负载HBcAg对慢性HBV感染患者树突状细胞的活化作用及功能影响

目的:探讨免疫耐受期慢性乙型肝炎(CHB)患者外周血树突状细胞(DC)负载HBcAg后细胞表型及免疫功能的改变。方法:从CHB免疫耐受期患者外周血分离培养DC,在DC成熟前,加入重组HBcAg表位肽,诱导HBV特异性DC分化成熟,流式细胞仪检测DC表面共刺激分子CD1a、CD80、CD83的表达水平,应用淋巴细胞增殖试验评估DC功能。结果:负载不同剂量的HBcAg后,DC细胞活化增强,DC细胞共刺激分子标志物CD1a、CD80以及CD83表达率均明显上升,且随着抗原负载剂量的增加,表达率进一步增加,各组之间差异具有统计学意义(均P<0.001);未负载HBcAg的DC细胞激活的淋巴细胞反应较弱,负载HBcAg的DC细胞刺激同种异体健康成人T淋巴细胞增殖能力增强,且随着负载抗原剂量的加大,T淋巴细胞的增殖能力进一步提高,与未负载抗原的对照组相比,差异具有统计学意义(F=428.14,P=0.000)。结论:体外负载HBcAg刺激CHB患者DC细胞可增强其有效抗原提呈能力,促进T淋巴细胞增殖。     

慢性乙型肝炎患者外周血树突状细胞HBV感染状态研究初报

有研究发现,树突状细胞(DC)的功能低下是引起慢性HBV感染的关键因素之一,治疗前DC功能状态对疫苗和细胞因子等治疗慢性HBv感染的效果有明显影响,我们通过在慢性HBv感染者PBMC中诱导分化DC,免疫组化法及原位杂交法检测DC内HBV标志(包括HBsAg、HBcAg和HBV DNA),以便为进一步研究DC的功能状态、导致HBV感染慢性化中DC     

慢性HBV感染者、健康人树突状细胞经HBsAg活化后的免疫效应差异

目的:研究慢性HBV感染者、健康人外周血树突状细胞(dendriticcells,DC)经HBsAg活化后的免疫功能的差异.方法:从慢性HBV感染者、健康人外周血中培养扩增DC和CIK,在DC成熟前加入纯的HBsAg刺激,并再与同一来源的CIK共同培养.用流式细胞仪检测DC、CIK表型,用ELISA法检测DC、CIK共培养上清液中的IL-12浓度,用CCK-8比色法测定DC诱导CIK对HepG2.2.15细胞的杀伤活性.结果:未经HBsAg致敏的健康人DC表面标志CDla、CD80及CD83明显高于慢性HBV感染者(P<0.05);经HBsAg致敏的健康人DC表面标志均高于慢性HBV感染者,差异具有统计学意义(P<0.01);慢性HBV感染者经HBsAg致敏的DC表面标志与未经HBsAg致敏无显著性差异.CIK细胞CD3、CD8、CD3、CD56的双阳性表达率,健康人HBsAg致敏者明显高于慢性HBV感染者及未经HBsAg致敏的健康人(P<0.01,P<0.05);慢性HBV感染组HBsAg致敏与未经HBsAg致敏者比较无显著性差异(P>0.05).HBsAg致敏DC诱导CIK对HepG2.2.15细胞的杀伤率,健康人显著高于慢性HBV感染者(P<0.01).健康人HBsAg致敏DC、CIK共培养上清液中的IL-12浓度显著高于慢性HBV感染者(P<0.001);慢性HBV感染者HBsAg致敏与未经HBsAg致敏IL-12含量差异不显著(P>0.05).结论:慢性HBV感染者、健康人DC经HBsAg活化后对HepG2.2.15细胞的免疫效应存在显著差异:健康人高于慢性HBV感染者.     

HBsAg、HBcAg活化树突状细胞治疗慢性乙型肝炎的体外研究

目的 研究慢性乙型肝炎患者外周血树突状细胞(DC)经HBsAg、HBcAg活化后的免疫功能.方法 从慢性乙型肝炎患者外周血中培养扩增DC,在DC成熟前,加入纯的HBsAg、HBcAg刺激,用流式细胞仪检测DC表型,用液闪计数仪观察DC对T细胞的增殖作用,用ELISA法检测混合淋巴细胞反应(MLR)中细胞因子的分泌水平.结果 经HBcAg刺激DC的CD86表达率为(92.14±5.12)%,明显高于HBsAg刺激组和未加抗原组(P＜0.01);经HBcAg刺激组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34259±3127,明显高于HBsAg刺激组(20258±2917)和单个核细胞组(3469±417),P＜0.01;经HBcAg刺激组DC MLR中IL-12浓度为(342±42.3)ng/L,分别高于HBsAg刺激组和未加抗原组(P＜0.01).结论 体外经HBcAg刺激DC可有效提呈抗原病毒,并可进一步刺激T细胞产生.     

