不同RF型类风湿性关节炎患者Th17相关细胞因子表达研究

目的：探讨类风湿因子阳性与阴性类风湿关节炎（rheumatoid arthritis,RA）患者外周血中辅助T细胞（Th17）及相关细胞因子白介素17（interleukin,IL-17）,白介素6（interleukin,IL-6）表达的差异。方法：收集RA患者51例,根据RF测定分为RF＋、RF-组,健康查体者（对照组）20例,采用流式细胞术检测受检者外周血单个核细胞（PBMC）的Th17细胞的百分率;以酶联免疫吸附法（ELISA）检测受检者血浆中IL-17,IL-6的水平。结果：RA患者CD4＋IL-17＋T细胞,IL-17、IL-6水平均高于对照组,RF因子阳性与阴性RA患者之间CD4＋IL-17＋T细胞,IL-17、IL-6表达水平均存在差异有统计学意义。结论：在RA中不同RF型免疫反应和炎症表达的不同,可能与Th17及相关细胞因子表达差异有关。     

新生隐球菌性脑膜炎患者外周血Th1/Th2及相关T淋巴细胞水平变化及意义

目的通过检测新生隐球菌性脑膜炎患者外周血中Th1及Th2类细胞及NKT细胞、T细胞的分布比例,从患者水平进一步了解机体T辅助淋巴细胞及相关T淋巴细胞在新生隐球菌感染过程中的作用机制。方法收集新生隐球菌性脑膜炎/脑膜脑炎的16例患者(HIV检测阴性)及作为对照组的10例正常健康人外周血标本,采用流式细胞技术检测标本中Th1及Th2类细胞及NKT细胞、γδT细胞的在外周血T淋巴细胞中的分布比例,并采用SPSS 11.0软件进行统计分析比较。结果隐脑患者(接受诱导治疗前)Th1细胞在CD4+T淋巴细胞中所占比例为(13.093±4.618)%,低于正常对照组的比例(19.582±4.949)%,而Th2细胞所占比例为(1.769±0.865)%,高于正常对照组的比例(0.584±0.202)%,差异皆有统计学意义(P0.01);隐脑患者经诱导治疗好转后Th1细胞在CD4+T淋巴细胞中所占比例为(16.069±1.516)%,较好转前的比例(13.589±2.101)%增加,Th2细胞所占比例为(1.499±0.274)%,较好转前的比例(1.673±0.290)%下降,但差异皆无统计学意义(P0.05);隐脑患者(接受诱导治疗前)NKT细胞在整体T淋巴细胞中所占比例为(11.848±2.936)%,低于正常对照组的比例(17.918±8.088)%,差异有统计学意义(P0.05),而隐脑患者的γδT细胞在整体T淋巴细胞中所占比例为(9.166±3.989)%,与正常对照组的比例(9.873±3.509)%对比差异无统计学意义(P0.05);隐脑患者经治疗好转后NKT细胞在T淋巴细胞中所占比例为(14.806±1.513)%,较好转前比例(11.830±1.350)%增加,γδT细胞所占比例为(6.984±0.843)%,则较好转前比例(8.094±2.037)%下降,但差异皆无统计学意义(P0.05)。结论新生隐球菌性脑膜炎患者存在Th1细胞减少,Th2细胞增加,及NKT细胞在T淋巴细胞中所占比例下降,其说明新生隐球菌性脑膜炎患者的细胞免疫功能明显低下;经治疗好转后的病例,Th1/Th2失衡状态改善,可能提示患者的细胞免疫功能恢复对疾病预后起重要作用。     

良性前列腺增生患者外周血Th17和Treg细胞比率的变化

目的:探讨良性前列腺增生患者外周血Th17和Treg细胞比率的变化。方法:选择33例良性前列腺增生患者及19例正常对照者为研究对象,采用流式细胞术检测和比较其外周血中T淋巴细胞亚群及Th17和Treg细胞占CD4~+T细胞的比率。结果:良性前列腺增生患者外周血Th17和Treg细胞占CD4~+T细胞的比率分别为1.58±0.71和1.76±0.83,Th17/Treg的比率为0.89±0.42。正常健康对照者外周血Th17和Treg细胞占CD4~+T细胞的比率分别为0.75±0.46和1.83±0.75,Th17/Treg的比率为0.41±0.32。良性前列腺增生患者外周血Th17占CD4~+T细胞的比率和Th17/Treg的比率明显高于正常健康对照者(P0.05)。结论:良性前列腺增生患者体内Th17细胞比率升高,Th17/Treg比率失衡,可能与良性前列腺增生的发生、发展有关。     

