T cells. Methods We followed the fate of OVA-specific CD8 + T cells in Tyk2-deficient ( Tyk2 -/- ) mice after infection with recombinant OVA-expressing BCG ( rBCGOVA ). Because the immunostimulatory BCG-derived peptides recognized by CD8 + T cells have not been defined, and the OVA is definite peptide for specific CD8 + T cells that has been accepted widely, therefore we examine the kinetics of the OVA-Ag-specific CD8 + T cell response after rBCG-OVA infection in mice.Tyk2-/- and wild type(Tyk2+/+ ) mice were inoculated with rBCG-OVA by intra-trachea( i. t. ), after the examination of bacterial growth in the lung and spleen, the population of CD8 + T cells were detected by FACS analysis, the epitope-specific CD8 + T cells were followed with tetrameric H-2Kb molecule folding with OVA257 264 peptide, and the kinetics of Ag-specific CD8 + Tc1 cells were detected by intracellular IFN-γ production in response to OVA257-264 peptide by cytokine FACS analysis. Results After rBCG-OVA challenge,the bacteria number in spleen and lung of Tyk2 -/- mice were significantly larger than those in Tyk2 +/+ mice on days 14, 21 and 49. Almost as same as that in Tyk2+/+ mice, the size of epitope-specific CD8+ T cella with OVA257-264/Kb-tetramer-positive and the CD8 +Tc1 (T eytotoxic 1 )cells positive for intracellular IFN-γ could proliferate to its peak on day 21, then contract and maintain to the memory phase in spleen and lung of Tyk2-/- mice, but the population of CD8+ T cells in spleen and lung of Tyk2 -/- mice were significantly smaller than those in Tyk2+/+ mice on days 21 and 49, the number of epitope-specific CD8+ T cells in spleen and lung of Tyk2 -/- mice were significantly decreased and the frequency of CD8 + Tc1 cells in spleen and lung of Tyk2 -/- mice significantly reduced on day 21,49 and 70 after rBCG-OVA infection. So correspond with the larger number of bacteria in Tyk2-/- mice than those in Tyk2 +/+ mice, the expansion of OVA257-264-specific CD8 + T cells and CD8+ Tc1 response were attenuated in Tyk2 -/- mice following rBCG-OVA infection. Conclusion These results suggest that the lack of Tyk2 signaling impairs the proliferation and difference of effector CD8 + T cell to rBCG-OVA infection and at least, is partly responsible for the susceptible to the rBCG-OVA infection.     

BMSCs移植通过T细胞免疫抑制缓解1型糖尿病小鼠发病实验研究

目的　探讨小鼠骨髓间充质干细胞（Bone marrow mesenchymal stem cells, BMSCs）移植缓解1型糖尿病小鼠（T1DM）发病及机制。 方法　BMSCs分离培养及鉴定;第3代培养的上清作为条件培养基,与2周龄NOD小鼠脾细胞共培养,FACS检测CD4+和CD8+T细胞增殖及活化;2周龄雌性NOD小鼠随机分为BMSCs组、PBS组、对照组,每组6只,对照组2周处死;BMSCs组和PBS组12周分别腹腔注射100 μl（2×107细胞）BMSCs或等体积PBS,监测小鼠血糖和体重至26周;HE、免疫组化、免疫荧光染色观察胰腺炎性细胞浸润;FACS检测CD4+和CD8+T细胞增殖及活化。 结果　成功分离培养BMSCs;BMSCs条件培养基抑制CD4+及CD8+T细胞的增殖及活化（P<0.05）;BMSCs组小鼠体重高于PBS组（P<0.05）,糖尿病发病率降低,炎性浸润减少（P<0.05）,脾脏CD4+和CD8+T细胞增殖及活化降低（P<0.05）。结论　BMSCs移植可能通过T细胞免疫抑制缓解T1DM小鼠发病。     

BMSCs移植通过T细胞免疫抑制缓解1型糖尿病小鼠发病实验研究

目的　探讨小鼠骨髓间充质干细胞（Bone marrow mesenchymal stem cells, BMSCs）移植缓解1型糖尿病小鼠（T1DM）发病及机制。 方法　BMSCs分离培养及鉴定;第3代培养的上清作为条件培养基,与2周龄NOD小鼠脾细胞共培养,FACS检测CD4+和CD8+T细胞增殖及活化;2周龄雌性NOD小鼠随机分为BMSCs组、PBS组、对照组,每组6只,对照组2周处死;BMSCs组和PBS组12周分别腹腔注射100 μl（2×107细胞）BMSCs或等体积PBS,监测小鼠血糖和体重至26周;HE、免疫组化、免疫荧光染色观察胰腺炎性细胞浸润;FACS检测CD4+和CD8+T细胞增殖及活化。 结果　成功分离培养BMSCs;BMSCs条件培养基抑制CD4+及CD8+T细胞的增殖及活化（P<0.05）;BMSCs组小鼠体重高于PBS组（P<0.05）,糖尿病发病率降低,炎性浸润减少（P<0.05）,脾脏CD4+和CD8+T细胞增殖及活化降低（P<0.05）。结论　BMSCs移植可能通过T细胞免疫抑制缓解T1DM小鼠发病。     

