甲状腺球蛋白基因外显子33单核苷酸多态性与Graves病停药后复发的相关性分析

目的:探究甲状腺球蛋白基因外显子33单核苷酸多态性(E33SNP)与Graves病(GD)复发的相关性,为临床预测GD抗甲状腺药物(ATD)治疗后的复发提供合理性依据。方法:选取健康对照者232例以及GD治疗后停药的患者243例,且根据GD停药患者的复发情况将观察组分为A、B、C 3个亚组:77例治疗后1年内复发者为A组,86例治疗后1~2年内复发者为B组,80例治疗后2年内未复发者为C组。利用RT-PCR检测对照组和观察组的E33SNP进行分型,对比分析对照组和观察组不同基因型的比率及观察组不同甲状腺球蛋白基因型患者的游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)、促甲状腺激素(TSH)和促甲状腺激素受体抗体(TRAb)水平,以及眼征、甲状腺肿大程度等临床资料,且对观察组不同基因型患者在治疗后2年内的累积有效率进行对比分析。结果:观察组与对照组E33SNP的基因型差异无统计学显著性,但观察组各个亚组间E33SNP基因型差异具有统计学显著性(P0.05)。对观察组的A、B、C 3个亚组间不同基因型患者各项甲状腺功能相关指标进行对比分析表明,不同基因型患者的TSH、FT3、FT4水平及甲状腺肿大程度的差异无统计学显著性,而TRAb水平和眼征发生率的差异具有统计学显著性(P0.05)。此外,E33SNP T/T型GD患者ATD治疗后2年内的累积有效率为61.8%,E33SNP T/C型患者为42.6%,E33SNP C/C型患者为21.3%,差异具有统计学显著性(P0.05)。结论:E33SNP C/C型GD患者停药后的TRAb水平以及眼征发生率明显偏高,在ATD治疗后更加容易复发,E33SNP T/T型患者则呈现相反的趋势,复发率明显偏低,因此E33SNP C/C型GD患者采用其它治疗方式或者联合治疗方式可能更加合理。     

Graves'病和桥本氏甲减患者外周血Th1、Th2、Th17、Treg细胞的变化

目的:探讨Graves'病和桥本氏甲减患者外周血中Th1、Th2、Th17细胞和CD4+CD25+FOXP3+调节性T细胞(Treg)的比例及其意义.方法:选择新诊断但未治疗的Graves'病(GD)患者25例、桥本氏甲减25例和正常对照者25例,通过流式细胞技术测定其外周血中Th1、Th2、Th17和CD4+CD25...     

尖锐湿疣患者外周血Thl/Th2及Tc1/Tc2亚群的检测及意义

目的 探讨辅助性T淋巴细胞(Th细胞)和细胞毒性T淋巴细胞(Tc细胞)的极化情况在尖锐湿疣(CA)发病机理中的作用及其与疾病复发的关系.方法 采用三色荧光抗体染色流式细胞术检测细胞内细胞因子的方法 ,检测30例CA患者和20名健康对照者外周血CD3+CD8-/IFN-γ+(Th1)、CD3+CD8-/IL4+(Th2)、CD3+CD8+/IFN-γ+(Tc1)和CD3+CD8+,IL-4+(Tc2)细胞比例.结果 与健康对照组比较,CA患者外周血中Thl细胞含量显著减少(P<0.01),Tc1细胞含量、Th1/Th2比值、Tc1/Tc2比值均减少(P<0.05);其中15例复发CA患者的Th1/Th2比值较正常对照显著减少(P<0.01),而Te1/Te2比值与健康对照组比较差异无统计学意义.结论 CA患者外周血Th1和Tc1细胞数减少,而Th2和Tc2细胞相对占优势,复发的CA患者这种失衡更趋严重.CA患者机体免疫状态可能存在Th1→Th2、Tc1→Tc2方向的漂移,这种漂移可能是人乳头瘤病毒(HPV)不易被有效清除或CA反复发生的一种免疫机制.     

