Changes in natural killer T cells subsets during therapy in type C hepatitis and hepatocellular carcinoma.

Natural killer T (NKT) cells share features of both classical T cells and NK cells. NKT are heterogenous populations, and recognize glycolipids associated with CD1d molecule. We investigated Th1/Th2 cytokine production as well as frequency and phenotype of circulating NKT cells in 14 healthy subjects and in patients during therapy with type C chronic hepatitis (CH; 14 cases) and hepatocellular carcinoma (HCC; 13 cases). Peripheral blood mononuclear cells (PBMC) were obtained before and 2 weeks later interferon (IFN)/ribavirin and radiofrequency ablation therapy for CH and HCC, respectively. PBMC were cultured for 10 days with alpha-galactosylceramide (alpha-GalCer) and interleukin-2 (IL-2). Frequencies and IFN-gamma/IL-4 production of NKT cells were analyzed using flow cytometry. Intrahepatic lymphocytes were analyzed in seven CH patients with liver biopsy specimen. Prevalence of circulating Valpha24+CD3+ T cells was 0.9+/-0.9% of PBMC for controls and increased to 8.5+/-8.9% (p<0.001) in response to alpha-GalCel. Similar frequency and expansion were noted in CH. The frequency increased during therapy. The prevalence in HCC tended to be high compared to controls and response to alpha-GalCel was well. Although frequency of Valpha24+Vbeta11+CD3+ T cells was low in all groups, the distribution pattern was similar to Valpha24+Vbeta11-CD3+ T cells. Prevalence of CD56+CD3+ T cells was low independent of therapy in CH (2-3%) compared to 5.0+/-4.0% of controls, although response to alpha-GalCel was not impaired. IFN-gamma production of Valpha24+CD3+ T cells did not differ among groups, but became greater after treatment in contrast to lowered IL-4 production. Frequencies of NKT populations were higher in liver than in peripheral blood. Our study suggests that CD1d-reactive T cells have distinct distribution in different populations and therapy for patients alters cytokine response of NKT cells.     

Predominance of type 1 (Th1) cytokine production in the liver of patients with HCV-associated mixed cryoglobulinemia vasculitis

BACKGROUND/AIMS: Patients with hepatitis C virus (HCV) mixed cryoglobulinemia (MC) vasculitis have a higher mortality rate and more frequent incidence of cirrhosis than their cryoglobulin-negative counterparts. To compare the cytokine profile of liver-infiltrating T cells in HCV-infected patients with or without MC vasculitis. METHODS: Hepatic biopsy specimens were obtained from HCV infected patients with and without MC vasculitis. Using intracellular staining and flow cytometry, we assessed the ability of freshly isolated liver T cells from these biopsies to produce IFN-gamma, TNF-alpha, IL-2, IL-4, and IL-10 in response to stimulation with PMA and ionomycin. RESULTS: HCV-MC vasculitis patients compared to HCV-MC negative controls have an enhanced hepatic T cells production of Th1-type cytokines [i.e. TNF-alpha(30.3 +/- 13% vs. 15.5 +/- 5%, P = 0.01), IL-2 (20.2 +/- 9% vs. 10 +/- 4%, P = 0.01) and IFN-gamma (22.2 +/- 11% vs. 9.4 +/- 4%, P = 0.008)], whereas IL-10, a representative Th2-type cytokine, was significantly lower (7.2 +/- 4% vs. 17 +/- 7%, P = 0.01). CONCLUSIONS: T cell from the liver of HCV-MC vasculitis patients display a significantly augmented liver Th1 profile compared to MC-negative controls. This enhanced production of type-1 cytokines may account for a more severe course of liver disease.     

原发性肝癌患者细胞免疫功能变化及其与转归的关系

目的了解原发性肝癌(肝癌)患者细胞免疫功能变化及与临床转归的关系。方法应用流式细胞术检测22例肝癌患者T淋巴细胞亚群及CD8 T淋巴细胞CD28共刺激分子表达，酶联免疫吸附试验及放射免疫法检测血清白细胞介素-2(IL-2)、转化生长因子β1(TGFβ1)及白细胞介素-6(IL-6)水平，并与慢性乙型肝炎、肝硬化及正常对照比较。结果肝癌患者CD8 CD28-T淋巴细胞较正常增高，CD8 CD28 T淋巴细胞显著降低。肝癌、肝硬化及慢性乙型肝炎患者CD4 T淋巴细胞、CD4 ／CD8 比值、IL-2水平较正常明显降低，CD8 T淋巴细胞、IL-6、TGFβ1水平升高；肝癌患者CD4 T淋巴细胞、CD4 ／CD8 比值、IL-2水平低于慢性乙型肝炎，IL-6、TGFβ1水平高于慢性乙型肝炎及肝硬化患者；血清IL-6及TGFβ1水平与肝癌分期相关。结论肝癌患者存在明显的细胞免疫功能紊乱和抗肿瘤免疫功能低下，观察这些指标的变化对估价肝癌患者的抗肿瘤免疫功能以及预后将有帮助。【     