调节T细胞和1型糖尿病

1型糖尿病是一种T细胞介导的胰岛β细胞进行性损伤的自身免疫病。在自身免疫过程中不仅有自身反应性T细胞的参与,也有可抑制免疫反应的自身反应调节性T细胞（Tregs）的参与,因而提高Tregs的反应性抑制自身免疫从而治疗糖尿病是很有希望的治疗策略。Tregs存在不同类型,它们的作用方式和表型还需进一步界定。其中抗原特异的诱导型Tregs可以在病变器官内发挥作用,通过细胞因子介导的“抑制旁观者”机制不依赖于抗原特异性地抑制自身反应性T细胞。而无抗原特异性的自然生成CD4+CD25+Tregs的应用将导致全身性的免疫调节和抑制,可能更适用于全身性而非器官特异性的自身免疫紊乱。因此应用抗原特异的诱导型Tregs治疗1型糖尿病可能更可取,但其成功尚有赖于对其最适诱导抗原、精确表型及体内作用机制的进一步了解。     

AIRE基因在NOD鼠相关组织及T1DM病人PBMC中的表达

目的：通过检测AIRE（Auto Immue REgulator）基因在NOD（Nonobese diabetic）鼠相关组织及1型糖尿病（Type 1 diabetes mellitus，T1DM）病人PBMC中的表达，从分子水平上探讨AIRE在免疫耐受中的作用。方法：运用半定量RT-PCR方法检测AIRE在BALB／c鼠（n=5）及NOD鼠（n=5）相关组织及T1DM病人的外周血单个核细胞（PBMC）中的表达情况，用β-aetin作为内参。结果：在BALB／c及NOD鼠中，Aire主要表达在免疫系统相关组织：胸腺、脾脏及淋巴结，另外在卵巢中也有表达，并且在NOD鼠中Aire含量与BALB／c鼠相比明显减少。部分T1DM病人PBMC上AIRE表达缺失。结论：＆IRE基因的表达异常与T1DM的发病具有一定的相关性。     

黄芪注射液对NOD小鼠CD4~+CD25~+CD127~(-/low)调节性T细胞的影响

目的通过对NOD小鼠应用大剂量的黄芪注射液,观察黄芪对于CD~4+CD25~+CD127~-调节性T细胞及相关细胞因子的影响情况。方法随机将20只NOD小鼠分成4组,每组5只,分别给予生理盐水、环磷酰胺注射液、黄芪注射液、黄芪联合环磷酰胺注射液各0.6 ml,连续给药2周后,取小鼠脾脏制成脾细胞悬液上流式细胞仪检测CD4~+CD25~+CD127~(-/low)调节性T细胞水平,取小鼠外周血采用酶联免疫吸附实验法(ELISA)、检测小鼠血液中的IL-10、IL-6的变化情况。结果调节性T细胞的检测结果为黄芪注射液组及联合用药组较空白对照组和环磷酰胺组有显著差别(P<0.05),而环磷酰胺组与空白对照组比较没有显著差别(P>0.05)。IL-10的检测结果为,黄芪注射液组与联合用药组较空白对照组和环磷酰胺组有统计学意义(P<0.05)。IL-6的检测结果为,其余3组均与空白对照组比较明显降低IL-6的含量(P<0.05)。结论大剂量的黄芪注射液产生免疫调节作用,可能是通过提高Treg细胞的水平而达到抑制自身反应性T细胞的增殖和分化,以及同时提高细胞因子IL-10的水平,降低IL-6的水平,抑制炎症反应,共同达到...     

