重组人MUC1-MBP融合蛋白诱导小鼠Th1活化

目的 检测重组MUC1-MBP融合蛋白对小鼠Th1细胞的活化作用.方法 通过生物活性法测定MUC1-MBP免疫鼠脾细胞培养上清液中IL-2、IFN和血清中TNF水平;通过免疫组织化学染色法检测CD4 T细胞在肿瘤组织中的浸润.结果 MUC1-MBP免疫鼠脾细胞分泌IL-2和IFN及血清TNF水平较对照组明显增高;MUC1-MBP诱导小鼠CD4 T细胞在肿瘤组织中浸润.结论 重组人MUC1-MBP融合蛋白可激活小鼠Th1细胞.     

HCV-NS3重组腺病毒转染树突状细胞体内诱导抗原特异性Th1应答的研究

目的:探讨表达丙型肝炎病毒(HCV)非结构蛋白(NS3)基因的重组腺病毒转染树突状细胞体内诱导特异性Th1细胞免疫应答。方法:分离培养小鼠骨髓树突状细胞,制备表达HCV-NS3蛋白的重组腺病毒转染树突状细胞(DC-AdNS3)疫苗,采用流式细胞术和免疫印迹法分析鉴定细胞及抗原蛋白表达。采用腹腔注射途径免疫接种BALB/c小鼠两次,每次间隔10天,3×105细胞/次。末次接种10天后,采用ELISPOT法和ELISA法分别测定脾NS3特异性分泌IFN-γ的T细胞反应以及细胞因子水平。结果:重组腺病毒转染DC可刺激DC成熟,同时可在DC内成功表达NS3蛋白。小鼠两次接种DC-AdNS3产生明显升高的分泌IFN-γ的T细胞反应(P<0·01),脾T细胞悬液内可测得高水平的IL-2和IFN-γ(P<0·01)以及显著降低的IL-10(P<0·05)。结论:DC-AdNS3疫苗可在BALB/c小鼠体内激发产生抗原特异性的Th1细胞免疫应答,为抗HCV感染的疫苗研究提供参考依据。     

Th1和Th2细胞在痢疫苗诱导小鼠产生粘膜免疫应答 …

目的 在初步了解ＣＤ４＾＋细胞参与痢疾疫苗诱导小鼠肠粘膜免疫应答的基础上，从细胞及分子水平进一步探索Ｔｈ１和Ｔｈ２细胞在小鼠肠粘膜对痢疾疫苗免疫应答中的调节作用。方法 以本室构建的福氏２ａ及宋内氏双价痢疾疫苗株ＦＳ－２１１７或ＦＳ－５４１６灌胃免疫小鼠，从细胞表型及几种细胞因子ｍＲＮＡ表达水平两方面观察小鼠肠粘膜中Ｐａｙｅｒ氏结（Ｐｅｙｅｒ’ｓ ｐａｔｃｈ，ＰＰ）及肠系膜巴结等免疫诱导部位Ｔｈ１和     

Th17细胞与Th1、Treg细胞关系的研究进展

CD4~+T细胞在不同细胞因子环境中可分化为Th1、Th2、Treg、Th17四种亚群,在一定条件下,各Th细胞亚群之间可以互相转化,从而使机体的免疫效应和免疫抑制处于精细而复杂的平衡状态。Th17细胞与自身免疫性疾病和肿瘤的发生发展关系密切,研究Th17细胞与Th1细胞、Treg细胞之间的相互关系,对于预防和治疗自身免疫性疾病和肿瘤等临床难题意义重大。     

Graves’病患者Th1/Th2免疫应答及性激素对Th1/Th2的影响（文献综述）

Graves’病（GD）是最常见的自身免疫性甲状腺疾病（AITD）,其发病机理以体液免疫异常为大家所熟知,但细胞因子在GD中的发病作用越来越受到国内外学者的重视,研究的焦点主要侧重于Th1／Th2细胞平衡紊乱。GD及其他自身免疫性疾病女性显著高发,提示性激素是影响机体免疫功能的重要因素。本文就近年来国内外对GD患者Th1／Th2免疫应答的研究及性激素对Thl／Th2平衡的影响作一综述。     

