UBE2T mediates the stemness properties of breast cancer cells through the mTOR signaling pathway

Objectives:This study aimed to reveal the role and possible mechanism of the ubiquitin-conjugating enzyme 2T(UBE2T)in the biological activities of breast cancer stem cells(BCSCs).Methods:The specific protein and gene expression were quantified by Western blotting and quantitative real-time polymerase chain reaction,the proportion of BCSCs was examined by flow cytometry,and the self-renewal and proliferation of BCSCs were verified by serial sphere formation and soft agar.Results:Increasing expression of UBE2T was drastically found in breast cancer than that in adjacent tissues.Furthermore,UBE2T overexpression significantly increased the proportion of BCSCs in breast cancer cells and promoted their self-renewal and proliferation.Silent UBE2T exhibited the opposite functions.UBE2T increased the levels of the mammalian target of rapamycin and the phosphorylated mammalian target of rapamycin.Mammalian target of rapamycin(mTOR)inhibitor rapamycin inhibited the function of UBE2T in BCSCs.Conclusion:UBE2T plays a role in BCSCs through mTOR pathway and may suggest a novel therapeutic strategy for breast cancer.

Populational change of CD4^(+)CD25^(+)Treg cells is responsible for the synergistic effect of the combination of RAMP2 with baicalin in treating recurrent spontaneous abortion mouse models

Background: The absence of a safe and effective therapy for recurrent spontaneous abortion due to a maternofetal failure in immunological tolerance remains an intractable clinical obstacle for surgeons. Recently, traditional Chinese medicine has become a feasible alternative for certain diseases, including recurrent spontaneous abortion. However, because of the complex composition of the traditional Chinese medicine formula, its action mechanism remains unclear. Methods: We selected two isolated active ingredients (RAMP and baicalin) from the traditional Chinese medicine formula and used an abortion-prone CBA/J × DBA/2 model to simulate human RSA and compared the changes in fetal resorption rate, Treg cell percentage, and relevant cytokines before and after combination therapy. In addition, The mechanisms were preliminarily discussed using in vitro differentiation models. Results: In CBA/J × DBA/2 abortion-prone mice, the combination therapy resulted in a lower embryo resorption rate compared to that obtained with individual delivery of either RAMP or baicalin, thereby playing an embryo-protective role through the increase in Treg cells for the maintenance of maternal-fetal immune tolerance. In in vitro primary cell differentiation experiments, the concentration of Treg cells significantly increased from 11% to 17.9% after the combination therapy compared to that of the single administration group. Conclusion: the synergistic effects of RAMP and baicalin were responsible for Treg differentiation. The present study provides a solid basis for improving the applicability of traditional Chinese herbs in the treatment of recurrent spontaneous abortion.

MBD2 promotes Th2 differentiation in ovalbumin-induced CD4+T cells

Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.

Adenosine 2A receptor contributes to the facilitation of postinfectious irritable bowel syndrome by γδ T cells via the PKA/CREB/NF-κB signaling pathway

BACKGROUND Immunological dysfunction-induced low-grade inflammation is regarded as one of the predominant pathogenetic mechanisms in post-infectious irritable bowel syndrome(PI-IBS).γδT cells play a crucial role in innate and adaptive immunity.Adenosine receptors expressed on the surface ofγδT cells participate in intestinal inflammation and immunity regulation.AIM To investigate the role ofγδT cell regulated by adenosine 2A receptor(A2AR)in PI-IBS.METHODS The PI-IBS mouse model has been established with Trichinella spiralis(T.spiralis)infection.The intestinal A2AR and A2AR inγδT cells were detected by immunohistochemistry,and the inflammatory cytokines were measured by western blot.The role of A2AR on the isolatedγδT cells,including proliferation,apoptosis,and cytokine production,were evaluated in vitro.Their A2AR expression was measured by western blot and reverse transcription polymerase chain reaction(RT-PCR).The animals were administered with A2AR agonist,or A2AR antagonist.Besides,γδT cells were also injected back into the animals,and the parameters described above were examined,as well as the clinical features.Furthermore,the A2AR-associated signaling pathway molecules were assessed by western blot and RT-PCR.RESULTS PI-IBS mice exhibited elevated ATP content and A2AR expression(P<0.05),and suppression of A2AR enhanced PI-IBS clinical characteristics,indicated by the abdominal withdrawal reflex and colon transportation test.PI-IBS was associated with an increase in intestinal T cells,and cytokine levels of interleukin-1(IL-1),IL-6,IL-17A,and interferon-α(IFN-α).Also,γδT cells expressed A2AR in vitro and generated IL-1,IL-6,IL-17A,and IFN-α,which can be controlled by A2AR agonist and antagonist.Mechanistic studies demonstrated that the A2AR antagonist improved the function ofγδT cells through the PKA/CREB/NF-κB signaling pathway.CONCLUSION Our results revealed that A2AR contributes to the facilitation of PI-IBS by regulating the function ofγδT cells via the PKA/CREB/NF-κB signaling pathway.