新生隐球菌通过Dectin-1上调单个核细胞miR-146a的表达

目的 研究miR-146a是否参与新生隐球菌感染免疫应答过程.方法 采用RT-PCR检测了6例新生隐球菌性脑膜炎患者和6名健康个体外周血单个核细胞（PBMC）中miR-146a的表达.以热灭活新生隐球菌刺激来自健康个体的PB-MC,并加入Dectin-1抑制剂昆布多糖,采用RT-PCR检测热灭活新生隐球菌和昆布多糖对PBMC中miR-146a表达的影响.结果 新生隐球菌性脑膜炎患者PBMC中miR-146a的表达较健康个体明显增高.热灭活新生隐球菌可以上调PBMC中miR-146a的表达,昆布多糖可以削弱其上调miR-146a表达的能力.结论 热灭活新生隐球菌可以通过Dectin-1受体上调miR-146a的表达.miR-146a参与了新生隐球菌感染免疫应答过程,值得进一步研究.     

CD44在隐球菌性脑膜炎发病机制中的作用研究

目的测定隐球菌性脑膜炎小鼠脑组织中CD44表达,探讨CD44在隐球菌性脑膜炎发病机制中的作用。方法隐球菌性脑膜炎免疫抑制小鼠为实验组,未接种隐球菌的免疫抑制小鼠为对照组,应用免疫组化法检测实验组6h、12h、24h、48h、72h、4d、7d小鼠脑组织CD44表达与对照组的变化。结果对照组小鼠脑组织CD44均匀分布在脑细胞膜上。实验组隐球菌作用小鼠48h、72h后,CD44在小鼠脑组织脑膜侧分布增加,而在脑实质侧CD44的分布明显减少。病灶周围的脑组织CD44分布也一侧增加,另一侧分布减少。结论隐球菌侵入小鼠脑组织后,隐球菌诱导CD44向脑组织的一侧移行,向脑膜方向聚集。病灶周围脑组织CD44分布也不均匀。说明隐球菌性脑膜炎的发生与CD44有密切的关系。     

不同RF型类风湿性关节炎患者Th17相关细胞因子表达研究

目的:探讨类风湿因子阳性与阴性类风湿关节炎(rheumatoid arthritis,RA)患者外周血中辅助T细胞(Th17)及相关细胞因子白介素17(interleukin,IL-17),白介素6(interleukin,IL-6)表达的差异。方法:收集RA患者51例,根据RF测定分为RF+、RF-组,健康查体者(对照组)20例,采用流式细胞术检测受检者外周血单个核细胞(PBMC)的Th17细胞的百分率;以酶联免疫吸附法(ELISA)检测受检者血浆中IL-17,IL-6的水平。结果:RA患者CD4+IL-17+T细胞,IL-17、IL-6水平均高于对照组,RF因子阳性与阴性RA患者之间CD4+IL-17+T细胞,IL-17、IL-6表达水平均存在差异有统计学意义。结论:在RA中不同RF型免疫反应和炎症表达的不同,可能与Th17及相关细胞因子表达差异有关。     

两性霉素B联合氟胞嘧啶与伏立康唑治疗艾滋病合并隐球菌脑膜炎的临床回顾性研究

目的观察两性霉素B联合氟胞嘧啶与伏立康唑治疗艾滋病合并隐球菌性脑膜炎的疗效和安全性,探讨艾滋病合并隐球菌性脑膜炎的新型治疗策略。方法采用回顾性病例对照研究,20例艾滋病合并隐球菌脑膜炎分别接受三联治疗（两性霉素B＋氟胞嘧啶＋伏立康唑,n=10）和传统的两联治疗（两性霉素B＋氟胞嘧啶,n=10）,比较两种治疗方案在降低脑脊液中隐球菌计数的幅度以及临床症状缓解、病死率等方面的差异以及不良反应发生情况。结果治疗2周后,三联治疗患者脑脊液中隐球菌计数下降率明显高于两联治疗患者（下降率分别为0.743和0.408,P=0.009）,三联治疗患者头痛明显减轻或消失的时间短于两联治疗者（分别为12 d和20 d,P=0.009）,两组在2周、4周、8周及12周时的病死率均无统计学差异。两种治疗方案的不良反应发生率相当。结论两性霉素B联合氟胞嘧啶与伏立康唑能有效降低脑脊液中隐球菌计数,可作为艾滋病合并隐球菌脑膜炎诱导期的治疗选择。     

