微囊化转mIFN-γ基因CHO细胞皮下移植促进荷瘤小鼠TH1/TH2的漂移

目的：通过观察微囊化转mIFN-γ基因CHO细胞皮下移植对荷瘤小鼠TH1／TH2类细胞因子的影响，进一步了解外源性IFN－γ对TH1／TH2平衡的调节作用，同时验证微囊化基因工程细胞移植治疗恶性肿瘤的可行性。方法：将mIFN-γ基因转染到正常细胞CHO，然后进行微囊化，移植到荷瘤小鼠的皮下，2周后，测定小鼠血清TH1和TH2类细胞因子IFN－γ、IL－2、IL－12、IL－4、IL－10的水平，并与对照组相比较。同时测量肿瘤的平均直径，以观察肿瘤的生长速度，并观察荷瘤小鼠的生存期。结果：经微囊化转mIFN-γ基因CHO细胞皮下移植干预治疗后，小鼠血清TH1类细胞因子IFN－γ、IL－2、IL－12水平明显升高，而TH2类细胞因子IL－4、IL－10变化不明显；小鼠肿瘤的生长速度明显减慢，生存期明显延长。结论：外源性IFN－γ促进荷瘤小鼠TH1细胞的分化，使TH1／TH2向着TH1方向漂移；微囊化基因工程细胞的移植，有望替代基因工程药物，成为治疗恶性肿瘤的又一新平台。     

TH1/TH2细胞因子与习惯性流产的关系研究

目的通过测定习惯性流产患者（RSA）及正常妊娠妇女外周血血清TH1/TH2细胞因子的含量,探讨TH1/TH2细胞因子与习惯性流产发生的关系。方法采用酶联免疫吸附法检测30例RSA患者,72例正常妊娠妇女血清IL-2、IL-4、IL-10、IFN-γ、TNF-α及TNF-β水平并比较两组之间的差异。结果RSA患者IL-2、IL-4、IL-10、IFN-γ、TNF-α血清水平显著高于正常妊娠妇女（P〈0.05）,TNF-β血清水平显著低于正常妊娠妇女（P〈0.05）。结论血清IL-2、IL-4、IL-10、IFN-γ、TNF-α水平可能在习惯性流产发生中起重要作用。     

DEN-2 NGC株感染BALB/c小鼠TH1/TH2类细胞因子产生的差异

目的通过观察2型登革病毒(DEN2)NGC株初次感染和再次感染BALB/c小鼠后小鼠血浆中TH1和TH2类细胞因子的产生及其动态变化,研究DEN2感染的免疫机制。方法用不同量的DEN2NGC株经皮下多点注射,建立BALB/c小鼠感染模型,间接ELISA法测定感染后不同时间小鼠血浆IL-2、IFN-γ、IL-4、IL-5、IL-6、IL-10的含量。结果(1)在初、再次感染阶段,DEN2NGC株各感染组产生IL-4、IFN-γ、IL-2、IL-5、IL-6、IL-10与正常对照组有显著差异(P<0·05)。①在初次感染阶段,NGC株各感染组血浆IL-2、IL-5、IL-6于第4、6、8天出现;在再次感染阶段,各感染组产生IL-2、IL-5、IL-6均于第1天迅速升高;IL-2水平于第4天达高峰(104448·46±13314·1pg/ml),维持5~7天后迅速下降;IL-5于48小时达到高峰(135·125±46·75pg/ml);各组小鼠IL-6产生的动态相似,在再次感染后第1天,达高峰(555·823±44·639pg/ml)后逐渐下降。②初次感染早期,IL-4水平明显升高,而IFN-γ处于较低水平;再次感染后第1天,IL-4水平达最高峰(4294·668±349·038pg/ml),IFN-γ则于第4天和第11天达较高水平,两者的产生彼消此长,且产生动态与感染病毒量有关。(2)在初、再次感染阶段,用不同量DEN2NGC株感染组产生细胞因子水平有差异。结论DEN2NGC株初次和再次感染BALB/c小鼠后,TH1和TH2应答均可发挥一定作用,二者相互影响,TH细胞及其产生的CK在DEN感染的发病机制中起重要作用。     