恩替卡韦对慢性乙型肝炎患者树突状细胞功能的体外影响

目的:研究恩替卡韦(ETV)对慢性乙型肝炎(CHB)患者外周血树突状细胞(dendritic cell,DC)功能的影响.方法:体外常规分离CHB患者及健康人外周血单个核细胞,诱导扩增后常规培养.第4天将其与一定浓度的恩替卡韦共培养,第8天收获DC进行细胞表型、同种异体混合淋巴细胞反应等相关检测.结果:细胞培养8 d时DC形态分化健康对照组优于CHB ETV处理组,CHB ETV处理组优于CHB组;CHB组CD1a(35.73±3.12 vs 62.31±5.22,P<0.01),CD80(28.19±1.64 vs 45.38±3.10,P<0.01),CD83(22.24±2.14 vs 40.63±7.21,P<0.01)及HLA-DR(36.74±0.98 vs 56.05±3.89,P<0.01)表达明显低于健康对照组,而ETV处理组与CHB组相比CD83 (27.41±9.23 vs 22.24±2.14,P<0.05),CD80(32.67±7.82 vs 28.19±1.64,P<0.05)及HLA-DR(40.84±5.57 vs 36.74±0.98,P<0.01)显著高表达;淋巴细胞增殖能力测定ETV处理组DC刺激同种异体T淋巴细胞增殖能力较CHB组增强(1.53±0.09 vs 1.45±0.12,P<0.05).结论:恩替卡韦作为治疗CHB的新一代核苷类药物,除了直接抑制乙肝病毒DNA合成外,也能够增强CHB患者外周血DC的功能,通过调节机体的免疫系统发挥间接抗病毒作用.     

慢性HBV感染者树突状细胞体外经HBsAg活化后的免疫效应

目的研究慢性HBV感染者、健康人的外周血树突状细胞(DC)经HBsAg活化后免疫功能的差异。方法从慢性HBV感染组及健康组外周血中培养扩增(以HBsAg刺激)DC和细胞因子诱导的杀伤细胞(CIK)。用流式细胞仪检测DC、CIK的表型,用酶联免疫吸附法(ELISA)检测DC、CIK上清液中的白细胞介素-12(IL-12)的浓度,用CCK-8比色法测定DC共培养的CIK对HepG2.2.15细胞的杀伤活性。结果经HBsAg致敏的健康组DC表面标志CD1a、CD80、HLA-DR及CD83阳性率明显高于慢性HBV感染组,差异具有统计学意义(P<0.01);慢性HBV感染组中经HBsAg致敏的DC表面标志阳性率与未经HBsAg致敏者相比差异无统计学意义(P>0.05)。健康组DC经HBsAg致敏后诱导的CIK对HepG2.2.15细胞的杀伤率显著高于慢性HBV感染组(P<0.01)。结论慢性HBV感染组与健康组DC经HBsAg活化后,对HepG2.2.15细胞的免疫效应差异有统计学意义,健康组高于慢性HBV感染组。     

高复制型慢性乙型肝炎患者肝内HBV复制指标与肝脏病变的关系

33例均为HBsAg、HBeAg及抗HBc三项阳性(部分伴HBcAg、DNA P或HBV DNA阳性)。病理报告慢性小叶性肝炎6例,慢性持续性肝炎27例.应用Southern Blot法共查肝内HBV DNA25例,其中阳性13例(52％),包括游离型9例,游离十整合型2例,整合型2例,应用双桥PAP法共查HBsAg及HBcAg23例,其中HBsAg阳性15例(65.2％),HBcAg阳性11例(43.8％)。提示肝内HBV DNA及HBcAg阳性组各项旰脏病变检出率在50％以上,项目(7项)比阴性组(5项)为高,并与血中DNAP、HBV DNA复制指标及ALT异常率相一致,肝内HBV DNA游离型及游离十整合型各项肝脏病变检出率也比整合型为高。因而,慢乙肝肝脏病变持续存在,与乙肝病毒复制有关,抗病毒治疗应予重视。     