Frequency of the C677T Polymorphism of MTHFR, G20210A of Prothrombin and R506Q of Factor V Leiden in Type 2 Diabetics in Abidjan

In Africa, the prevalence of diabetes is escalating and remains a concern due to the numerous complications it causes. Vascular damage associated with diabetes leads to a prothrombotic state observed in diabetic individuals. Diabetes is a complex and multifactorial disease involving genetic components. With the aim of preventing complications and contributing to an efficient management of diabetes, we investigated genes likely to lead to a risk of thrombosis, in particular the C677T of MTHFR, G20210A of prothrombin, and R506Q of factor V Leiden in type 2 diabetics in Abidjan receiving ambulatory care. A descriptive cross-sectional study was carried out on consenting type 2 diabetic patients. Mutation detection was carried out using the PCR-RFLP method employing restriction enzymes. Hemostasis tests (fibrinogen, D-dimers, fibrin monomers, and von Willebrand factor) were performed using citrate tubes on the Stage? Star Max automated system. Plasminogen activator inhibitor was assayed by ELISA method, and biochemical parameters were determined using the COBAS C311. The study population consisted of 45 diabetic patients, 51.1% of whom presented vascular complications, mainly neuropathy. Disturbances in hemostasis parameters were observed, with 15.5% of patients showing an increase in fibrin monomers. Mutation analysis revealed an absence of factor V mutation (factor V Leiden) and of G20210A mutation of the prothrombin gene. However, 15.6% of subjects had a heterozygous C677T mutation of MTHFR, with 57% of them being anemic. The exploration of biological and genetic factors associated with thrombotic risk is of significant interest in the optimal management of African type 2 diabetics.

肿瘤相关性贫血与SARS-CoV-2特异性记忆CD8^(+)T细胞丰度的相关性分析

目的 探讨肿瘤相关性贫血对外周血中新型冠状病毒特异性记忆CD8^(+)T细胞丰度的影响。方法 纳入2023年6-7月在火箭军特色医学中心住院治疗的HLA-A*11∶01阳性中晚期实体瘤患者34例。根据血红蛋白浓度将患者分为无贫血组(HGB≥120 g/L,n=14)、轻度贫血组(120 g/L> HGB≥90 g/L,n=14)与中重度贫血组(HGB<90 g/L,n=6)。分离外周血单个核细胞,利用新冠病毒抗原表位刺激并扩增记忆CD8^(+)T细胞,使用ELISPOT筛选阳性表位,构建Tetramer。利用扩增后细胞检测Tetramer+细胞比例、数量及ELISPOT斑点数,用以反映新冠病毒特异性记忆CD8^(+)T细胞丰度。分析患者血红蛋白浓度与Tetramer+细胞比例、数量及ELISPOT斑点数的相关性。分析不同贫血组Tetramer+细胞比例、数量及ELISPOT斑点数的差异。结果 34例患者中男性25例,女性9例;年龄36~78岁,中位年龄57岁。患者扩增后Tetramer+细胞比例、数量及ELISPOT斑点数与血红蛋白浓度均呈正相关关系(P<0.05);与无贫血组相比,中重度贫血组患者Tetramer+细胞比例、数量及ELISPOT斑点数均明显降低(P<0.05)。结论 肿瘤患者外周血新冠病毒特异性记忆CD8^(+)T细胞的丰度与血红蛋白浓度正相关,提示肿瘤相关性贫血可能通过降低外周血中记忆CD8^(+)T细胞的丰度导致患者病毒易感性增高。