强直性脊柱炎TH亚群激活及T细胞活化状态研究

目的 :研究强直性脊柱炎 (AS)患者TH1 TH2细胞激活状态及T细胞活化状况 ,探讨其发病机理。方法 :运用流式细胞仪 (CBA)法检测 35例AS患者TH1(INF γ、TNF α、IL 2 )、TH2 (IL 10、IL 5、IL 4 )细胞因子水平以及外周血淋巴细胞CD3 、CD4 、CD8 T细胞、B细胞 (CD19 )、NK细胞 (CD16 5 6 )和CD3 HLA DR 、CD4 HLA DR 、CD8 HLA DR T细胞百分率 ,并与健康对照组比较。结果 :AS患者血浆TNF α水平、IL 2水平均显著低于健康对照组 (P <0 0 1,P <0 0 5 ) ,IL 10水平显著高于健康对照组 (P <0 0 5 )。CD3 、CD3 CD8 T细胞百分率显著低于健康对照。CD8 HLA DR T细胞百分率均显著低于健康对照 (P <0 0 5 ) ,CD4 HLA DR T显著高于健康对照 (P <0 0 5 )。结论 :AS患者血浆低水平的TNF α、IL 2和高水平的IL 10提示其体内存在着TH1 TH2平衡的偏移 ;TH1激活程度低下 ,而TH2激活程度增强 ,细胞因子水平的改变尤以TH1细胞因子TNF α改变特别明显。AS患者在多个层面存在细胞免疫功能紊乱     

Methods for the in vitro determination of an individual disposition towards TH1- or TH2-reactivity by the application of appropriate stimulatory antigens

In this study we performed several methods for the determination of cytokines (RT-PCR for the demonstration of cytokine mRNA and flow cytometry for the analysis of intracellular cytokines) and compared them with a recently established test system stimulating peripheral blood mononuclear cells (PBMC) with TH1- and TH2-relevant recall antigens and analysing type 1 and type 2 cytokines by ELISA. Aim of the study was therefore to evaluate the reliability of TH1/TH2 cytokine profiles in two individuals with different types of an allergic/atopic disposition: one of them showed a strong TH1/type 1-mediated tuberculin-reaction (subject A), the other (subject B) revealed elevated IgE-levels and eosinophil counts (TH2/type 2-mediated). PBMC were incubated with the type 1-antigen purified protein derivative (PPD) and the type 2-antigen tetanus-toxoid (TT) for seven days. From the comparison of ELISA with RT-PCR and flow cytometry-analysis it became evident that all three methods allowed the definition of subject A as a 'type 1-responder'. Subject B showed a pure type 2-response in the ELISA method; PCR and flow cytometry analysis revealed the simultaneous production of type 1- and type 2-cytokines resulting in a mixed type 1/type 2-profile. Active immunization of subject A with TT at the end of the observation period of 12 months resulted in a transient shift from type 1- to a mixed type 1/type 2-profile (simultaneous PPD-induced IFN-gamma- and TT-induced IL-5 production). From this pilot study based on clear cut clinical criteria concerning either a humoral or cellular immunological reactivity towards allergens/antigens it is suggested that the determination of type 1/type 2-cytokines by ELISA in supernatants of PBMC stimulated with type 1/type 2-relevant antigens is a useful approach for a better classification of 'type1-' or 'type 2-responder'.     