Th1和Th2细胞在机体免疫中的作用及调节

在小鼠和人体内ＣＤ＿４￣＋Ｔ淋巴细胞可区分为Ｔｈ＿１细胞和Ｔｈ＿２细胞，在机体免疫中其作用各有不同。Ｔｈ＿１细胞主要分泌ＩＬ－２，ＩＦＮ－ｒ，刺激细胞免疫反应；Ｔｈ＿２细胞则以分泌ＩＬ－４，ＩＬ－１０为主，刺激体液免疫反应。ＩＬ－２、ＩＦＮ－ｒ促进Ｔｈ＿１细胞分化，抑制Ｔｈ＿２细胞分化；而ＩＬ－４、ＩＬ－１０促进Ｔｈ＿２细胞分化，抑制Ｔｈ＿１细胞分化。     

急性MCMV感染对小鼠脾Th1/Th2/Th17细胞亚群分化的影响

目的:在整体水平观察小鼠巨细胞病毒(MCMV)感染对小鼠脾Th1/Th2/Th17细胞亚群分化及其主要的效应性细胞因子(IFN-γ、IL-4、IL-17A)表达的影响.方法:建立MCMV感染模型,8只BALB/c小鼠分别于接种MCMV Smith株后3天和14天各处死4只;另设8只接种唾液腺匀浆的模拟感染小鼠作为对照.用空斑形成试验测定肝、脾和唾液腺组织病毒滴度;流式细胞术检测脾T淋巴细胞中Th1(CD4+ IFN-γ+)、Th2(CD4+ IL-4+)、Th17(CD4+IL-17A+)细胞比例,双抗体夹心ELISA法检测脾细胞培养上清中病毒特异性IFN-γ、IL-4、IL-17A水平.结果:MCMV感染早期肝、脾和唾液腺组织中病毒呈低水平复制,而感染后14天仅在唾液腺组织呈高水平复制;Th1细胞比例及病毒特异性IFN-γ主要在MCMV感染早期呈显著升高(P ＜0.01);Th2细胞及IL-4均无明显表达及改变;Th17细胞及病毒特异性IL-17A则主要在感染后14天升高(P＜0.05).结论:MCMV感染早期,机体通过上调Th1细胞分化比例及IFN-γ的表达发挥抗病毒效应,而MCMV诱导Th17细胞分化及IL-17A的高表达可能是MCMV感染致宿主特异性细胞免疫功能失调并逃避机体特异性细胞免疫攻击的原因之一.     

Th1细胞是诱导器官特异性自身免疫病的主要原因

ＣＤ４＋Ｔ辅助细胞（Ｔｈ）根据其分泌细胞因子的种类,而分为不同的细胞亚群。虽然这些细胞亚群最初是在体外从小鼠Ｔ细胞克隆分离的［１］,目前已有许多证据表明,在小鼠、大鼠和人体内同样存在有类似的Ｔ细胞亚群。Ｔｈ细胞可分为三个亚群,Ｔｈ１,Ｔｈ２和Ｔｈ０。Ｔｈ１细胞主要分泌ＩＬ２,ＩＦＮγ和ＴＮＦα。它的功能主要是激活巨噬细胞,介导迟发型超敏反应（ＤＴＨ）和促使合成ＩｇＧ２ａ抗体。Ｔｈ１细胞所介导的免疫反应,在抗感染免疫、器官移植排斥反应和自身免疫病的诱发过程中有重要的调节作用。Ｔｈ２细胞分泌Ｉ…     

HBcAg/HBeAg对慢性乙型肝炎患者PBMC中Th1/Th2类细胞应答的影响

目的 探讨ＨＢｃＡｇ／ＨＢｅＡｇ对慢性乙型肝炎患者ＰＢＭＣ中Ｔｈ１／Ｔｈ２类细胞应答的影响。方法 用套式ＰＣＲ法检测６４便慢性ＨＢＶ感染者ＰＢＭＣ中ＨＶＢ ＤＮＡ;分别用ＰＨＡ、ＨＢｃＡｇ和ＨＢｅＡｇ体外培养;ＥＬＩＳＡ法检测ＰＢＭＣ产生Ｔｈ１类细胞因子（ＩＬ－２、ＩＦＮ－γ）和Ｔｈ２类细胞因子（ＩＬ－４、ＩＬ－１０）的含量。结果 表明ＨＢＶ ＤＮＡ阳性组和阴性组相比,无论是在ＰＨＡ还是在ＨＢｃＡ     