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 byγ9TCR,BTN3A1 and BTN2A1.The self-activation of Vγ9Vδ2 T cells,which leads to self-killing,can therefore participate in the failure ofγδT cell-based therapies with exogenous PAgs and should be taken into account.

Antigen-presenting effects of effector memory Vγ9Vδ2 T cells in rheumatoid arthritis

Rheumatoid arthritis is an autoimmune disease that primarily affects the limbs, but the pathogenic mechanism remains unclear. 78 T cells, a T-cell subpopulation, are characterized by multiple biological functions and associated with a variety of diseases. This study investigated the antigen-presenting effects of γδ2 cells and their relationship with rheumatoid arthritis development. We found that Vγ9Vδ2 T cells （the predominant subtype of γδ T cells in peripheral blood） were activated by isopentenyl pyrophosphate to continuously proliferate and differentiate into effector memory cells. The effector memory Vγ9Vδ2 T cells exhibited phenotypic characteristics of specific antigen-presenting cells, including high HLA-DR and CD80/86 expression. These Vγ9Vδ2 T cells could present soluble antigens and synthetic peptides to CD4＋ T cells. Vγ9Vδ2 T cells with different phenotypes showed different cytokine secretion patterns. Effector memoryVγ9Vδ2 T cells simultaneously secreted not only interferon （IFN）-γbut also IL-17. The peripheral blood and joint synovial fluid from RA patients contained numerous heterogeneous γδ T cells that were predominantly effector memory Vγ9Vδ2 T cells with the ability to secrete inflammatory factors. We also found that γδ T cells had a similar antigen-presenting capability to B cells. These results suggest that during the development of rheumatoid arthritis, 78 T cells can aggravate immune dysfunction and produce abnormal immune damage by secreting cytokines and inducing inflammatory cells to participate in synergistic inflammatory responses. Furthermore, γδ T cells can behave similarly to B cells to present viral peptides and autoantigen peptides to CD4＋ T cells, thus sustaining CD4＋ T-cell activation.

成人接种2剂新冠灭活疫苗12个月后T细胞免疫应答特点

目的评估成年人接种新型冠状病毒(SARS-CoV-2)灭活疫苗12个月后不同抗原特异性T细胞免疫应答的特点。方法解放军总医院第五医学中心2022年4-6月招募15名健康成年人,于接种2剂新冠灭活疫苗12个月后采集其静脉血标本,以基于多色流式细胞术的活化诱导标记法(AIM)检测SARS-CoV-2抗原特异性T淋巴细胞水平,并分析其记忆表型与亚群分化特点。结果成年人接种2剂灭活疫苗12个月后,90%以上个体可检测到Spike及Non-spike特异性CD4^(+)T细胞反应(S:14/15,P=0.0001;NS:15/15,P<0.0001);80%个体检测到Spike及Non-spike特异性CD8^(+)T细胞反应(S:12/15,P=0.0463;NS:12/15,P=0.0806)。抗原特异性CD4^(+)T细胞主要表现为中央记忆细胞(CM)、1型效应记忆细胞(EM1)记忆表型,以及1/17型辅助性T细胞(Th1/17)、2型辅助性T细胞(Th2)辅助表型。结论灭活疫苗接种后能诱导广泛且持久的抗原特异性CD4^(+)T细胞反应,可能是当前国内新型冠状病毒感染重症化比例较低的关键因素。

Vlultifunctional immune responses of HMBPP-specific Jy2Vδ2 T cells in M. tuberculosis and other infections