变应性鼻炎患者外周血中Tr1/Th3细胞的检测和分析

目的:检测变应性鼻炎(Allergic rhinitis,AR)患者和健康对照者外周血中IL-10+CD4+T细胞、TGF-β+CD4+T细胞(分别代表Tr1细胞和Th3细胞的特性)的比例,并探讨其在AR发病中的意义,为AR的治疗提供临床参考。方法:分离19例对粉尘螨过敏的AR患者和19例健康对照者外周血单个核细胞(PBMCs),采用流式细胞术分别检测外周血中IL-10+CD4+T细胞、TGF-β+CD4+T细胞的比例。结果:同健康对照者相比,AR患者外周血中IL-10+CD4+T细胞的比例显著降低[(1.66±0.48)%vs.(3.80.92)%,t=-9.08,P0.01)],AR患者外周血中TGF-β+CD4+T细胞的比例降低[(1.92±0.54)%vs.(4.76±1.12)%,t=-9.94,P0.01)]。结论:外周血中IL-10+CD4+T(Tr1)细胞比例的降低可能是AR发病的一个重要因素,提高AR患者外周血中分泌IL-10的Tr1细胞的比例可能在AR的治疗中具有重要意义。外周血中TGF-β1+CD4+T(Th3)细胞的比例显著降低,可能是AR发病的一个重要因素。但TGF-β1与AR关系的研究较少,特别是外周血中TGF-β1水平与AR的关系研究较少,需进一步研究。     

乳杆菌肽聚糖对β-乳球蛋白致敏小鼠脾细胞Th1/Th2及Treg/Th17失衡的体外影响

【目的】观察嗜酸乳杆菌完整肽聚糖(Whole peptidoglycan,WPG)对致敏脾淋巴细胞Th1/Th2及Treg/Th17平衡的体外调节作用。【方法】通过腹腔注射β-乳球蛋白(β-Lg)建立BALB/c小鼠牛乳过敏模型。造模成功后,分离致敏小鼠的脾淋巴细胞并分别与不同剂量的WPG共同孵育,酶联免疫法检测细胞上清液中抗体(总IgE和特异性IgE),Th1/Th2及Treg/Th17相关细胞因子(IFN-γ,IL-4,TGF-β,IL-17)水平,流式细胞术检测脾淋巴细胞中CD3+、CD4+和CD8+的百分含量,荧光定量PCR法检测过敏小鼠脾细胞中Th1/Th2及Treg/Th17相关转录因子T-bet、GATA-3、Foxp3和RORγt mRNA的表达量。【结果】WPG体外刺激致敏脾细胞后可显著抑制IgE的产生,上调CD3+、CD4+细胞数及CD4+/CD8+比值,下调Th2型因子(IL-4,GATA-3mRNA)和Th17型因子(IL-17,RORγt mRNA)的表达,且与过敏组相比,中、高剂量WPG的作用效果显著(P0.05);另外,WPG体外作用还上调了Th1型因子(IFN-γ,T-bet mRNA)及Treg型因子(TGF-β,Foxp3 mRNA)的表达,且具有剂量依赖性。【结论】嗜酸乳杆菌WPG体外刺激可有效纠正致敏脾淋巴细胞的Th1/Th2及Treg/Th17失衡。     

21例侵袭性肺曲霉病患者Th以及Treg细胞的检测

目的：分析侵袭性肺曲霉病患者辅助性T细胞（Th）以及调节性T细胞（Treg）在外周血中单个核细胞中的表达情况及其临床相关性,探讨Th和Treg细胞介导的免疫反应在侵袭性肺曲霉病中的作用。方法分离21例侵袭性肺曲霉病患者及19例健康人外周血的单个核细胞,采用流式细胞术分析Th1、Th2、Th17、Treg细胞群的表达情况,Real-timePCR方法检测相关转录因子T-bet、GATA-3、RORγt以及Foxp3的表达,ELISA法检测血清中相关细胞因子IFN-γ、IL-4、IL-17以及TGF-β的表达。结果与健康人对照组相比,侵袭性肺曲霉病患者Th1细胞以及Treg细胞占CD4＋T细胞的比例较之对照组明显降低;Th1、Th17、Treg细胞相关转录因子T-bet、RORγt、Foxp3以及相关细胞因子IFN-γ、IL-17A、TGF-β与对照组相比表达明显降低。结论IPA患者的Th1、Th17以及Treg细胞所介导的免疫反应受抑制。     