2型糖尿病患者外周血CD8+CD25+Foxp3+Treg细胞检测及意义

目的探讨外周血CD8~+CD25~+Foxp3~+Treg(CD8~+Treg)细胞比例在2型糖尿病(T2DM)疾病过程中的作用。方法选取T2DM患者90例。根据尿微量白蛋白(MAL),分为45例T2DM伴正常蛋白尿患者(NMAU,MAL30 mg/24 h),45例T2DM伴微量蛋白尿患者(MAU,MAL 30～300 mg/24 h),同时选择门诊体检健康人员45名作为对照组(HC)。通过特定蛋白仪检测患者mAlb浓度;流式细胞仪检测CD8~+Treg细胞比例;Luminex200检测血浆白细胞介素(IL)-8、1β、6和肿瘤坏死因子(TNF)-α浓度。结果与HC组[4.43(3.26～5.75)%]相比,NMAU组[4.06(3.15～5.43)%]和MAU组[3.52(2.30～4.87)%]患者CD8~+Treg细胞比例降低,且MAU组降低更明显(P0.05);与HC组相比,MAU组和NMAU组患者血浆细胞因子TNF-α、IL-8、IL-6和IL-1β浓度均升高(P0.05);MAU组患者CD8~+Treg比例与血清细胞因子TNF-α(r=-0.444 8,P0.01)、IL-8(r=-0.341 2,P0.05)、IL-6(r=-0.365 3,P0.05)和IL-1β(r=-0.319 2,P0.05)呈负相关;Logistic回归分析显示CD8~+Treg对T2DM患者可能具有保护作用。结论通过调控T2DM患者外周血CD8~+Treg细胞比例,有可能减缓糖尿病的进程。     

T cells interact with T cells via CD40-CD154 to promote autoimmunity in type 1 diabetes

We have investigated the role of CD40 signaling in islet-reactive, diabetogenic CD4(+) Th1 T-cell clones. Using multispectral flow cytometry, we showed that CD40 and CD154 are co-expressed and form complexes on the surface of activated T cells. We also demonstrate that activated Tcells can transactivate CD4(+) CD40(+) T cells through the CD40-CD154 pathway. To investigate the role of CD40 signaling on Th1 cells, we used the diabetogenic clone BDC-5.2.9 retrovirally transduced with a truncated form of the CD40 molecule to produce a CD40 dominant-negative T-cell clone. Upon challenge with antigen in vitro, the production of IFN-#x003B3; by BDC-5.2.9 CD40DN was greatly reduced and, in vivo, the dominant-negative variant was unable to induce diabetes. Transduction with the CD40DN vector was also effective in preventing transfer of disease by primary NOD CD4(+) T cells. Ex vivo analysis of pancreatic infiltrates after transfer of BDC-5.2.9 CD40DN cells revealed an overall reduction of cell numbers and cytokine production by both T cells and macrophages. These data indicate that CD40 is an important signaling molecule on autoreactive CD4(+) T cells and contributes to their pathogenic effector function.     

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.     

Limited effect of CpG ODN in preventing type 1 diabetes in NOD mice

Type 1 diabetes is considered as Th1 cell mediated autoimmune disease and the suppression of Th1 cells or the activation of Th2 cells has been regarded as a plausible immunologic intervention for the prevention of type 1 diabetogenesis in a rodent model. CpG ODN is an immunostimulatory sequence primarily present in bacterial DNA, viral DNA and BCG. CpG ODN is conventionally classified as a Th1 cell activator, which has been clinically applied to cancer, allergy and infectious disease. Recently, there was a promising report of that CpG ODN administration suppressed the development of type 1 diabetes in NOD mice by inducing Th2 cell mediated cytokine. However, the antidiabetogenic effect of CpG ODN on NOD mice is controversial. Thus, two studies were serially undertaken with various kinds of CpG motif to find a more optimal sequence and administration method. In the first study, CpG ODN was vaccinated four times and pancreatic inflammation and the quantity of serum insulin subsequently evaluated. In the second study, the amounts of IFN gamma and IL-4 in sera were measured as representative cytokines of Th1 and Th2 cells, respectively. As a result, vaccination or continuous injection of CpG ODN failed to show a preventive effect on type 1 diabetogenesis in NOD mice. Structural differences of CpG ODN also had no affect on the result. CpG ODN also consistently showed affect on the pancreatic pathology. The productions of IFN gamma and IL-4 were detected only in the K and D type CpG ODN administration groups. Comparison of the two cytokines leads to the conclusion that CpG ODN generated a Th1-weighted response in both study groups. It was assumed that CpG ODN failed to produce Th2-weighted cytokine milieu, which can overcome the genetically determined phenotype of NOD mice. Given these results, it was concluded that the immunotherapeutic application of CpG ODN on Type 1 diabetes had clear limitations.     