Expression of intracellular Th1 and Th2 cytokines in women with recurrent spontaneous abortion,implantation failures after IVF/ET or normal pregnancy

PROBLEM: We aimed to investigate absolute counts of intracellular T helper 1 (Th1) and Th2 cytokine expressing T-cell subpopulations in women with three or more recurrent spontaneous abortions (RSA), multiple implantation failures after in-vitro fertilization and embryo transfer (IVF/ET) (three or more) or during normal pregnancy. METHOD OF STUDY: Absolute cell counts and percentages of CD3+, CD3+/CD4+, and CD3+/CD8+ T-cell populations expressing intracellular cytokines [interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4 and IL-10] was studied by four-color flow cytometry in 15 RSA and 13 implantation failure patients. Eighteen fertile non-pregnant and 47 normal pregnant women were also compared with regard to intracellular cytokine expression. RESULTS: Interleukin-10 producing CD3+/CD8+ T-cell counts were significantly lower in women with RSA (P < 0.05) and implantation failures (P < 0.05), and TNF-alpha producing CD3+/CD4+ T-cell counts were higher in women with RSA (P < 0.05) and implantation failures (P < 0.005) than those of non-pregnant fertile controls. During normal pregnancies, first trimester IL-4 expressing CD3+, CD3+/CD4+ T-cell counts (P < 0.05) and IFN-gamma expressing CD3+ T-cell counts (P < 0.05) were significantly higher than those of third trimester (P < 0.05). First trimester TNF-alpha expressing CD3+/CD8+ T-cell counts were significantly higher than those of second and third trimester women (P < 0.05). However, there are no differences in cytokine expression between non-pregnant and first trimester pregnant women. CONCLUSION: Absolute counts of IFN-gamma, IL-4, and TNF-alpha expressing T cells decrease with the progress of gestation (third trimester) during normal pregnancies. In women with implantation failures, absolute cell counts of TNF-alpha expressing CD3+/ 4- cells reflects the presence of dominant Th1 immune response. A significantly increased Th1 cytokine expression may be the underlying immune etiology for reproductive failures.     

Lack of evidence for the Th2 predominance in patients with chronic hepatitis C

A T helper (Th)1 to Th2 shift has been proposed to be a critical pathogenic determinant in chronic hepatitis C. Here, we evaluated mitogen-induced and hepatitis C virus (HCV) core antigen-induced cytokine production in 28 patients with biopsy-proven chronic hepatitis C. Flow cytometry demonstrated that after mitogenic stimulation the percentage of Th2 cells (IL-4 + or IL-13 +) and Th0 cells (IFN-gamma/IL-4 + or IL-2/IL-13 +) did not differ between patients and controls. In contrast, the percentage of Th1 cells (IFN-gamma + or IL-2 +) was significantly increased in CD4 +, CD8 +, 'naive'-CD45RA + and 'memory'-CD45RO + T-cell subsets from patients versus controls. Similar results were obtained by ELISA testing supernatants from mitogen-stimulated, unfractionated peripheral blood mononuclear cell (PBMC) cultures. Interferon-alpha treatment was associated with a reduction in the mitogen-induced Th1 cytokine response in those patients who cleared their plasma HCV-RNA. Analysis of cytokine expression by CD4 + T cells after HCV core antigen stimulation in a subgroup of 13 chronic hepatitis C patients demonstrated no cytokine response in 74% of these patients and an IFN-gamma-restricted response in 26%. Finally, no Th2 shift was found in lipopolysaccharide-stimulated monocytes. These data indicate that a Th1 to Th2 shift does not occur in chronic hepatitis C.     

Change in the Th1/Th2 Phenotype of Memory T-Cell Clones from Rheumatoid Arthritis Synovium

The stability of established memory T helper (Th)1/Th2 cells in chronic inflammatory diseases is not clear, and a shift of the cytokine balance could control chronic inflammation. In order to study the regulation of the Th phenotype of memory T cells, polyclonal T-cell lines and clones with a Th1, Th0 or Th2 phenotype were developed from rheumatoid synovial tissue. Th1 [interleukin (IL)-12 + anti-IL-4] and Th2 (IL-4 + anti-IL-12) promoting environments and IL-2 were used to manipulate the cytokine profile. Polyclonal T-cell lines of predominantly Th1 type could be shifted to produce Th2 cytokines, and polyclonal Th2/Th0 lines could be shifted to produce Th1 cytokines. However, this shift was due to an amplification of CD8+ T cells with a memory phenotype and a loss of the CD4+ T cells, giving Tc2 or Tc1 profiles, respectively. Th2 clones cultured repeatedly with IL-2 switched to either a Th0 or a Th1 phenotype, while both Th1 and Th0 memory clones kept a stable phenotype. Addition of Th2-promoting conditions strongly reduced the production of both interferon-gamma and IL-17, while Th1-promoting conditions increased the production of these cytokines. These results demonstrate that RA Th2 clones readily switch, while Th1 and Th0 clones are stable. However, induction of Th2 cytokines can be obtained in polyclonal polarized memory T cells due to amplification of Tc2 cells.     