补肾清毒法对慢性乙型肝炎患者外周血树突状细胞表型和功能的影响

目的:观察研究慢性乙型肝炎(CHB)患者外周血树突状细胞(DC)表型及功能的变化,探讨补肾清毒法治疗CHB的作用机制。方法:筛选血清HBV DNA>105拷贝/mL且无其他肝脏疾病及自身免疫性疾病的CHB患者49例。其中补肾清毒法治疗组28例,核苷类似物治疗组21例,疗程均为24周。治疗前后均从患者外周血分离单个核细胞,体外诱导培养DC。用流式细胞仪检测DC表型HLA-DR、CD80、CD83、CD86的表达,MTT法检测DC的自体淋巴细胞反应,ELISA法测定IL-12、IL-10的水平。结果:CHB患者DC的扩增数量、速度低于正常人。CHB患者治疗后HLA-DR、CD80、CD83、CD86的表达均高于治疗前,补肾清毒法组HLA-DR、CD80、CD86表达率高于核苷类似物组,差异有统计学意义(P<0.05)。两组患者经治疗后,DC自体混合淋巴反应中刺激能力较治疗前升高,但仍低于健康人水平,差异有统计学意义(P<0.05)。CHB患者和正常人DC上清液中IL-10的量,各组间差异无统计学意义。混合淋巴细胞反应上清液中IL-l2的量两组经治疗后,较治疗前升高,差异有统计学意义(P<0.05)。结论:补肾清毒法能改善DC的表型表达率和抗原递呈能力及其分泌功能,部分恢复CHB患者免疫功能。     

慢性乙型肝炎肝组织内HBsAg、HBcAg的表达及临床研究进展

一直以来临床将血清乙型肝炎e抗原(HBeAg)、乙肝病毒DNA(HBV DNA)阳性作为乙肝病毒复制的标志,随着肝穿活检及抗病毒治疗的研究进展,肝活检组织中乙肝表面抗原(HBsAg)和乙肝核心抗原(HBcAg)的表达模式与血清乙型肝炎病毒(HBV)DNA定量、肝组织炎症活动度分级及纤维化分期之间关系的临床研究日益增多,本文就HBsAg和HBcAg在肝组织的表达模式及临床研究进展综述如下.     

HBsAg和HBcAg联合负载DC与CIK共培养细胞体系的建立和功能评估

目的观察在不同HBV抗原负载下诱导的树突状细胞(DC)同细胞因子诱导的杀伤细胞(CIK)共培养后CIK的功能。方法将2015年1月-2016年6月在南京军区福州总医院就诊的13例慢性乙型肝炎患者作为研究对象,分离13例慢性乙型肝炎患者的外周血单个核细胞,并培养DC和CIK,设为CIK单独培养组、DC+CIK共培养组、DC+CIK+HBsAg共培养组、DC+CIK+HBcAg共培养组、DC+CIK+HBsAg+HBcAg共培养组,培养完成后,用ELISPOT方法测定CIK产生IFNγ的能力,用HepG2.2.15细胞作为靶细胞,测定CIK的杀伤功能。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果不同CIK试验组产IFNγ的水平差异有统计学意义(F=29.84,P0.001),其中以DC+CIK+HBsAg+HBcAg组产生的IFNγ水平最高;CIK杀伤率组间比较差异有统计学意义(F=14.77,P0.001),其中以DC+CIK+HBsAg+HBcAg最为突出。结论 HBsAg和HBcAg联合负载的DC与CIK共培养可能是治疗慢性乙型肝炎有效的细胞体系。     

Restoration in vitro of impaired T-cell responses in patients with chronic hepatitis B by autologous dendritic cells loaded with hepatitis B virus proteins (R2)