Th1型和Th2型细胞因子在口腔扁平苔藓患者外周血中的表达及临床意义

目的:研究口腔扁平苔藓(OLP1)患者Th1型和Th2型细胞因子的表达及临床意义。方法:选取2013年1月至2014年3月于我院就诊的21例充血糜烂型及14例光滑型OLP患者为研究对象,18例正常人为对照组,采用密度梯度离心法对各组外周血单个核细胞进行分离,酶联免疫吸附剂测定(ELISA)法对各组外周血单个核细胞中的IL-4和IFN-gamma的表达进行检测,逆转录-聚合酶链反应法对各组血清中IL-4 m RNA和IFN-gamma m RNA的表达进行检测。结果:与正常对照组相比,OLP患者IL-4m RNA及蛋白的表达均增高,而IFN-gamma m RNA及蛋白的表达则降低,差异均有显著统计学意义(均P0.01)。充血糜烂型及光滑型OLP患者组间比较发现,IL-4 m RNA和IFN-gamma m RNA及蛋白的表达差异无统计学意义(P0.05)。结论:OLP发病机制与Th1与Th2的表达失衡有关,为临床治疗提供参考。     

Direct correlation between Th1 and Th17 responses in immunity to Brucella infection

Th1 cells play a central role in immunity to brucellosis, while the exact role of Th17 cells has remained unknown. This study aimed to evaluate the peripheral distributions of Th1 and Th17 cells and serum levels of IFN-γ, IL-17A and IL-22 cytokines in brucellosis patients. One hundred patients (36 acute, 41 under-treatment and 23 relapsed) and 30 age- and sex-matched healthy controls were included. The frequencies of Th1 and Th17 cells were determined by flow cytometric analysis. Serum levels of IFN-γ, IL-17A and IL-22 were measured by multi-analyte flow assay. Increased frequencies of Th1 and Th17 cells were observed in acute and relapsed brucellosis versus under-treatment patients and healthy controls (P < 0.05). The mean serum levels of IFN-γ were significantly elevated in acute and relapsed groups compared to under-treatment patients (P = 0.002 and P = 0.01 respectively). Acute patients showed higher levels of IL-22 than under-treatment (P = 0.008). Direct correlations were found between increased frequencies of Th1 and Th17 cells in acute and relapsed patients (P = 0.007 and P = 0.001 respectively) and between IL-17A and IL-22 in both groups of patients. Our findings indicate a cooperative role for Th1 and Th17 cells in immunity to brucellosis which is more evident during acute and relapse phases of brucellosis.     

The prevalence of Th17 cells in patients with gastric cancer

Th17 cells have emerged as an important mediator in inflammatory and autoimmune diseases. However, recent studies suggest a potential impact of Th17 cells on tumor. The current study was designed to investigate the possible involvement of Th17 cells in gastric cancer. Compared with healthy volunteers, patients with gastric cancer had a higher proportion of Th17 cells in peripheral blood. Notably, the increased prevalence of Th17 cells was associated with clinical stage. In addition, increased populations of Th17 cells were present in tumor-draining lymph nodes with advanced disease. Furthermore, the mRNA expression levels of Th17-related factors (IL-17, IL-23p19, and RORC) in tumor tissues and the serum concentrations of IL-17 and IL-23 cytokines were significantly increased in patients with advanced gastric cancer. The results indicate that Th17 cells may contribute to gastric cancer pathogenesis.     

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n = 15) and inactive disease (n = 19) and healthy age- and gender-matched controls (n = 15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-β and FoxP3 mRNA. Ιn active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.     

Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome

Background

Immunological mechanisms are increasingly recognized in the progression of myelodysplastic syndrome (MDS). Early-stage MDS (E-MDS) is characterized by autoimmune-mediated myelosuppression whereas late-stage MDS (L-MDS) involves immune evasion, giving dysplastic cells growth potential to progress into acute myeloid leukemia. T-helper (Th) 22 is involved in the pathogenesis of inflammatory autoimmunity and tumorigenesis. The roles of Th22 cells in the pathophysiology of E-MDS and L-MDS remain unsettled.