自身免疫甲状腺疾病患者甲状腺自身抗体和淋巴细胞亚群临床免疫相关性比较

报告120例Graves 病(GD)和30例桥本氏甲状腺炎(HT)患者甲状腺自身抗体和淋巴细胞亚群的实验结果。GD 未治组TRAb、TMA 和TGA 阳性率分别为88.9%、61.1%和57.8%;HT 未治组阳性率分别为20.0%.80.0%和70.0%。GD 和HT 未治组CD_3~+、CD_(?)~+明显下降,CD_4~+/CD_(?)~+比值和CD_(20)~+显著上升(P<0.01)。GD 缓解组以上各指标恢复正常。GD 未治组TRAb 含量与CD_(8)~+的负相关性强于TRAb 与CD_(20)~+的正相关性。提示CD_8~+和TRAb 的测定是确定Graves 病及其治疗效果和预后的重要指标。     

Predominance of Th2-promoting dendritic cells in early human pregnancy decidua

Dendritic cells (DCs) are specialized antigen-presenting cells required for the priming and activation of T cells and promote the differentiation of na?ve CD4+ T cells toward the T helper cell type 1 (Th1) or Th2 phenotype. Here, we describe the characterization of CD45+CD3-CD14-CD16-CD19-CD20-CD56-HLA-DRbright DCs from early human pregnancy decidua by flow cytometry. The percentage of DCs to mononuclear cells (leukocytes) in the decidua was significantly higher than that in the peripheral blood. Moreover, decidual DCs expressed costimulatory molecules such as CD80 and CD86 and a mature marker such as CD83 on their surface. The percentage of CD11c+CD123- myeloid DCs in the decidua was significantly higher than that in the peripheral blood. Conversely, the ratio of CD11c-CD123+ lymphoid DCs in the decidua was significantly lower than that in the peripheral blood. The number of interleukin (IL)-12-producing cells in the total DC population and the myeloid DCs in the decidua was significantly lower than that in the peripheral blood. IL-12 secretion by activated decidual myeloid DCs was significantly lower than that by peripheral DCs. Na?ve CD4+ T cells primed with decidual myeloid DCs led to a higher percentage of Th2 cells in comparison with that with peripheral myeloid DCs. This finding was abolished by exogenous IL-12 administration with decidual myeloid DCs. Thus, the DCs in the decidua could regulate the Th1/Th2 balance to maintain a Th2-dominant state, leading to maintenance of pregnancy.     

Th1 and Th2 cells help CD8 T-cell responses

Help from CD4 T cells is often important for the establishment of primary and memory CD8 T-cell responses. However, it has yet to be determined whether T helper polarization affects the delivery of help and/or whether responding CD8 T cells helped by Th1 or Th2 cells express distinct effector properties. To address these issues, we compared CD8 T-cell responses in the context of Th1 or Th2 help by injecting dendritic cells copulsed with the major histocompatibility complex class I-restricted OVA peptide plus, respectively, bacterial or helminth antigens. We found that Th2 cells, like Th1 cells, can help primary and long-lived memory CD8 T-cell responses. Experiments in interleukin-12 (IL-12)-/- and IL-4-/- mice, in which polarized Th1 or Th2 responses, respectively, fail to develop, indicate that the underlying basis of CD4 help is independent of attributes acquired as a response to polarization.     

Association of Graves’ Disease and Prevalence of Circulating IFN-γ-producing CD28<Superscript>−</Superscript> T Cells

Background  Peripheral blood CD4⁺ and CD8⁺ T-cell subsets lacking surface CD28 have been suggested to predispose patients to immune-mediated disorders. Materials and Methods  To determine the role of CD28⁻ T-cell subset in Graves’ disease (GD), we characterized peripheral blood CD4⁺CD28⁻ and CD8⁺CD28⁻ T cell from early onset GD patients. Results and Discussion  GD patients had significantly higher percentages of CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells than did healthy donors. Both CD28⁻ T cells expressed mostly CD45RO, suggesting that they are activated and/or are memory T cells. GD patient-derived CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells produced more intracellular IFN-γ than their counterparts from healthy donors. Furthermore, CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells from GD patients with Graves’ ophthalmopathy (GO) secreted higher level of intracellular IFN-γ than those CD28⁻ T cells from GD patients without GO. Retrospective analysis showed that the increased levels of CD4⁺CD28⁻ T cells and their IFN-γ-producing subgroups were positively correlated to the serum anti-thyrotropin receptor (TSHR) autoantibodies (TRAb). Our observations suggest that increased IFN-γ-producing CD28⁻ T cells in GD patients may play an important role in the pathogenesis of GD. Zhiping Sun and Weixue Zhong contributed equally to this paper.     