BACKGROUND: The purpose of the present paper was to investigate dendritic cell (DC) and T-cell functions in patients with chronic hepatitis B (CHB) and determine whether therapeutic DC vaccines could restore T-cell function in those patients in vitro. METHODS: Twelve patients with CHB and 10 normal control subjects with positivity for antibodies to hepatitis B surface and core antigens (anti-HBs and anti-HBc positivity) were enrolled in the present study. Phenotype analysis and allogeneic mixed lymphocyte reaction assay of DC from CHB patients and normal controls were made in the absence or presence of a cocktail of cytokines: interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)), IL-6 and tumor necrosis factor-alpha (TNF-alpha). Autologous T-cell proliferation assays and the enzyme-linked immunospot (ELISPOT) method for detecting interferon-gamma (IFN-gamma)-producing CD8(+) T cells were used to evaluate the efficacy of DC loaded in vitro with HBsAg or HBcAg. RESULTS: The DC from CHB patients had a lower expression of costimulatory molecules CD80, CD86 and impaired allogeneic mixed lymphocyte reaction capacity compared to those from normal controls. However, the impaired DC function could be restored partially by cytokine cocktail supplemented in vitro. Mature DC loaded with HBsAg or HBcAg showed a greater capacity for autologous T-cell proliferation and antigen-specific IFN-gamma production than immature DC. Moreover, as a DC -loading antigen, HBcAg was more immunogenic than HBsAg. CONCLUSIONS: The impaired function of DC in patients with CHB may be restored by supplementation in vitro with a cocktail of cytokines, and therapeutic DC vaccines might be effective to treat CHB infection in humans.     

Therapeutic potential of a combined hepatitis B virus surface and core antigen vaccine in patients with chronic hepatitis B

Purpose

The safety and clinical efficacy of a vaccine containing both hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) (HBsAg/HBcAg) were evaluated in patients with chronic hepatitis B (CHB).

Methods

Eighteen patients with CHB were administered a vaccine containing 100 μg of HBsAg and 100 μg of HBcAg. The vaccine was administered ten times at 2-weekly intervals, the first five times via the nasal route only and the subsequent five times via both nasal and subcutaneous routes. The safety and efficacy of this therapeutic approach were assessed by periodic assessment of the patients’ general condition, viral kinetics, and biochemical parameters during treatment and 24 and 48 weeks after therapy. The production of cytokines by peripheral blood mononuclear cells (PBMC) and antigen-pulsed dendritic cells (DC) was evaluated to assess the immunomodulatory effects of the HBsAg/HBcAg vaccine in CHB patients.

Results

The HBsAg/HBcAg vaccine was safe in all patients. No flare of HBV DNA or alanine aminotransferase (ALT) was recorded in any patient. Sustained HBV DNA negativity and persistently normalized ALT were detected in 9 (50 %) and 18 (100 %) patients with CHB, respectively. PBMC and HBsAg/HBcAg-pulsed DCs from HBsAg/HBcAg-vaccinated CHB patients produced significantly higher levels of various cytokines [interleukin 1β (IL-1β), IL-6, IL-8, IL-12, and tumor necrosis factor α (TNF-α)] than those from control unvaccinated CHB patients (p < 0.05) after stimulation with HBsAg/HBcAg in vitro.

Conclusion

HBsAg/HBcAg vaccine seems a safe and efficient therapeutic approach for patients with CHB.     

Chronic hepatitis B: The interplay between intrahepatic lymphocyte population and viral antigens in relation to liver damage

In Chronic hepatitis B (CHB) infection, virus and immune response interplay is thought to be responsible for pathogenesis. Yet, the impact of each immune cell population and viral protein expression in liver damage is still unknown. Our aim was to study the interplay between intrahepatic immune response and viral activity in relation to CHB liver damage. Immunostaining was performed in 29 liver biopsies from untreated CHB patients to characterize liver infiltrate [Th (CD4+), CTL (CD8+), Treg (FoxP3+), Th17 (IL‐17A+) and Th1 (T‐bet+)] and viral antigen expression (HBsAg and HBcAg). Inflammatory activity and fibrosis were assessed using the HAI and METAVIR scoring system. All studied populations were identified in the portal‐periportal (P‐P) areas with a CD4+ lymphocyte predominance, while only CD8+ and FoxP3+ cells were observed in the intralobular area. Both P‐P CD4+ and intralobular CD8+ cell frequencies were increased among severe hepatitis cases. Concerning HBsAg and HBcAg expression, a mutually exclusive pattern was observed. HBcAg was mainly detected among HBeAg‐positive patients and was associated with hepatitis severity and higher frequency of P‐P FoxP3+, intralobular CD8+ and FoxP3+ cells. HBsAg was identified among HBeAg‐negative cases with less severe hepatitis grade and lower frequency of P‐P CD4+ and intralobular FoxP3+ lymphocytes. In conclusion, the HBV antigen profile expression seen during CHB infection may be reflecting different stages of viral replication which impacts the host immune response and liver damage process. While HBcAg might be an inducer of a regulatory microenvironment, the intralobular CTL population seemed to have a key role in hepatitis severity.     