Vy2Vδ2 T （also known as Vy9Vδ2 T） cells exist only in primates, and in humans represent a major yδ T-cell sub-population in the total population of circulating yδ T cells. Results from recent studies suggest that while （E）-4-hydroxy-3-methyl-but-2-enyl pyrophosphate （HMBPP） phosphoantigen from Mycobacterium tuberculosis （Mtb） and other microbes activates and expands primate Vy2Vδ2 T cells, the Vy2Vδ2 T-cell receptor （TCR） recognizes and binds to HMBPP on antigen-presenting cells （APC）. In response to HMBPP stimulus, Vy2V82 TCRs array to form signaling-related nanoclusters or nanodomains during the activation of Vy2V82 T cells. Primary infections with H MBPP-producing pathogens drive the evolution of multieffector functional responses in Vy2Vδ2 T cells, although Vy2V82 T cells display different patterns of responses during the acute and chronic phases of Mtb infection and in other infections. Expanded Vy2Vδ2 T cells in primary Mtb infection can exhibit a broader TCR repertoire and a greater clonal response than previously assumed, with different distribution patterns of Vδ,2Vδ2 T-cell clones in lymphoid and non-lymphoid compartments. Emerging in vivo data suggest that HMBPP activation of Vy2W2 T cells appears to impact other immune cells during infection.

梅毒血清固定患者中NOD样受体蛋白3和Toll样受体4表达与Th1/Th2相关细胞因子的相关性研究

目的探究梅毒血清固定患者中NOD样受体蛋白3(NLRP3)、Toll样受体4(TLR4)表达与辅助性T细胞1/辅助性T细胞2(Th1/Th2)相关细胞因子的相关性。方法选取2021年2月至2022年4月川北医学院附属三台医院收治的197例梅毒患者作为研究对象。将进行驱梅治疗后血清转阴者纳入转阴组(n=88),未进行驱梅治疗者纳入梅毒组(n=45),接受驱梅治疗后血清固定者纳入固定组(n=64)。另选取同期进行体检的健康人作为对照组(n=53)。采用实时荧光定量聚合酶链反应检测外周血单个核细胞(PBMCs)中NLRP3、TLR4 mRNA相对表达水平;采用酶联免疫吸附试验法检测Th1/Th2相关细胞因子的表达水平;采用Pearson法分析TLR4、NLRP3 mRNA与Th1/Th2相关细胞因子的相关性。结果各组PBMCs中TLR4、NLRP3 mRNA表达水平比较,梅毒组>转阴组>对照组>固定组,差异具有统计学意义(P<0.05);各组白介素(IL)-2、γ干扰素(IFN-γ)水平比较,对照组>转阴组>梅毒组>固定组,差异具有统计学意义(P<0.05);各组IL-1β、IL-4、IL-10及IL-18水平比较,对照组<转阴组<梅毒组<固定组,差异具有统计学意义(P<0.05);梅毒血清固定患者TLR4、NLRP3 mRNA表达与IFN-γ、IL-2呈正相关,与IL-1β、IL-4、IL-10、IL-18呈负相关(P<0.05)。结论梅毒血清固定患者NLRP3、TLR4 mRNA表达水平显著降低,且与Th1/Th2相关细胞因子密切相关。

Rapamycin ameliorates experimental autoimmune uveoretinitis by inhibiting Th1/Th2/Th17 cells and upregulating CD4+CD25+ Foxp3 regulatory T cells

· AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptides1169 to 1191 of the interphotoreceptor binding protein(IRBP). Rapamycin(0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4 +CD25 + regulatory T cells(Tregs)from rat spleen were detected by flow cytometry.·RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro.Rapamycin also significantly increased TGF-β1production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore,rapamycin decreased the ratio of Th17 cells/CD4 +T cells and upregulated Tregs in EAU, as detected by flow cytometry.·CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU.Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.