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.     

Th17细胞与Graves’病

Th17细胞是近几年研究发现的一类不同于Th1和Th2细胞亚群的新型CD4^＋效应T细胞。该类细胞是由天然T细胞前体分化而来,具有独立分化和发育调节机制,在其分化过程中需要IL-6和转化生长因子。B、转录因子RORα及STAT3等的参与,主要分泌IL-17A、IL-17F、IL-22等多种细胞因子,并参与多种炎症、自身免疫性疾病的发生和发展。Graves’病（GD）是一种器官特异性自身免疫病,Th2细胞介导的体液免疫在其发病中起着重要作用。近来有研究提示IL-23／Th17轴亦参与GD的发展。因此了解Th17细胞分化的影响因素、产生的细胞因子以及在免疫性疾病GD中的作用具有重要的临床意义。     

Effect of intravenous immunoglobulin treatment on the Th1/Th2 balance in women with recurrent spontaneous abortions

PROBLEM: The way by which intravenous immunoglobulin (IvIg) acts to prevent immunlogically mediated recurrent spontaneous abortions (RSA) has not been clarified. In the present study, a possible effect of IvIg on the T helper cell (Th1/Th2) balance was investigated in abortions of either alloimmune or autoimmune abnormalities. METHOD OF STUDY: The study included 21 women treated with IvIg before conception because of a history of RSA characterized by alloimmune abnormalities (n = 15) or associated with anti-phospholipid antibodies (APA) (n = 6). Peripheral blood samples, collected before and 5 days after the first IvIg infusion, were stimulated, and Th1 and Th2 cells were detected by flow-cytometric analysis using a combination of monoclonal antibodies against T-cell surface markers and intracellular interferon (IFN)-gamma and interleukin (IL)-4. The percentage of IFN-gamma-producing (Th1) and IL-4-producing (Th2) cells and the Th1/Th2 ratio were compared between pre- and post-infusion samples. RESULTS: A decrease of Th1 percentage in 66.6% of the cases and a concurrent Th2 percentage increase (47.61%) resulted in a decrease in the Th1/Th2 ratio in most of the cases (76.1%) (p < 0.01). Similar results were found in Group A (Th1/Th2 decreased in 60% of the cases, p < 0.05), while in Group B the effect of IvIg was not clear (Th1/Th2 increased in three and decreased in another three cases). CONCLUSION: Our finding suggests that IvIg administration in women with alloimmune RSA enhances Th2 polarization. This is not always the case with APA-associated abortions.     

Th1/Th2细胞对再生障碍性贫血CD34+细胞体外扩增和集落形成的影响

目的探讨Th1/Th2细胞平衡偏离及平衡回复对重型再生障碍性贫血(sAA)骨髓CD34 细胞体外扩增和集落生成的影响。方法以1例确诊的sAA患者为研究对象。(1)分离骨髓单个核细胞,用免疫磁珠法富集CD34 细胞、CD4 (Th)细胞。(2)以流式细胞术(FCM)检测CD4 细胞中Th1、Th2细胞比例。(3)扩增CD34 细胞并再次富集以获足量CD34 细胞,分为4组对照组;Th细胞作用组;Th细胞 IFN-γ作用组;Th细胞 IL-4作用组。(4)各组扩增培养10d,继以集落生成试验,测定各组CD34 细胞扩增率和集落产率。(5)患者经免疫抑制治疗后随访,用FCM监测Th1/Th2细胞比。结果(1)患者经5个月治疗获缓解。(2)缓解前、后Th1/Th2比分别是22.47和12.27,正常对照组为8.98±4.45。(3)CD34 细胞扩增率,以对照组最高,其次为Th细胞 IL-4组、Th细胞组,Th细胞 IFN-γ组的最低。(4)各组CD34 细胞集落产率与其扩增率数值平行相关,即对照组最多,其次为Th细胞 IL-4组、Th细胞组,Th细胞 IFN-γ组的最少。结论Th1细胞反应亢进直接抑制sAA患者CD34 细胞在体外的自我更新和增殖分化,IL-4能拮抗这种造血抑制效应,这可能是通过调节Th1/Th2平衡而间接实现的。     