ORIGINAL ARTICLE: TH1 – TH2 Response and the Atopy Risk in Patients with Reproduction Failure

Problem Enhanced TH2 activity is characteristic for atopic diseases and is observed also in physiological pregnancy. The immune causes of repeated pregnancy losses and/or repeated in vitro fertilization failure may be associated with TH2 hypoactivity. The association with frequency of atopic diseases is unclear. Method of Study Intracellular production of IL‐4 and IFN‐γ by peripheral CD4+ T lymphocytes was studied, as well as serum levels of total and allergen specific IgE. Simultaneously skin prick tests with inhalant allergens were performed, and clinical features of atopy were registered by means of a questionnaire. Results Lower intracellular production of IL‐4 by peripheral CD4+ T cells and lower frequency of elevated total and allergen specific IgE were found in women with reproduction failure compared to controls, as well as lower frequency of some symptoms possibly associated with atopy. Conclusion Our study showed the presence of TH2 hypoactivity in women with reproduction failure, which may be associated with lower occurrence of atopic diseases.     

TH2 activated cells prevent experimental autoimmune uveoretinitis,a TH1-dependent autoimmune disease

Mercuric chloride (HgCl₂) injections protect (Lewis x Brown-Norway) F1 (F1) rats against experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal S antigen (S-Ag); in contrast HgCl₂-injected F1 rats develop EAU following transfer of lymph node (LN) cells from rats immunized with S-Ag alone. In the present study we demonstrate that the ability of LN cells from rats protected against EAU to transfer the disease into naive F1 rats was considerably reduced. These LN cells neither produced interleukin (IL)-2 nor (interferon (IFN)-γ but exhibited mRNA for IL-4. In contrast, LN cells from diseased rats easily transferred EAU into naive F1 rats, produced significant IL-2 and IFN-γ levels but barely exhibited mRNA for IL-4. Furthermore protected rats predominantly produced IgG2b anti-S-Ag antibodies, while diseased rats produced IgG2b anti-S-Ag antibodies and the increase in expression of MHC class II molecules on B cells was higher in protected rats than in diseased rats. These data suggest that (1) to exert a protective effect, HgCl₂ must act at an early stage of differentiation of precursors of S-Ag specific T cells, and (2) this effect is related to the preferential activation of TH2 cells to the detriment of uveitogenic TH1 cells. Finally, these results indicate that activation of TH2 cells protect from a TH1-dependent autoimmune disease.     

Balanced TH1 and TH2 immunopotentiating effects of silicates partly containing nanoparticles present in calcined serpentine

Calcined Serpentine (CS) is used in various formulations of alternative systems of medicine as a tonic to vital organs and as an anti-inflammatory agent. The process of calcination or incineration is believed to render non-toxic, gently absorbable, adaptable and digestible properties to the mineral compounds. The present study characterized CS and also evaluated its immunostimulatory potential. CS was characterized by using transmission electron microscopy (TEM), X-ray powder diffraction, atomic absorption spectroscopy and CHNS analysis. The characterized CS was further evaluated for its immunomodulatory potential in Swiss mice. X-Ray diffraction analysis revealed that the CS contained silicates of magnesium, calcium and iron as major minerals. Elemental composition and heavy metal analyses showed a presence of various inorganic elements/heavy metals, albeit at levels well below daily permissive intake values. TEM analysis of the test CS revealed a presence of nano particles with an average size of 10–20?nm (≈ 26% of total material). Oral administration of CS to mice at 50, 75, 100 or 200?μg/kg body weight for 10 days led to enhanced levels of total IgG, IgG₁, IgG_2a and IgG_2b in ovalbumin-immunized mice as well as ex vivo lymphocyte proliferation and levels of T_H1 (IL-2, IFNγ) and T_H2 (IL-4, IL-10) cytokines produced by their cultured splenocytes. Similarly, CS treatment resulted in enhanced delayed-type hypersensitivity responses in GRBC-primed hosts. CS also activated host peritoneal macrophages, as indicated by increases in phagocytic activity and in TLR-2, CD80 and CD86 expression. The CS did not affect liver, kidney and spleen histology. Taken together, the results indicated that absorbed CS was stimulatory of host cell-mediated immune responses. It is hypothesized for now that the immunomodulatory effect of CS may have been due, in part, to a presence of nanoparticles on the CS; further study is required to validate this viewpoint.     