免疫检查点T细胞激活抑制物免疫球蛋白可变区结构域、人内源性逆转录病毒-H长端重复相关蛋白2在子宫内膜癌中的表达及临床意义

目的分析免疫检查点T细胞激活抑制物免疫球蛋白可变区结构域(VISTA)、人内源性逆转录病毒-H长端重复相关蛋白2(HHLA2)在子宫内膜癌(EC)中的表达及临床意义。方法选取2017年9月至2019年10月唐山市妇幼保健院收治的120例的EC患者作为研究对象,收集其的EC组织和癌旁正常组织。检测EC组织和癌旁正常组织VISTA、HHLA2的表达;采用Spearman分析免疫检查点VISTA、HHLA2的相关性;Kaplan-Meier分析VISTA、HHLA2与预后的关系;Cox回归分析影响EC患者预后的危险因素。结果EC组织中VISTA、HHLA2阳性表达率显著高于正常癌旁组织(P<0.05)。Spearman相关性结果显示EC组织中VISTA与HHLA2表达呈正相关性(r=0.587,P<0.05)。EC组织中VISTA、HHLA2表达与临床病理特征有关(P<0.05)。Kaplan-Meier曲线结果显示VISTA阳性表达患者3年生存率低于阴性表达患者(χ^(2)=11.864,P<0.05),HHLA2阳性表达患者3年生存率低于阴性表达患者(χ^(2)=4.975,P<0.05)。Cox回归分析结果显示VISTA和HHLA2是影响EC患者预后的危险因素(P<0.05)。结论免疫检查点VISTA、HHLA2在EC中高表达,其是影响EC预后的危险因素,二者可能是有价值的预后标志物。

Cyclooxygenase-2 Blockade Inhibits Accumulation and Function of Myeloid-derived Suppressor Cells and Restores T Cell Response after Traumatic Stress

Myeloid-derived suppressor cells （MDSCs） play a crucial role in T cell dysfunction, which is related to poor outcome in patients with severe trauma. Cyclooxygenase-2 （Cox-2） contributes to immune disorder in trauma and infection via production of prostaglandin E2. However, the role of Cox-2 in the accumulation and function of MDSCs after traumatic stress has not been fully elucidated. In the present study, we treated murine trauma model with NS398, a selective Cox-2 inhibitor. Then the percentages of CD1 lb＋/Gr-l＋ cells, proliferation and apoptosis of CD4＋ T cells were determined. Ar- ginase activity and arginase-1 （Arg-1） protein expression of splenic CDllb＋/Gr-I＋ cells, and de- layed-type hypersensitivity （DTH） response were analyzed. The results showed that Cox-2 blockade significantly decreased the percentages of CD1 lb＋/Gr-l＋ cells in the spleen and bone marrow 48 and 72 h after traumatic stress. NS398 inhibited arginase activity and down-regulated the Arg-1 expression of splenic CD1 lb＋/Gr-l＋ ceils. Moreover, NS398 could promote proliferation and inhibit apoptosis of CD4＋ T cells. It also restored DTH response of traumatic mice. Taken together, our data revealed that Cox-2 might play a pivotal role in the accumulation and function of MDSC after traumatic stress.

Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

AIM:To investigate the role of nuclear factor of activated T cell 2(NFAT2),the major NFAT protein in peripheral T cells,in sustained T cell activation and intractable inflammation in human ulcerative colitis(UC). METHODS:We used two-dimensional gel-electrophoresis, immunohistochemistry,double immunohistochemical staining,and confocal microscopy to inspect the expression of NFAT2 in 107,15,48 and 5 cases of UC, Crohn's disease(CD),non-specific colitis,and 5 healthy individuals,respectively. RESULTS:Up-regulation with profound nucleo- translocation/activation of NFAT2 of lamina propria mononuclear cells(LPMC)of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC,as compared to CD or NC(P<0.001,Kruskal- Wallis test).Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T,but was less prominent in CD4+T cells or CD20+B cells.It was strongly associated with the disease activity,including endoscopic stage (τ=0.2145,P=0.0281)and histologic grade(τ=0.4167, P<0.001). CONCLUSION:We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis.Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity.Since activation of NFAT2is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways,our results not only provide new insights into the mechanism for sustained intractable inflammation,but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.