CD8+ regulatory T cells are responsible for GAD-IgG gene-transferred tolerance induction in NOD mice

Our previous studies demonstrated that lipopolysaccharide (LPS)-stimulated splenocytes, retrovirally transduced with a glutamate decarboxylate 65 (GAD) and immunoglobulin G (IgG) fusion construct, can protect non-obese diabetic (NOD) mice from diabetes by inducing GAD-specific tolerance, and also that there are increased numbers of CD4(+) regulatory T cells (Tregs) in GAD-IgG-treated NOD mice. However, little is known about the role of CD8(+) Tregs in GAD-IgG gene-transferred tolerance induction in NOD mice. Here, we found that GAD-IgG-transduced splenocytes induced an increase in the number of CD8(+) Foxp3(+) Tregs in vitro. Using a T-cell depletion assay, we found that, compared with undepleted groups, NOD recipients transfused with CD8(-) or CD8(-) CD25(-) GAD-IgG-transduced splenocytes showed a decrease in the percentage of CD8(+) Foxp3(+) T cells, a high incidence of diabetes, serious insulitis, GAD-specific hyperresponsiveness at both the cellular and humoral levels, and changes in cytokine expression. These results indicate that CD8(+) Tregs, which were induced in vitro by GAD-IgG-transduced splenocytes, were also responsible for GAD-IgG gene-transferred tolerance induction in NOD mice.     

IL‐Y,a synthetic member of the IL‐12 cytokine family,suppresses the development of type 1 diabetes in NOD mice

The IL‐12 family of heterodimeric cytokines, consisting of IL‐12, IL‐23, IL‐27, and IL‐35, has important roles in regulating the immune response. IL‐12 family members are comprised of a heterodimer consisting of α and β chains: IL‐12 (p40 and p35), IL‐23 (p40 and p19), IL‐27 (Ebi3 and p28), and IL‐35 (Ebi3 and p35). Given the combinatorial nature of the IL‐12 family, we generated adenoviral vectors expressing two putative IL‐12 family members not yet found naturally, termed IL‐X (Ebi3 and p19) and IL‐Y (p40 and p28), as single‐chain molecules. Single chain IL‐Y (scIL‐Y), but not scIL‐X, was able to stimulate significantly a unique cytokine/chemokine expression profile as well as activate STAT3 in mice, in part, through a pathway involving IL‐27Rα in splenocytes. Adenoviral‐mediated, intratumoral delivery of scIL‐Y increased tumor growth in contrast to the anti‐tumor effects of scIL‐12 and scIL‐23. Similarly, treatment of prediabetic NOD mice by intravenous injection of Ad.scIL‐Y prevented the onset of hyperglycemia. Analysis of cells from Ad.scIL‐Y‐treated NOD mice demonstrated that scIL‐Y reduced expression of inflammatory mediators such as IFN‐γ. Our data demonstrate that a novel, synthetic member of the IL‐12 family, termed IL‐Y, confers unique immunosuppressive effects in two different disease models and thus could have therapeutic applications.     

Interferon-tau inhibits the development of diabetes in NOD mice

Interferon-alpha (IFN-α) inhibits the development of diabetes in animal models of autoimmune diabetes. However, the mechanism of the action is not fully understood and drug toxicity could limit its potential clinical utility. Interferon-tau (IFN-τ) is another type 1 interferon, which has less toxicity but may have different biologic activity than IFN-α. This study explores the effect of IFN-τ on the diabetic process in non-obese diabetic (NOD) mice. IFN-τ by intraperitoneal, subcutaneous, or oral routes of administration decreased the development of spontaneous diabetes in NOD mice. Islet inflammation was decreased 50%. IFN-τ administration to recipient mice prevented the development of passively transferred and cyclophosphamide accelerated diabetes. IFN-τ treatment also decreased anti-islet effector activity of NOD splenic cells. Immunoregulatory activity of splenic cells was augmented by IFN-τ administration as was the number of splenic CD25⁺CD4⁺ cells. Concanavalin A (Con A)-induced release of IFN-γ was decreased in spleen cells from IFN-τ treated mice. In conclusion, IFN-τ inhibits spontaneous autoimmune diabetes and passively transferred diabetes in the NOD mouse. This diabetes sparing activity may be due to an induction of regulatory cells, possibly CD25⁺CD4⁺ T cells, which in turn inhibit anti-islet effector cell activity and the development of insulitis and diabetes. Due to the lower drug toxicity, IFN-τ could be a better drug candidate than IFN-α for experimental clinical trials.     