TH1型/TH2型细胞因子对滋养层及蜕膜内分泌功能的影响

目的　探讨辅助T细胞 (TH) 1型 / 2型细胞因子对人绒毛组织及蜕膜组织内分泌功能的影响。方法　应用新鲜绒毛组织和蜕膜组织建立体外分泌人绒毛膜促性腺激素 (hCG)及催乳素(PRL)的模型。hCG及PRL的分泌水平应用放免法 (RIA)进行分析测定。结果　TH1型细胞因子γ干扰素 (IFN γ)在一定的浓度范围内 (10～ 10 0 0ng/ml)对绒毛组织分泌hCG有明显的抑制作用 (P <0 .0 1) ;而TH2型细胞因子白细胞介素 4 (IL 4 ) (1～ 10ng/ml)则有明显的促进作用 (P <0 .0 1)。TH1型细胞因子IFN γ低浓度时 (1～ 10ng/ml)对蜕膜组织分泌PRL有刺激作用 (P <0 .0 5 ) ,高浓度时 (10 0～10 0 0ng/ml)则有抑制作用 (P <0 .0 1) ;TH2型细胞因子IL 4对PRL分泌的调节作用与对hCG分泌的作用相类似。结论　TH1型 /TH2型细胞因子可能通过影响滋养层及蜕膜内分泌功能而在早期妊娠中起重要的免疫调节作用。     

细胞因子信号抑制因子在支气管哮喘TH1/TH2细胞失衡中的作用

目的 探讨细胞因子信号抑制因子（SOCS）在支气管哮喘（简称哮喘）患儿TH细胞亚群（TH1／TH2）功能失衡中的作用，以及转录因子T-bet和GATA3与TH1／TH2反应的关系。方法 观察20例哮喘患儿及相同数量同龄对照，采用流式细胞术（FCM）检测哮喘患者外周血CD4^＋T淋巴细胞浆中白细胞介素4（IL-4）和γ干扰素（INF-γ）的表达，逆转录-聚合酶链反应（RT-PCR）和荧光定量聚合酶链反应（real time PCR）测定淋巴细胞SOCS3、SOCS5、T-bet、GATA3 mRNA表达水平。结果 急性发作期哮喘患儿TH1细胞比例显著下降，TH2细胞显著增高（P〈0．01）；哮喘患儿淋巴细胞SOCS3、GATA3 mRNA表达水平显著高于正常同龄对照（P〈0．01），SOCS5、T-bet mRNA表达显著下降（P〈0．01）。结论 SOCS3、SOCS5、T-bet和GATA3表达异常与哮喘患儿TH1／TH2失衡有关，其中SOCS3和SOCS5表达异常可能是重要的因素之一。     

CD40L blockade prevents autoimmune encephalomyelitis and hampers TH1 but not TH2 pathway of T cell differentiation

 Although it is well established that CD40 and its ligand (CD40L) play pivotal roles in the development of humoral immunity, their roles in cell-mediated immunity and cell-mediated autoimmune diseases are not well defined. We report here that CD40:CD40L interaction is crucial for the development of experimental autoimmune encephalomyelitis (EAE), a prototype TH1-cell mediated autoimmune disease. Specific blockade of CD40L at the time of immunization markedly suppressed the incidence, mortality, day of onset, and clinical scores of EAE in (PLJ×SJL) F1 mice. Importantly, the disease suppression was not associated with anergy or deletion of autoreactive T cells but was accompanied by a drastic alteration of their cytokine profiles. The production of interferon (IFN)-γ was markedly suppressed while that of interleukin (IL)-4 enhanced. These results suggest that CD40:CD40L interaction plays important roles in the differentiation of autoreactive TH1 versus TH2 cells in vivo, and that CD40L blockade is effective in preventing autoimmune encephalomyelitis. Received: 7 November 1996 / Accepted: 3 April 1997     