Design and Methods

We studied 37 MDS patients (E-MDS, n = 17; L-MDS, n = 20) and 20 healthy controls to characterize their peripheral blood (PB), as well as 25 MDS patients and 10 healthy controls to characterize their bone marrow(BM). The expression of Interleukin-22 (IL-22), IL-17 or interferon gamma (IFN-γ) was examined in E-MDS, L-MDS patients and controls by flow cytometry. The mRNA expression levels of RAR-related orphan receptor C (RORC), IL-6, tumor necrosis factor alpha (TNF-α) and IL-23 in peripheral blood mononuclear cells (PBMCs) were determined by real-time quantitative polymerase chain reaction. The levels of IL-22 and IL-17 both in PB and BM plasma were examined by enzyme-linked immunosorbent assay.

Results

In E-MDS, peripheral Th17 cells were significantly elevated and correlated with peripheral Th22 cells compared with healthy controls and L-MDS. Significantly higher levels of peripheral Th22 expansion, mRNA expression of IL-6, TNF-α and lower level of RORC mRNA expression were observed in L-MDS compared with E-MDS. No statistical difference was found in IL-23 mRNA expression or plasma IL-22, IL-17 levels among E-MDS, L-MDS and controls.

Conclusions

Our data demonstrated that L-MDS cohort had increased frequencies of peripheral Th22 cells and higher mRNA expression levels of IL-6 and TNF-α, indicating that Th22 cells along with Th17 cells or not are involved in the dynamic immune responses of MDS.     

The number of CD161 positive Th17 cells are decreased in head and neck cancer patients

Background

Despite lots of research efforts, the pathology of head and neck cancer remains elusive. Accumulating evidence suggests that the innate and adaptive immunity plays an important role in HNSCC (Head and Neck Squamous Cell Carcinoma) development. Recently, a new T helper cell subset additional to the classical Th1 and Th2 cells was identified called Th17 cells, due to their secretion of IL-17. However, Th17 cells also produce additional proinflammatory cytokines and many other cytokines are involved in their differentiation and expansion. It was shown that Th17 cells play a prominent role in host defense but are also associated with the development of autoimmune diseases. The role of Th17 cells in cancer pathogenesis remains nebulous.

Methods

Th17 cells of peripheral blood, primary tumors and metastatic lymph nodes were FACS analyzed for their CD161 expression. Supernatants of the permanent HNSCC cell line BHY were used to induce Th17 cells by HNSCC tumor mileu.

Results

Here we show that Th17 cells from patients with HNSCC downregulate the Th17 cell surface receptor CD161 in peripheral blood as well as in primary tumors and especially in metastatic lymph nodes.

Conclusion

We have showed for the first time alterations of Th17 cell phenotype in HNSCC patients.     

Increased frequencies of Th22 cells as well as Th17 cells in the peripheral blood of patients with ankylosing spondylitis and rheumatoid arthritis

Background

T-helper (Th) 22 is involved in the pathogenesis of inflammatory diseases. The roles of Th22 cells in the pathophysiological of ankylosing spondylitis (AS) and rheumatoid arthritis (RA) remain unsettled. So we examined the frequencies of Th22 cells, Th17 cells and Th1 cells in peripheral blood (PB) from patients with AS and patients with RA compared with both healthy controls as well as patients with osteoarthritis.

Design and Methods

We studied 32 AS patients, 20 RA patients, 10 OA patients and 20 healthy controls. The expression of IL-22, IL-17 and IFN-γ were examined in AS, RA, OA patients and healthy controls by flow cytometry. Plasma IL-22 and IL-17 levels were examined by enzyme-linked immunosorbent assay.

Results

Th22 cells, Th17 cells and interleukin-22 were significantly elevated in AS and RA patients compared with OA patients and healthy controls. Moreover, Th22 cells showed positive correlation with Th17 cells as well as interleukin-22 in AS and RA patients. However, positive correlation between IL-22 and Th17 cells was only found in AS patients not in RA patients. In addition, the percentages of both Th22 cells and Th17 cells correlated positively with disease activity only in RA patients not in AS patients.

Conclusions

The frequencies of both Th22 cells and Th17 cells were elevated in PB from patients with AS and patients with RA. These findings suggest that Th22 cells and Th17 cells may be implicated in the pathogenesis of AS and RA, and Th22 cells and Th17 cells may be reasonable cellular targets for therapeutic intervention.