During the early prediabetic period in NOD mice,the pathogenic CD8(+) T-cell population comprises multiple antigenic specificities

In the NOD mouse model of type 1 diabetes, major histocompatibilitycomplex (MHC) class I-restricted CD8(+) T cells are essential for disease development. However, the extent of diversity of their antigenic specificities during early pathogenesis remains unclear. An insulin-derived peptide was recently identified as the epitope for the NOD-derived diabetogenic T-cell clone G9C8. To explore the possibility that the early pathogenic CD8(+) T-cell population comprises additional antigenic specificities, we employed the T-cell clones AI4 and NY8.3, both of which are pathogenic and represent specificities present in early insulitic lesions. The clones responded to distinct fractions of chromatographically separated class I MHC-bound peptides purified from NOD-derived NIT-1 beta cells, and neither clone recognized the insulin-derived peptide. NIT-1 cells represent an unlimited peptide source that will allow for the future isolation and sequencing of the novel multiple epitopes targeted early in the autoimmune response by pathogenic CD8(+) T cells.     

Rapamycin prevents the onset of insulin-dependent diabetes mellitus (IDDM) in NOD mice.

The effect of the immunosuppressive agent rapamycin (RAPA) was assessed in the non-obese diabetic (NOD) mouse which is an autoimmune model of IDDM. RAPA was prepared in a vehicle of 8% cremophor EL/2% ethanol and investigated in two studies. NOD/MrK female mice (six per group, study no. 1; 10 per group, study no. 2) were dosed three times per week p.o. by gavage from 56 to 170 days of age (study no. 1) or from 64 to 176 days of age (study no. 2). Mice treated with RAPA at 0.6 mg/kg, 6 mg/kg, or 12 mg/kg maintained normal plasma glucose through 170 or 176 days of age with 10%, 0%, and 0% incidence of diabetes respectively. In contrast, naive, vehicle-treated, or RAPA 0.06 mg/kg-treated mice exhibited elevated plasma glucose and disease incidence typical for female NOD mice. Mice which became diabetic had elevated levels of beta-hydroxybutyrate, triglycerides and cholesterol. These plasma lipid concentrations were positively correlated with the duration of hyperglycaemia (r = 0.85, 0.87 and 0.84 respectively). Outside of its ability to prevent diabetes, RAPA itself did not affect the lipid profile of the mice. Intervention therapy with RAPA was ineffective at reversing the course of disease after IDDM onset under these experimental conditions. Finally, we report here that prophylactic treatment with RAPA was able to protect against IDDM development in some RAPA-treated mice 41 weeks after cessation of treatment. These data show that orally administered RAPA is effective in preventing onset of disease in the NOD mouse, a relevant model of autoimmune type I diabetes in man.     

Short-term subcutaneous insulin treatment delays but does not prevent diabetes in NOD mice

Despite encouraging results in the NOD mouse, type 1 diabetes prevention trials using subcutaneous insulin have been unsuccessful. To explain these discrepancies, 3-week-old NOD mice were treated for 7 weeks with subcutaneous insulin at two different doses: a high dose (0.5 U/mouse) used in previous mouse studies; and a low dose (0.005 U/mouse) equivalent to that used in human trials. Effects on insulitis and diabetes were monitored along with immune and metabolic modifications. Low-dose insulin did not have any effect on disease incidence. High-dose treatment delayed but did not prevent diabetes, with reduced insulitis reappearing once insulin discontinued. This effect was not associated with significant immune changes in islet infiltrates, either in terms of cell composition or frequency and IFN-γ secretion of islet-reactive CD8(+) T cells recognizing the immunodominant epitopes insulin B(15-23) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214). Delayed diabetes and insulitis were associated with lower blood glucose and endogenous C-peptide levels, which rapidly returned to normal upon treatment discontinuation. In conclusion, high- but not low-dose prophylactic insulin treatment delays diabetes onset and is associated with metabolic changes suggestive of β-cell "rest" which do not persist beyond treatment. These findings have important implications for designing insulin-based prevention trials.     

A comprehensive guide to antibody and T-cell responses in type 1 diabetes

Type 1 diabetes (T1D) is an organ-specific autoimmune disease in which the insulin-producing beta cells in the pancreatic islets are selectively eliminated. T cells specific for beta-cell antigens are the mediators of this precise cellular destruction. However, antibodies to beta-cell proteins are also generated and may be used for predicting disease in at-risk populations. Over the past two decades, numerous beta-cell proteins and lipids have been implicated as autoantigens in patients or in non-obese diabetic (NOD) mice, a well-studied animal model of T1D. Here, we present a review of these antigens, accompanied by their T-cell epitopes, where known, and a discussion of our current understanding of why particular self-proteins become disease-inciting antigens. Although two dozen beta-cell antigens have been identified to date, few of these have been confirmed to be recognized by pathogenic T cells early in the disease process. Further identification and characterization of initiating beta-cell antigens targeted by pathogenic T cells should be a priority for future studies.