Direct ex vivo analysis of dendritic cells in patients with hepatocellular carcinoma

AIM: To analyze the phenotype and function of dendritic cells (DC) from patients with hepatocellular carcinoma (HCC) in order to understand their role in this disease. METHODS: Myeloid dendritic cells were enumerated in peripheral blood of HCC patients. CD80, CD83, CD86 and HLA-DR expression on naive and stimulated myeloid dendritic cells from peripheral blood were analyzed. Myeloid dendritic cells were isolated from peripheral blood and their function was tested. Phagocytosis was analyzed using FITC-dextran beads, peptide specific stimulation, the capacity to stimulate allogeneic T cells and secretion of cytokines upon poly dI:dC was tested. RESULTS: Myeloid dendritic cells were reduced in patients with HCC. No differences in CD80, CD83, CD86 and HLA-DR expression were found on naive and stimulated myeloid dendritic cells from HCC patients and healthy controls. Normal phagocytosis or stimulation of peptide specific T cells was observed in contrast to an impaired allo-stimulatory capacity and a reduced IL-12 secretion. CONCLUSION: Impaired IL-12 production of mDCs in patients could lead to an impaired stimulatory capacity of naive T cells suggesting that IL-12 directed therapies may enhance tumor specific immune responses in HCC patients.     

慢性乙型肝炎患者树突状细胞功能与单个核细胞乙型肝炎病毒共价闭合环状DNA的关系

目的 探讨慢性乙型肝炎(CHB)患者外周血单个核细胞(PBMC)及树突状细胞(DC)内HBV共价闭合环状DNA(HBV cccDNA)的存在状况,DC成熟度及功能状态与DC或PBMC中HBV cccDNA载量的关系.方法 分离29例CHB患者和10例健康对照者的PBMC,用重组人粒细胞-巨噬细胞集落刺激因子(GM-CS...     

Reduced expression and functional impairment of Toll-like receptor 2 on dendritic cells in chronic hepatitis C virus infection.

BACKGROUND: Dendritic cells (DCs) utilize Toll-like receptors (TLRs) to sense virus and initiate immune responses. We aimed at elucidating the roles of TLRs on DCs in hepatitis C virus (HCV) infection. METHODS: Monocyte-derived DCs were obtained from 32 healthy volunteers (HV) and 30 chronically HCV-infected patients (CH). TLR2, TLR3 and TLR4 expressions on immature DCs were quantified by real-time quantitative RT-PCR. We stimulated DCs with specific TLR ligands and examined DC maturation, cytokine production and ability to stimulate allogeneic CD4(+) T cells. RESULTS: TLR2 expression on immature DCs was lower in the CH group, whereas those of TLR3 or TLR4 were not different between the groups. Each TLR ligand induced DC maturation and stimulated them to release comparable levels of IL-12p70, IL-6, IL-10, TNF-alpha and IFN-beta between the groups. TLR2 and TLR4 ligands enhanced DC ability to stimulate T cell proliferation, with the degree due to the TLR2 ligand being lower in the CH group. CONCLUSIONS: In HCV infection, the TLR2 expression on DCs is reduced and TLR2-stimulated DCs show lesser ability to proliferate T cells than healthy counterparts, suggesting that the TLR2 system is involved in HCV-induced immunopathogenesis.     

Effects of lamivudine on the function of dendritic cells derived from patients with chronic hepatitis B virus infection

AIM: To investigate if the nucleoside analogue lamivudine (LAM), a potent inhibitor of HBV replication, could restore the function of dendritic cells derived from patients with chronic hepatitis B (CHB) in an Asian population. METHODS: Dendritic cells (DCs) derived from mononuclearcytes of patients with chronic HBV infection were cultured in the presence of IL-4, granulocyte-macrophage colony-stimulating factors (GM-CSF) and gradient concentrations of LAM (0-2 mmol/L). Cell morphology was observed under light microscopy. Cell surface molecules, including HLA-DR, CD80, CD83, and CD1α, were analyzed with flow cytometry. The concentrations of IL-6 and IL-12 in the supernatant were assayed by ELISA. T cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT). RESULTS: The expression of CD1α on DC treated with 0.5 mmol/L LAM (LAM-DC 0.5 mmol/L) was significantly higher than that of DC untreated with LAM (54.1 ± 4.21 vs 33.57 ± 3.14, P < 0.05), and so was the expression of CD83 (20.24 ± 2.51 vs 12.83 ± 2.12, P < 0.05) as well as the expression of HLA-DR (74.5 ± 5.16 vs 52.8 ± 2.51, P < 0.05). Compared with control group, LAM-DC group (0.5 mmol/L) secreted significantly more IL-12 (910 ± 91.5 vs 268 ± 34.3 pg/mL, P < 0.05), had lower levels of IL-6 in the culture supernatant (28 ± 2.6 vs 55 ± 7.36 pg/mL, P < 0.05), markedly enhanced the stimulatory capacity in the allogeneic mixed leukocyte reaction (MLR) (1.87 ± 0.6 vs 1.24 ± 0.51, P < 0.05).CONCLUSION: The lower expression of phenotypic molecules and impaired allogeneic mixed lymphocyte reaction function of dendritic cells derived from patients with HBV infection could be restored in vitro by incubation with LAM.     