CpG寡核苷酸联合HBsAg对慢性乙型肝炎患者树突细胞功能及核因子活性的影响

目的 研究含CpG寡核苷酸(CpG-ODN)联合重组HBsAg对慢性乙型肝炎(CHB)患者外周血树突状细胞(DC)表型和功能及其对核因子-κB和激活蛋白-1活性的影响.方法 以重组细胞因子联合诱导扩增CHB患者外周血单个核细胞得到DC;以CpG-ODN和HBsAg及肿瘤坏死因子α刺激DC,评价其表型、功能和胞核核因子-κB、激活蛋白-1活性.结果 CpG-ODN联合HBsAg明显提高CHB患者DC表面分子人类白细胞抗原-DR表达、白细胞介素-12分泌以及刺激同种T淋巴细胞增殖的能力,尤其能提高CD1a的表达.上述免疫佐剂能增强DC细胞核因子κB活性,同时抑制激活蛋白-1活性.结论 CpG-ODN与hTNF α一样能够促进CHB患者外周血DC成熟;其作用机制可能是选择性激活或抑制DC胞内不同核因子活性.     

Entecavir up-regulates dendritic cell function in patients with chronic hepatitis B

AIM： To investigate the in vitro effect of entecavir （ETV on the function of dendritic cells （DCs） derived from chronic hepatitis B （CHB） patients. METHODS： Mononuclear cells were isolated from peripheral blood of patients with CHB. DCs wer incubated with RPMI-1640 medium supplemented wit fetal bovine serum, IL-4, granulocyte-macrophag colony-stimulating factor （GM-CSF）. DCs were treate with or without ETV on the fourth day. Cell surfac molecules, including CD1a, CD80, CD83 and HLA-DR were assessed by flow cytometry. Concentrations of IL- and IL-12 in the supernatant were assayed by enzyme linked immunosorbent assay （ELISA）. The ability of th generated DCs to stimulate lymphocyte proliferation wa observed. RESULTS： Compared with CHB control group, th expression levels of CD1a （29.07 ± 3.20 vs 26.85 ± 2.80 CD83 （25.66 ± 3.19 vs 23.21 ± 3.10）, CD80 （28.00 ± 2.7 vs 25.75 ± 2.51） and HLA-DR （41.96 ± 3.81 vs 32.20 ± 3.04） in ETV-treated group were higher （P 〈 0.05）. ETV treated group secreted significantly more IL-12 （157.6 ± 26.85 pg/mL vs 132.60 ± 22.00 pg/mL （P 〈 0.05） an had a lower level of IL-6 in the culture supernatant （83.0 ± 13.88 pg/mL vs 93.60 ± 13.61 pg/mL, P 〈 0.05） tha CHB control group. The ability of DCs to stimulate th proliferation of allogeneic lymphocytes was increase in ETV-treated group compared with CHB control grou （1.53 ± 0.09 vs 1.42 ± 0.08, P 〈 0.05）.CONCLUSION： Entecavir can enhance the biological activity of DCs derived from CHB patients.     

慢性乙型肝炎患者肠源性内毒素血症与树突状细胞表型及功能的关系

目的 探讨慢性乙型肝炎(CHB)患者树突状细胞(DC)与肠源性内毒素血症(IETM)的关系.方法 CHB患者80例,健康对照者21例,采集外周血,测定血浆内毒素含量、ALT、TBil.根据血浆内毒素水平,将患者分为内毒素阳性组和阴性组.同时用重组人粒细胞巨噬细胞集落刺激因子、重组人白细胞介素-4、酪氨酸激酶受体3配体和TNF-a体外诱导、培养CHB患者DC,采用流式细胞仪检测DC表型,混合淋巴细胞反应检测DC刺激T淋巴细胞的能力,用ELISA检测DC分泌细胞因子的水平.多组间比较采用单因素方差分析.结果 CHB患者DC表达CD83、CD80、CD86和人类白细胞抗原(HLA)-DR分子的水平及诱导同种异体混合T淋巴细胞增殖的能力均明显低于健康对照组.内毒素阳性组患者表达CD83、CD80、CD86、HLA-DR水平及诱导T淋巴细胞增殖的能力分别为(8.25±3.63)%、(10.63±4.52)%、(36.61±16.16)%、(61.65±14.33)%、0.812±0.311,明显低于内毒素阴性组的(11.39±4.35)%、(13.56±5.13)%、(45.90±15.35)%、(70.35±18.89)%、1.153±0.324(F=5.123、4.213、3.714、3.323、3.125,均P＜0.05).培养至第9天,CHB患者DC分泌IL-12和IFN-γ分别为(16.99±6.74)pg/mL和(10.52±4.19)pg/mL,明显低于健康者的(44.51±14.56)pg/mL和(17.94±5.86)pg/mL.内毒素阳性组患者IL-12水平为(13.14±5.71)pg/mL,明显低于内毒素阴性组的(20.98±9.03)pg/mL(F=3.225,P=0.016).IFN-γ水平在内毒素阳性组为(9.46±3.24)pg/mL,与阴性组的(11.54±5.20)pg/mL比较,差异无统计学意义(F=2.003,P=0.076).结论 IETM是导致CHB患者体内DC功能异常的原因之一.