Th细胞的活化与树突状细胞

树突状细胞 (dendritic ,DC)是体内功能最强的专职抗原提呈细胞 (APC) ,具有很强的抗原呈递能力 ,并能有效激发T细胞应答。DC在免疫学及肿瘤学乃至其它各学科中的作用也日益受到重视。选择性激活Th1和Th2是许多免疫反应发生的关键 ,而Th的分化由DC所控制 ,本文就免疫应答中DC和T细胞相互作用方面的最新研究作一综述     

Potential role for Toll-like receptor 4 in mediating Escherichia coli maltose-binding protein activation of dendritic cells

The Escherichia coli maltose-binding protein (MBP) is used to increase the stability and solubility of proteins in bacterial protein expression systems and is increasingly being used to facilitate the production and delivery of subunit vaccines against various pathogenic bacteria and viruses. The MBP tag is presumed inert, with minimum effects on the bioactivity of the tagged protein or its biodistribution. However, few studies have characterized the immunological attributes of MBP. Here, we analyze the phenotypic and functional outcomes of MBP-treated dendritic cells (DCs) and show that MBP induces DC activation and production of proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, IL-8, tumor necrosis factor alpha, and IL-12p70) within 24 h and strongly increases Ikappabeta phosphorylation in treated cells. Interestingly, phosphorylation of Ikappabeta was largely abrogated by the addition of anti-human Toll-like receptor 4 (TLR4) antibodies, indicating that MBP activates signaling for DC maturation via TLR4. Consistent with this hypothesis, MBP activated the TLR4-expressing cell line 293-hTLR4A but not control cultures to secrete IL-8. The observed data were independent of lipopolysaccharide contamination and support a role for TLR4 in mediating the effects of MBP. These results provide insight into a mechanism by which MBP might enhance immune responses to vaccine fusion proteins.     

Multiple interleukin-18 injections promote both mouse Th1 and Th2 responses after sublethal Escherichia coli infection

Interleukin (IL)-18 is considered to induce exclusively the Th1 immune response but not the Th2 response in the presence of adequate IL-12 stimulation in bacterial infections. However, we demonstrate herein that multiple IL-18 injections to the mice not only enhance the early Th1 response but also stimulate the Th2 response later after viable Escherichia coli infection. Multiple IL-18 injections (three alternate-day injections) raised the serum interferon (IFN)-gamma level at 6 h and serum Th2 cytokine levels, such as IL-4, IL-10 and IL-13, at 48 h after infection, while a single IL-18 injection increased only the serum IFN-gamma level. Depletion of mouse CD4+ cells suppressed the IL-18-induced Th2 cytokines, IL-4, IL-10 and IL-13. In contrast, depletion of natural killer (NK)1.1+ cells reduced the IFN-gamma and IL-13 levels. Moreover, multiple IL-18 injections up-regulated the serum IgM level at 72 h after infection while a single IL-18 injection did not. Interestingly, neutralization of IL-4 but not IFN-gamma partially suppressed the increased serum IgM. Liver mononuclear cells (MNCs) from the mice treated with multiple IL-18 injections significantly increased more production of not only IFN-gamma but also Th2 cytokines and IgM by in vitro lipopolysaccharide (LPS) stimulation than those from the phosphate-buffered saline (PBS)-treated mice, while liver MNCs from the single IL-18-injected mice also increased IFN-gamma production but significantly suppressed IL-4 and IgM production compared to those from the PBS-treated mice. Our findings suggest that multiple injections of IL-18 up-regulate both the cellular and humoral innate immunities, thereby enhancing host defence against bacterial infections.     