Cellular Oxidative Damage of HEK293T Cells Induced by Combination of CdCl_2 and Nano-TiO_2

This study investigated the conjoined cellular oxidative damage of human embryo kidney 293T（HEK293T） cells induced by cadmium chloride（CdCl2） and nanometer titanium dioxide（nano-TiO2）.RT-PCR technique was used to detect the expressions of Heme oxygenase-1（HO-1） and 8-oxoguanine DNA glycosylase（OGG1）.The activities of superoxide dismutase（SOD） and catalase enzyme（CAT） and concentrations of reactive oxygen species（ROS） and maldondialdehyde（MDA） were measured by different approaches.The results showed that CdCl2 and nano-TiO2 at a low concen-tration of 0.75 total toxic unit（TU） exerted an additive effects on HO-1 gene expression,CAT activities and MDA concentrations.When the total TU was increased to 1 or 1.25 TU,the interaction was syner-getic.Moreover,the mixture with high proportion of CdCl2 produced an additive effect on the OGG1 gene expression,and the interaction was changed to be synergetic when the concentration of CdCl2 was lower than or equal to that of nano-TiO2.Synergetic effects of CdCl2 and nano-TiO2 on cellular oxida-tive damage of HEK293T cells were found as indicated by the changes in the SOD activities and ROS concentrations.It was concluded that CdCl2 and nano-TiO2 exerts synergistic effects on the cellular oxidative damage of HEK293T cells,and the sensitivity of these indicators of oxidative damage varies with the proportion of CdCl2 and nano-TiO2 in the mixture.

Inhibitory actions of mibefradil on steroidogenesis in mouse Leydig cells： involvement of Ca^2＋ entry via the T-type Ca^2＋ channel

Intracellular cAMP and Ca^2＋ are involved in the regulation of steroidogenic activity in Leydig cells, which coordinate responses to luteinizing hormone （LH） and human ehorionic gonadotropin （hCG）. However, the identification of Ca^2＋ entry implicated in Leydig cell steroidogenesis is not well defined. The objective of this study was to identify the type of Ca^2＋ channel that affects Leydig cell steroidogenesis. In vitro steroidogenesis in the freshly dissociated Leydig cells of mice was induced by hCG incubation. The effects of mibefradil （a putative T-type Ca^2＋ channel blocker） on steroidogenesis were assessed using reverse transcription （RT）-polymerase chain reaction analysis for the steroidogenic acute regulatory protein （STAR） mRNA expression and testosterone production using radioimmunoassay. In the presence of 1.0 mmol L-1 extracellular Ca^2＋, hCG at 1 to 100 IU noticeably elevated both StAR mRNA level and testosterone secretion （P 〈 0.05）, and the stimulatory effects of hCG were markedly diminished by mibefradil in a dose-dependent manner （P 〈 0.05）. Moreover; the hCG-induced increase in testosterone production was completely removed when external Ca^2＋ was omitted, implying that Ca entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca^24- currents seen at a concentration range that nearly paralleled those inhibiting steroidogenesis. Collectively, Our data provide evidence that hCG-stimulated steroidogenesis is mediated at least in part by Ca^2＋ entry carried out by the T-type Ca^2＋ channel in the Leydig cells of mice.

Adenosine triphosphate-sensitive potassium channel opener protects PC12 cells against hypoxia-induced apoptosis through PI3K/Akt and Bcl-2 signaling pathways