CD+4 CD+25调节性T细胞抑制持续性丙型肝炎病毒感染患者CD+4T细胞反应

目的探讨CD+4 CD+25调节性T细胞(CD+4 CD+25Treg细胞)在持续性HCV感染患者CD+4 T细胞下调中的意义.方法流式细胞术检测慢性丙型肝炎患者外周血中CD+4 CD+25Treg细胞的数量以及细胞内因子的合成;与正常人或患者CD+4 CD-25 T细胞共同培养,检测其抑制功能;RT-PCR检测Foxp3的mRNA表达.结果 CD+4 CD+25Treg细胞约占慢性丙型肝炎患者外周血中CD+4 T细胞的(13.5±1.8)%,高于正常对照(5.3±0.8)% (P=0.004);主要合成IL-10,高表达Foxp3;CD+4 CD+25Treg细胞显著抑制CD+4 T细胞的增殖,以及合成IFNγ,并且抑制活性较正常人增高(P=0.034),这种作用不依赖IL-10和转化生长因子β.结论持续性HCV感染患者CD+4 CD+25Treg细胞表达增加,抑制活性增强,特异性抑制Th1反应.     

慢性丙型肝炎患者树突状细胞功能的研究

目的　探讨树突状细胞 (DC)与慢性丙型肝炎患者体内病毒持续感染的关系。方法　从外周血分离单核细胞 ,用含粒细胞、巨噬细胞集落刺激因子 (GM CSF) ,白细胞介素 (IL) 4的AIM V无血清培养基诱导培养DC。观察DC外部形态和超微结构 ,检测DC表型 ,进行混合淋巴细胞反应(MLR) ,检测DC诱导同种异体静止T细胞增殖的能力和上清中细胞因子水平。结果　实验组DCCD86表达率为 (5 2 4± 13 3) % ,明显低于对照组的 (83 7± 15 8) % (P <0 0 1) ;实验组DC诱导同种异体静止T细胞增殖的能力每分钟液闪计数值 (cpm)为 192 4 5± 2 788,明显低于对照组的 2 6 5 2 9± 32 18(P <0 0 1) ;实验组MLR中IL 12p40 平均值为 (40 4± 10 3)ng/L ,干扰素γ平均值为 (7891± 82 1)ng/L ,分别低于对照组 (80 2± 2 0 5 )ng/L和 (13490± 15 5 4 )ng/L(P <0 0 0 5 )。结论　慢性丙型肝炎患者DC功能低下 ,提示与HCV持续感染有关。     

过敏性哮喘患者树突细胞表型及分泌细胞因子的研究

目的观察过敏性哮喘患者树突细胞(DC)表达表面分子(CD1a、CD83、CD40、CD86)和分泌细胞因子(IL12和IL10)的情况,及对原始T细胞分化的影响。方法分别取过敏性哮喘患者(9例)和健康对照者(14例)外周血培养成熟DC。另取无哮喘家族史的新生儿脐血分离得到原始T细胞。将2组DC和原始T细胞共同培养。流式细胞仪测DC表面协同刺激分子CD1a、CD83、CD40、CD86的表达。ELISA测DC分泌的IL12、IL10及T细胞分泌的IFNγ、IL4的含量。结果哮喘组DC表达CD86分子比对照组显著升高(P<001)。哮喘组DC分泌IL12、IL12p40和IL10较对照组显著减少(P<001,P<005)。哮喘组T细胞释放IFNγ较对照组减少(P<005),释放IL4较对照组显著增多(P<001)。哮喘组IL12与IFNγ呈正相关(r=0758,P<005),与IL4呈负相关(r=-0756,P<005);IL10与IL4呈负相关(r=-0685,P<005);IL12与IL10呈正相关(r=0926,P<001)。结论过敏性哮喘患者DC存在缺陷,使原始T细胞向Th2优势分化,IL4等的释放增加,且不能有效地形成T细胞耐受,共同导致过敏性哮喘的发生。