Multidrug-resistance-associated protein 1 (Mrp1) is probably not required for murine Th cell activation

Previously, we have demonstrated that multidrug-resistance-associated protein 1 (Mrp1) represents an activation marker for murine Th1 cells and is constitutively expressed by Th2 cells. Using the inhibitor MK571, we and others also suggested that Mrp1 is necessary for Th cell activation. However, herein, we show that Mrp1-deficient Th cells can be differentiated to a similar extent to Th1 and Th2 cells in vitro and, upon re-stimulation, produce comparable amounts of IL-2, IFNgamma and IL-4. Mrp1-deficient mice are equally susceptible than wild-type mice to infection with the protozoan parasite Leishmania major, a well-respected model for in vivo Th1 and Th2 cell differentiation. Intriguingly, MK571 is able to completely block activation of Mrp1-deficient Th cells. Most likely, therefore, the molecule relevant for Th cell activation which is blocked by MK571 is different from Mrp1. While these results are compatible with our previously reported data on Mrp1 expression, they contradict our previous conclusions about Mrp1 function in murine Th1 cells as well as those published in a very recent report in this journal on human Th cells.     

Outer membrane protein A of Escherichia coli O157:H7 stimulates dendritic cell activation

Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved. While OmpA was required for bacterial binding to polarized Caco-2 cells, it was not needed for the induction of cytokine production by Caco-2 cells or for human DC migration across polarized cells.     

Effect of type 1 piliation on in vitro killing of Escherichia coli by mouse peritoneal macrophages.

Escherichia coli K-12 mutants possessing defined lesions affecting type 1 pilus production, receptor binding, or length were examined for their ability to resist killing by mouse peritoneal macrophages in vitro. Mutants were mixed pairwise at known ratios in wells containing macrophages, and after incubation, the ratio of the survivors was assayed. The difference in phagocytic killing between type 1 piliated cells and isogenic nonpiliated cells was significant, the piliated cells being approximately threefold more resistant. Pilus length had little effect upon survival, as the long-piliated mutants were no more resistant to killing than the normal-length parents. Interestingly, the receptor-binding function of type 1 pili was most important in effecting resistance, as mutants lacking the ability to bind receptor were killed as effectively as nonpiliated mutants. These data are consistent with the notion that pili actually impede killing by macrophages rather than serve as passive physical barriers to uptake.     

Dectin-1 isoforms contribute to distinct Th1/Th17 cell activation in mucosal candidiasis

The recognition of β-glucans by dectin-1 has been shown to mediate cell activation, cytokine production and a variety of antifungal responses. Here, we report that the functional activity of dectin-1 in mucosal immunity to Candida albicans is influenced by the genetic background of the host. Dectin-1 was required for the proper control of gastrointestinal and vaginal candidiasis in C57BL/6, but not BALB/c mice; in fact, the latter showed increased resistance in the absence of dectin-1. The susceptibility of dectin-1-deficient C57BL/6 mice to infection was associated with defects in IL-17A and aryl hydrocarbon receptor-dependent IL-22 production and in adaptive Th1 responses. In contrast, the resistance of dectin-1-deficient BALB/c mice was associated with increased IL-17A and IL-22 production and the skewing towards Th1/Treg immune responses that provide immunological memory. Disparate canonical/noncanonical NF-κB signaling pathways downstream of dectin-1 were activated in the two different mouse strains. Thus, the net activity of dectin-1 in antifungal mucosal immunity is dependent on the host''s genetic background, which affects both the innate cytokine production and the adaptive Th1/Th17 cell activation upon dectin-1 signaling.     

Characterization of the E. coli glucose permease fused to the maltose-binding protein

The ptsG gene that encodes the major glucose transporter of Escherichia coli, II Glc, was inserted into a pMALE-amp r expression vector down-stream of the malE gene which encodes the E. coli maltose-binding protein (MBP). II Glc-MBP in the 2 h high speed supernatant of cell lysates eluted from a gel filtration column showing two activity peaks. The glucose-6-phosphate-dependent transphosphorylation (TP) activity of the membrane bound oligomeric peak 1 showed substrate inhibition while that of the soluble monomeric peak 2 did not. Purification of peak 2 yielded activity with weak substrate inhibition, and further gel filtration analyses showed that upon purification, some of the monomeric II Glc-MBP associated to higher molecular size forms. Assays of the phosphoenolpyruvate-dependent and transphosphorylation reactions showed that the specific activity of the purified enzyme from peak 1 was approximately double that from peak 2. The results show that the monomeric II Glc-MBP exhibits no substrate inhibition although the oligomeric form does. Purification promotes subunit association, an increase in catalytic activity, and restoration of substrate inhibition.     