Abstract：

Objective To investigate the relationship between dendritic cell (DC)and intestinal endotoxemia in patients with chronic hepatitis B (CHB).Methods Peripheral blood were collected from CHB patients (n = 80)and healthy controls (n = 21 ).Plasma endotoxin (ET)levels,liver function (alanine transaminase,total bilirubin)were detected.According to plasma ET concentration,all CHB patients were divided into two groups:ET positive and ET negative.The peripheral blood mononuclear cells (PBMCs)were isolated and then cultured with recombinant human granulocyte-macrophage colony-stimulating factor ( rhGM-CSF),recombinant human interleukin-4 ( rhIL-4 ),FMS-related tyrosine kinase 3 ligand (Flt3L)and tumor necrosis factor-alpha (TNF-α)to derive DC.The phenotypic patterns were characterized by flow cytometry.The proliferation of T lymphocytes was evaluated with mixed leukocytes reaction (MLR)and the levels of IL-12 and interferon-γ (IFN-γ)produced by DC were analyzed with enzyme-linked immunosorbent assay (ELISA).Comparisons among the two groups and healthy control group were done by single factor analysis of variance.Results Compared to healthy controls,the expressions of CD83,CD80,CD86,human leucocyte antigen (HLA)-DR and the proliferation of allogeneic T lymphocytes by DC were all significantly reduced in CHB patient groups.The expressions of CD83,CD80,CD86,HLA-DR and the activation of proliferation in ET positive subjects were lower than those in ET negative subjects [CD83 (8.25±3.63)% vs(11.39±4.35)% ,CD80 (10.63±4.52)% vs (13.56±5.13)%,CD86 (36.61±16.16)% vs (45.90±15.35)%,HLA-DR (61.65±14.33)% vs (70.35±18.89)%,the activation of proliferation0.812±0.311 vs 1.153±0.324; F=5.123,4.213,3.714,3.323 and 3.125,respectively; all P＜0.05].After cultured for 9 days,the secretions of IL-12 and IFN-γ by DC were significantly lower in CHB patients than in healthy controls [IL-12 (16.99± 6.74)pg/mL vs (44.51±14.56)pg/mL,IFN-γ (10.52±4.19)pg/mL vs (17.94±5.86)pg/mL].The level of IL-12 in the ET positive group was significantly lower than that ET negative group [( 13.14 ±5.71)pg/mL vs (20.98 ± 9.03)pg/mL; F= 3.225,P = 0.016].The level of IFN-γ was not different between two groups [(9.46 ± 3.24)pg/mL vs (11.54 ± 5.20)pg/mL; F = 2.003,P =0.076].Conclusion The intestinal endotoxemia may play a role in DC dysfunction in CHB patients.     