Although previous studies have shown the neuroprotective effects of the adenosine triphosphate （ATP）-sensitive potassium （KATP） channel opener against ischemic neuronal damage, little is known about the mechanisms involved. Phosphatidylinositol-3 kinase （PI3K）/v-akt murine thy-moma viral oncogene homolog （Akt） and Bcl-2 are thought to be important factors that mediate neuroprotection. The present study investigated the effects of KATP openers on hypoxia-induced PC12 cell apoptosis, as well as mRNA and protein expression of Akt and Bcl-2. Results demon-strated that pretreatment of PC12 cells with pinacidil, a KATP opener, resulted in decreased PC12 cell apoptosis following hypoxia, as detected by Annexin-V fluorescein isothiocyanate/ propidium iodide double staining flow cytometry. In addition, mRNA and protein expression of phosphorylated Akt （p-Akt） and Bcl-2 increased, as detected by immunofluorescence, Western blot analysis, and reverse-transcription polymerase chain reaction. The protective effect of this preconditioning was attenuated by glipizide, a selective KATP blocker. These results demonstrate for the first time that the protective mechanisms of KATP openers on PC12 cell apoptosis following hypoxia could result from activation of the PI3K/Akt signaling pathway, which further activates expression of the downstream Bcl-2 gene.

Effect on T cell subsets and function of isletβ cells of levemir combined with acarbose in elder patients with early-onset type 2 Diabetes Mellitus

Objective:To discuss the effect of the combined therapy of levemir and acarbose on T cell subsets and function of isletβ cells in elder patients with early-onset type 2 Diabetes Mellitus. Methods:According to the number parity of entry sequence, 100 cases of elder patients with early-onset type 2 Diabetes Mellitus are divided into the control group and the observation group of 50 cases. The control group was treated with novolin and acarbose, the observation group was given subcutaneous injection of levemir and acarbose treatment. Compare the T cell subsets and function of isletβ cells in two group of patients before the treatment (T0), treatment for 4 weeks (T1) ,treatment for 8 weeks (T2).Results:(1) The levels of T0, T1, T2CD3+, CD4+, CD4+/CD8+ were increased in both groups, and CD8+ decreased. Among them, the levels of T1, T2CD3+, CD4+, CD4+/CD8+ of the observation group were obviously higher than the control group, the level of CD8+ was lowly than the control group, the difference was statistically significant;(2) In the stage of T0, T1, T2, the levels of FPG, HbA1c, HOMA-IR were showed a downward trend, the levels of FIns, HOMA-B were increased. In these two groups, the levels of T1, T2FPG, HbA1c, HOMA-IR of the observation group were lower than the control group, and the levels of FIns, HOMA-B were higher than the control group, the difference was statistically significant;(3) In the control group occurred 3 cases of hypoglycemia, and the incidence of adverse reactions was 6%. However, in the observation group no occurred adverse reactions, the difference was statistically significant.Conclusions:The combined therapy of levemir and acarbose in elder patients with early-onset type 2 Diabetes Mellitus, It helps to improve immune function, protect the isletβ-cell function.

Study on T lymphocyte subsets and NK cells in patients with Graves' disease combined with type 2 diabetes

Objective To investigate changes in T lymphocyte subsets and NK cells in patients with simple Graves’ disease(GD)and Graves’ disease combined with type 2 diabetes mellitus(GD/T2DM).Methods Fifteen cases of GD/T2DM were selected from our hospital from November 2001 to November 2004.Before and after therapy thyroid function,thyroglobulin antibody(TGA),thyroid microsomal antibody(TMA)and blood glucose level were measured,and T lymphocyte subsets(CD3,CD4,CD8,CD4/CD8)and NK cells(CD56)were measured by immunofluorescence double labeling monoclonal antibody and flow cytometry,respectively.At the same time,comparison was made with simple GD(15 cases),T2DM(15 cases)and healthy control(20 cases).Results Before therapy,CD4/CD8,CD4 and NK cells in GD/T2DM were less than normal,and there was no significant difference in comparison with simple GD(P<0.05).In T2DM group,only CD4/CD8 and CD4 were less than those of healthy controls(P<0.05).When thyroid function recovered after 1 to 3 months of methimazole treatment in both GD/T2DM and simple GD groups,various indexes recovered,which were more obvious in simple GD.Conclusion Immune hypofunction of GD may be the key to the immune abnormality of GD/T2DM,which is more significant than that of simple GD or T2DM.The recovery of thyroid function and immune abnormality is not consistent,and the recovery of GD is more significant than that of GD/T2DM.