Th1/Th2 lymphocyte balance in patients with aplastic anemia

Activated T cell plays an important role in the pathogenesis of aplastic anemia (AA). CD4+ T cells are divided into Th1 cells producing hematopoietic inhibitory cytokines like interferon-gamma and Th2 cells producing interleukin-4. We investigated the Th1/Th2 cell ratio in the peripheral blood of AA patients treated with immunosuppressive therapy (IST). There were 10 patients who responded well to IST (responders) and 3 patients who were refractory to IST (non-responders). Th1 cells were lower in responders than in non-responders (16.2+/-2.4% vs. 28.8+/-5.5%, respectively, p<0.05), whereas Th2 cells did not differ. The Th1/Th2 ratio was also significantly lower in responders than in non-responders, being 13.2+/-1.5 and 40.4+/-5.1 (p<0.001), respectively. In three responders, the Th1/Th2 ratio was declined according to the hematological recovery (from 10.6 to 8.3, 16.3 to 10.9 and 11.8 to 9.5). Our results suggest that Th1 lymphocytes are more predominant in AA, and it may be very useful to monitor the Th1/Th2 ratio during IST.     

Effects of hydrocortisone on the differentiation of human T helper 2 cells

We investigated the effects of the neuroendocrine modulator hydrocortisone (HC) on Th2 differentiation of human naive CD4+ T cells with and without CD28 co-stimulation. Human naive CD4+ T cells were isolated and purified from umbilical cord blood mononuclear cells from full-term newborn infants. CD4+ T cells were treated with different concentrations of HC under Th0 or Th2 culture conditions. Th0 conditions included stimulation by immobilized monoclonal antibodies (mAbs) against CD3 and CD28; Th2 conditions were the same + rhIL-4. Parallel cultures excluded the CD28 mAb. Th1 (IL-2, INF-γ)- and Th2 (IL-4, IL-5)-type cytokines were quantified in culture supernatants by ELISA and within cells by flow cytometry. For both Th0 and Th2 culture conditions, HC significantly inhibited Th1 cytokines' release (IL-2 and INF-γ). For Th0 culture conditions, HC slightly increased IL-4 expression (Th2 cytokine). However, for Th2 culture conditions, HC inhibited the IL4-induced production of IL-4. Although the absolute cytokine amounts were decreased, absence of CD28 co-stimulation did not alter these 'trends'. Our findings indicate that HC can alter the Th1/Th2 balance by inhibiting the production of Th1-type cytokines. HC can also diminish the extensive Th2 differentiation induced by IL-4.     

Th9细胞与自身免疫性疾病

Th1、Th2、Th17和调节性T细胞（Treg）亚群是CD4＋T细胞亚群中的重要成员,其参与了人类及动物自身免疫性疾病的发病过程.既往认为,IL-9是由CD4＋Th2细胞分泌的细胞因子,是机体免疫应答中重要的调节因子.最近研究表明,机体内可能存在着一群新型的具有分泌IL-9和IL-10能力的CD4＋Th细胞亚群,称之为＂Th9＂细胞.该细胞亚群与自身免疫性疾病的相关性尚不清楚.     

Distribution of Th1, Th2, and Th0 and the Th1/Th2 cell ratios in human peripheral and endometrial T cells

PROBLEM: To examine whether normal pregnancy involves type 2 T-helper (Th2) immune condition or not. METHOD OF STUDY: We measured the percentage of Th0, Th1, and Th2 and the Th1/Th2 cell ratios of human peripheral blood and endometrial T cells using flow cytometry, which can analyze both the surface marker CD3, and intracellular cytokines, interleukin 4 (IL-4) and interferon gamma (IFNgamma). RESULTS: No significant differences were found in the percentages of Th1, Th2, and Th0 and the Th1/Th2 cell ratios in the peripheral blood T cells of nonpregnant women and women in early pregnancy. On the other hand, the percentage of Th1 cells was highest during the proliferative phase of the endometrium, followed by the secretory phase and early pregnancy decidua. The percentage of Th2 cells was highest in early pregnancy decidua and lowest during the proliferative phase of the endometrium. The Th1/Th2 ratio was 147.48+/-96.68 during the proliferative phase of the endometrium, 37.74+/-21.33 during the secretory phase, and 1.31+/-0.48 in the early pregnancy decidua. CONCLUSIONS: These data indicate that Th1 cells predominate in the nonpregnant endometrium, especially during the proliferative phase, while Th2 cells predominate in early pregnancy decidua.