Polarized TH1 responses by liposome-entrapped allergen and its potential in immunotherapy of allergic disorders

BACKGROUND: The conventional immunotherapy used for treating allergic individuals may at times lead to varying degrees of anaphylactic reaction. Liposomes have been proposed as a vehicle for safe and effective allergen-specific immunotherapy as multiple injections of liposome-entrapped allergen (LEA) have been shown to reduce specific IgE response and induce specific IgG response in BALB/c mice. OBJECTIVE AND METHODS: To elucidate the effect of LEA on polarization of T-cell responses, its effect on the relative production of TH1/TH2 type cytokines (namely IL-2, IL-4 and IFN-gamma by commercially available ELISA kits and immunoglobulin profile as measured by ELISA) were studied. Histamine release on challenge of immunized mice was measured to examine the efficacy of LEA in preventing anaphylactic reactions. RESULTS: Measurement of cytokine levels in serum and spleen cell culture supernatants of BALB/c mice injected repeatedly with either free allergen (FA) or LEA (three mice per group) indicate that LEA preferentially induces a TH1-type of response dominated by IFN-gamma and IL-2 production. Further, it was also shown that immunization of mice with FA or LEA and subsequent challenge with a large dose of the sensitizing allergen leads to fatal systemic reactions in 50-65% of the animals treated with FA, whereas no mortality was observed in mice injected with LEA. Analysis of IgG subclasses in sera of mice immunized with LEA revealed a sixfold higher production of IgG1 antibodies than mice immunized with FA. Serum IgG2a, IgG2b, IgG3 and IgM responses were also enhanced in the group of mice immunized with LEA in comparison with mice injected with FA. CONCLUSION: The results indicate that LEA confers protection against anaphylaxis to mice due to their ability to induce a high IFN-gamma:IL-4 ratio which may lead to decreased synthesis of IgE and reduced histamine release on challenge with FA.     

恶性淋巴瘤患者TH 1/TH 2细胞因子表达水平的研究

目的 探讨恶性淋巴瘤患者血清中TH1/TH2细胞因子变化及其临床意义,为肿瘤的免疫治疗提供实验依据.方法 用流式细胞小球微阵列术(cytometric bead array,CBA)检测92例恶性淋巴瘤患者及70例健康人群血清中γ干扰素(IFN-γ)、肿瘤坏死因子-α仪(TNF-α)、白细胞介素(IL-2、IL-4、IL-5、IL-10)表达水平.结果 92例恶性淋巴瘤患者血清中TH1型细胞因子的水平分别为:IFN-γ(34.26±33.4g)pg/ml、TNF-α(8.17±10.09)pg/ml、IL-2(3.74 4±1.72)pg/ml;TH2型细胞因子的水平分别为:IL-10(6.28±8.56)pg/ml、IL-5(3.53±3.20)pg/ml、IL-4(6.22±7.13)pg/ml.除TNF-α表达水平降低外,其余5项均明显高于健康体检组,差异有统计学意义(P＜0.01).TH1细胞因子IL-2与TH2细胞因子IL-4的比值明显下降(0.78±O.44),与健康体检组(1.09±0.45)比较差异有统计学意义(P＜0.01).IL-10与疾病的进展相关,Ⅲ/Ⅳ期恶性淋巴瘤患者的表达水平为(9.58±13.96)pg/ml,Ⅰ/Ⅱ期的表达水平为(4.77±3.50)pg/ml,二者比较差异有统计学意义(P＜0.01).IFN-γ在大于60岁的恶性淋巴瘤患者中表达水平明显降低,与其他年龄段恶性淋巴瘤患者比较差异有统计学意义(P ＜0.05).结论 恶性淋巴瘤患者血清中TH1/TH2细胞因子平衡失调,检测TH1/TH2细胞因子可作为评价淋巴瘤临床进展及预后指标.TH1/TH2平衡向TH2方向漂移,这可能是肿瘤细胞发生免疫逃逸,从而导致肿瘤的发生或者转移的原因之一.     