不同病毒载量的慢性乙型肝炎患者外周血树突状细胞的表型和功能

目的观察研究不同HBV DNA载量的CHB患者外周血DC表型及功能变化,探讨HBV在DC成熟障碍中的作用机制。方法筛选血清HBV DNA>10~5拷贝/ml且无其他肝脏疾病及自身免疫性疾病的CHB患者28例。所有病例予核苷类似物抗病毒治疗24周。治疗前后测定外周血HBV DNA并经肝穿刺明确肝组织病理状态。将治疗前患者设为高载量组(HBV DNA>10~5拷贝/ml),共28例;治疗后患者设为低载量组(HBV DNA<10~3拷贝/ml),共25例。从患者外周血分离单核细胞,体外诱导培养DC。用流式细胞仪测定DC表型,MTT法测定DC对同种异体淋巴细胞的刺激增殖作用,ELISA法测定DC分泌IL-12和IL-10的量。同时以10名健康人作对照。结果DC在体外经细胞因子的刺激可明显增殖,但CHB患者DC的扩增数量、速度低于正常人。DC表面标记:正常人的HLA-DR、CD86、CD80和CD83表达阳性率均大于80%,显著高于两组CHB患者;不同HBV DNA载量的两组CHB患者间差异无统计学意义。两组CHB患者的DE在混合淋巴细胞反应中的刺激能力差异无统计学意义,但明显低于正常对照组。CHB患者和正常人DC上清液中IL-10的量,各组间差异无统计学意义;高载量组与低载量组纯DC培养和混合淋巴细胞反应上清液中IL-12量的差异无统计学意义,而分别明显低于正常人DC。结论在HBV持续感染期间,CHB患者DC的表型变化及功能下调与外周血HBV DNA载量间差异无统计学意义。     

Immunohistochemical investigation of hepatitis B virus associated antigens,HLA antigens and lymphocyte subsets in type B chronic hepatitis

HLA antigens, hepatitis B virus (HBV)-assodated antigens and lymphocyte subsets in liver tissue from 35 patients with HBs antigenemia were studied using an immunoperoxidase double staining method arid immunoelectron microscopy in order to clarify the immune mechanism of hepatocyte lysis in type B hepatitis. Immune light and electron microscopy using monoclonal antibodies to lymphocyte subsets revealed that infiltrating lymphocytes in the areas of piecemeal necrosis and focal necrosis were predominantly CD8-positive, showing direct contact with hepatocytes. In contrast, CD4(+) cells were infrequently observed in necrotizing inflammatory lesions. HLA-A,B,C antigens were mainly found on hepatocytes in areas of piecemeal necrosis and focal necrosis, in association with CD8(+) lymphocyte infiltration. HLA-DR antigens were demonstrated on a few hepatocytes in the same lesions. In cases of CAH with serum HBeAg positive, HLA-A,B,C, antigens and HBV antigens simultaneously demonstrated on the same hepatocytes. Especially, hepatocytes expressing both HLA-A,B,C antigen and HBsAg on the plasma membrane showed direct contact with CD8(+) lymphocytes. This finding fullfilled the morphological requirements for HBsAg as a target antigen. On the other hand, HBcAg was hardly demonstrated in the liver cell membrane but was demonstrated mainly in the cytoplasm. Compared with the nuclear localization of HBcAg in cases of NSR, cytoplasmic localization of this antigen may be associated with membranous expression of new antigens induced by HBV infection.     

Immunohistochemical investigation of hepatitis B virus associated antigens, HLA antigens and lymphocyte subsets in type B chronic hepatitis

HLA antigens, hepatitis B virus (HBV)-associated antigens and lymphocyte subsets in liver tissue from 35 patients with HBs antigenemia were studied using an immunoperoxidase double staining method and immunoelectron microscopy in order to clarify the immune mechanism of hepatocyte lysis in type B hepatitis. Immune light and electron microscopy using monoclonal antibodies to lymphocyte subsets revealed that infiltrating lymphocytes in the areas of piecemeal necrosis and focal necrosis were predominantly CD8-positive, showing direct contact with hepatocytes. In contrast, CD4(+) cells were infrequently observed in necrotizing inflammatory lesions. HLA-A,B,C antigens were mainly found on hepatocytes in areas of piecemeal necrosis and focal necrosis, in association with CD8(+) lymphocyte infiltration. HLA-DR antigens were demonstrated on a few hepatocytes in the same lesions. In cases of CAH with serum HBeAg positive, HLA-A,B,C, antigens and HBV antigens simultaneously demonstrated on the same hepatocytes. Especially, hepatocytes expressing both HLA-A,B,C antigen and HBsAg on the plasma membrane showed direct contact with CD8(+)lymphocytes. This finding fulfilled the morphological requirements for HBsAg as a target antigen. On the other hand, HBcAg was hardly demonstrated in the liver cell membrane but was demonstrated mainly in the cytoplasm. Compared with the nuclear localization of HBcAg in cases of NSR, cytoplasmic localization of this antigen may be associated with membranous expression of new antigens induced by HBV infection.     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.