川芎嗪对小鼠哮喘模型Th17细胞的调控作用以及对Th17、Treg细胞平衡的影响

目的：研究川芎嗪对小鼠哮喘模型外周血中Th17、Treg细胞比例以及特征性细胞因子IL-17、IL-10水平的影响。方法：雄性BALB/c小鼠随机分成四组：正常对照组、哮喘模型组、川芎嗪治疗组以及激素治疗组。 OVA诱导激发哮喘,治疗组小鼠在第0、7、14天以及每次雾化吸入前30 min腹腔注射川芎嗪或地塞米松注射液,正常对照组用生理盐水替代OVA进行腹腔注射以及雾化吸入。小鼠的肺组织HE染色,分离外周血淋巴细胞做流式细胞学检测并ELISA法检测血清中IL-17、IL-10的水平。结果：哮喘模型组造模成功,川芎嗪治疗组及激素治疗组哮喘表现较轻微。 HE染色显示哮喘模型组小鼠肺组织在支气管以及小血管周围发现大量的嗜酸性粒细胞、中性粒细胞、巨噬细胞等炎性细胞浸润;而川芎嗪治疗组和激素治疗组小鼠的肺组织切片中仅发现少量的炎性细胞。流式细胞仪检测显示哮喘组小鼠的Treg细胞较正常组小鼠比例明显降低,而Th17细胞占CD4＋T细胞显著升高;川芎嗪治疗组小鼠以及激素治疗组小鼠的变化趋势一致,Treg细胞和Th17细胞的比例趋于正常。 ELISA结果显示哮喘组小鼠的IL-17的水平显著高于正常组,IL-10的水平较正常组显著降低,川芎嗪治疗组小鼠的IL-17水平较哮喘模型组明显降低,而IL-10的水平显著升高,激素治疗组小鼠的变化趋势与川芎嗪治疗组一致。结论：在OVA诱导小鼠哮喘模型中,川芎嗪可以通过增强Treg细胞的功能,增加Treg细胞的数量,进而抑制Th17细胞的数量以及功能,减轻Th17细胞的应答,降低IL-17细胞因子的分泌,从而起到预防/控制哮喘发作的作用。     

Effects of PCBs 52 and 77 on Th1/Th2 balance in mouse thymocyte cell cultures

Polychlorinated biphenyls (PCBs) are industrial chemicals that have been released into the environment, resulting in widespread and persistent contamination. PCBs exist as 209 different congeners depending on the chlorine substitution on the biphenyl rings, and the physical properties and toxic effects of different PCB congeners are structure-dependent. We have tested an ortho-substituted, noncoplanar congener, 2,2',5,5'-tetrachlorobiphenyl (PCB 52), and a non-ortho-substituted coplanar congener, 3,3',4,4'-tetrachlorobiphenyl (PCB 77), for their effects on the T helper 1 (Th1) and T helper 2 (Th2) lymphocyte balance. The experiments were performed in 10 microg/ml concanavalin A (Con A)-stimulated and nonstimulated thymocytes for determination of cytokine production profiles. Interferon-gamma (produced by Th1 cells) and interleukin (IL)-10 (produced by Th2 cells) concentrations were measured in the supernatants at 12 and 24 hr after treatment with PCBs. PCBs 52 and 77 caused significant increases in interferon-gamma levels at 12 and 24 hr in both Con A-stimulated and nonstimulated media. IL-10 levels were significantly reduced at two interval periods by PCBs 52 and 77 in cultures with and without Con A. Our results show that the cytokine production profile was significantly shifted to Th1 by both ortho-substituted and coplanar PCB congeners in mouse thymocyte cultures.