Immunohistochemical Study of Netrin-1 in the Spinal Cord with Rat Experimental Autoimmune Encephalomyelitis

To investigate whether netrin-1 is involved in autoimmune injury of the central nervous system, the expression of netrin-1 protein was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis revealed significantly increased content of netrin-1 in the spinal cords of rats at the peak stage of EAE, as compared with the levels in normal control animals (p < 0.01). Immunohistochemistry detected the netrin-1 protein in neurons, oligodendrocytes, astrocytes and vascular endothelial cells in the spinal cords of normal controls. In EAE-affected spinal cords, netrin-1 immunoreactivity was detected in infiltrating inflammatory cells at the peak stage as well as in neurons, oligodendrocytes and astrocytes. These results suggest that netrin-1 is transiently increased in rat EAE lesions, where it contributes to the modulation of rat acute EAE.     

乙型肝炎病毒感染者血清TH1/TH2型细胞因子水平及意义

TH1 型细胞因子主要促进细胞免疫 ,在抗病毒免疫中具有重要作用。TH 细胞亚群失衡将严重扰乱体液免疫和细胞免疫。本研究通过检测不同乙型肝炎病毒 (HBV)感染状态下血清TH1 TH2 型细胞因子水平 ,来探讨其在HBV感染中的作用、与慢性乙型肝炎发病的关系及其作为慢性HBV感染者常规临床检测的可能性等。1　对象与方法1.1　研究对象　HBV感染者 38例 (男 2 4例 ,女 11例 ,18～4 1岁 ) ,其中急性乙型肝炎 1例、慢性乙型肝炎 14例、慢性无症状携带者 (AsC) 2 0例、AsC重叠甲型肝炎病毒 (HAV)、戊型肝炎病毒 (HEV)感染各 1例、急性甲型…     

CSF1PO、TH01和TPOX复合扩增系统的研究

目的研究CSF1PO、TH01和TPOX复合扩增最佳体系。方法设计正交实验进行聚合酶链式反应(PCR),应用聚丙烯酰胺凝胶电泳分离显带技术检测扩增片段。结果 最佳反应体系组成为:10 × PCRbuffer2.5μl、0.72 mmol/L dNTP、2.5 mmoL/L MgCl2、2.0 U Taq酶,各基因座引物终浓度(μmol/L)CSF1PO:0.4,TH01:0.4,TPOX:0.6。结论获得复合扩增条件各反应因素较为满意的参数。     

Decreased Secretion of Th2 Cytokines Precedes Up-regulated and Delayed Secretion of Th1 Cytokines in Activated Peripheral Blood Mononuclear Cells from Patients with Insulin-Dependent Diabetes Mellitus

Recent evidence suggests that autoimmune animal diabetes is associated with an imbalance between the Th1 and Th2 arms of the cellular immune system. However, limited data is available regarding the Th1/Th2 imbalance in human Insulin dependent diabetes mellitus (IDDM) patients. Therefore, we examined the peak levels, secretory pattern and total cytokine production (calculated as the area under the curve, AUC) of the Th1 cytokines, IL-2 and IFN-γ, and Th2 cytokines, IL-4 and IL-10, from stimulated peripheral blood mononuclear cells, from 17 IDDM patients and 24 normal controls. In contrast to controls, diabetic patients were characterized by an early, uniformly low secretion of Th2 cytokines, followed by a late increased secretion of Th1 cytokines. This resulted in significant differences in secretory patterns of IFN-γIL-2, IL-4 and IL-10 between the two groups;P<0.001,P<0.005,P<0.005 andP<0.001, respectively. No correlation was found in the diabetic patients between any profiles of the cytokines and their various clinical parameters, including age, gender, disease duration, insulin requirements or glycated hemoglobin levels. In conclusion, our data provides the first comprehensive evidence for an independent and persistent impairment of both Th1 and Th2 cytokine secretory patterns in IDDM patients.