抗病毒治疗对CHB患者外周血TCRβ链CDR3谱系的影响

目的:分析慢性乙型肝炎(CHB)患者抗病毒治疗前后T淋巴细胞受体(TCR)β链各家族互补决定区3(CDR3)谱系的变化,了解抗病毒治疗前后T细胞应答的变化。方法:采用荧光定量PCR(FQ-PCR)溶解曲线法对11例CHB患者抗病毒治疗前后外周血单个核细胞(PBMC)T淋巴细胞受体β链可变区(TCR BV)基因各家族CDR3谱系进行分析,了解抗病毒治疗前后T细胞应答的变化。分析TCR CDR3谱系与CHB患者血清病毒载量的关系。结果:11例CHB患者抗病毒治疗后多个TCR CDR3谱系均出现了不同程度偏移。低病毒载量组(HBV DNA≤104拷贝/ml)治疗后谱型偏移率显著高于高病毒载量组(HBV DNA≥105拷贝/ml)。结论:CHB患者抗病毒治疗后单寡谱型偏移率与治疗前血清HBV DNA的水平相关。     

慢性乙型肝炎患者抗病毒治疗前后HBeAg特异性T细胞应答特征研究

目的研究慢性乙型肝炎(CHB)患者抗病毒治疗前后T细胞对HBV特异性抗原蛋白(HBeAg)的免疫应答特征。方法分别收集22例慢性乙型肝炎患者抗病毒治疗前、治疗后3月、6月和12月的外周血,以HBeAg蛋白刺激外周血单个核细胞,采用酶联免疫斑点技术(ELISPOT)检测产生IFN-γ的HBeAg特异性T细胞的频率和强度。结果 1)CHB患者抗病毒治疗前和治疗后3月、6月、12月总的T细胞反应频率分别为31.8%(7/22)、50.0%(11/22)、77.3%(17/22)和95.5%(21/22),治疗后12月反应频率明显高于治疗前和治疗后3月(P0.05),治疗后6月反应频率明显高于治疗前(P0.05),核苷类似物治疗组与干扰素治疗组治疗后T细胞反应频率均无明显差异(P0.05)。2)治疗后12月T细胞的反应强度明显高于治疗前、治疗后3月及治疗后6月(P0.05)。核苷类似物治疗组与干扰素治疗组均分别得出上述相同的结果。3)将患者T细胞的反应强度与HBV DNA载量(取对数值表示)做Spearman秩相关分析,rs=-0.1860,P=0.041,P0.05,认为患者T细胞对HBeAg蛋白的反应强度与HBV DNA载量存在负相关关系。结论 HBeAg可以刺激CHB患者产生特异性T细胞应答,且随着抗病毒治疗时间的延长,患者HBeAg特异性T细胞的应答频率和应答强度均有所增强,且与血清HBV DNA水平呈负相关关系。     

CHB患者CD4+ T细胞ICOS表达及其与干扰素治疗的相互关系

目的 研究慢性乙型肝炎(CHB)患者经干扰素治疗前后外周血CD4+T细胞上可诱导共刺激分子( inducible costimulator,ICOS)表达水平的变化情况.方法 CHB患者56例,其中聚乙二醇(PEG)干扰素α2a治疗28例,拉米夫定治疗28例,以健康志愿者20例为正常对照组.采集两治疗组治疗前及治疗后24、48周的患者外周血,以流式细胞技术检测ICOS+ CD4+T细胞在外周血单个核细胞(peripheral blood mononuclear cells,PBMC)中的频数变化;采用real-time PCR法检测患者HBV DNA载量变化.结果 CHB患者CD4+T细胞ICOS表达水平明显高于正常对照者(P＜0.001).干扰素治疗后患者ICOS表达水平下降,同拉米夫定组差异有统计学意义(P＜0.05).干扰素治疗后患者ICOS改变值同HBV DNA载量变化值呈正相关(r=0.972,P＜0.001),而拉米夫定组则未发现相关性(r=-0.101,P=0.608).结论 CHB患者存在着细胞免疫紊乱,其CD4+T细胞ICOS的表达升高.干扰素可下调CD4+T细胞ICOS的表达,纠正细胞免疫偏移,发挥抗HBV作用.     

拉米夫定对慢性乙型肝炎患者CTL活性的影响

目的 探讨拉米夫定治疗是否会影响慢性乙型肝炎患者（CHB）HBV特异性细胞毒T淋巴细胞（CTL）活性。方法 选择34例HLA-A2 CHB患者，其中19例患者行干扰素-a治疗，另15例患者用拉米夫定治疗。在治疗前和治疗后1、3、6个月后分离外周血单核细胞（PBMC），经体外诱导扩增，以HepG2.2.15细胞和HepG2细胞作靶细胞，乳酸脱氢酶释放法检测其细胞毒活性。结果 在接受干扰素-a或拉米夫定治疗后，CHB患者HBV特异性CTL活性均能得到不同程度的恢复。两种治疗方法比较显示，干扰素-a治疗组CTL活性增强持续的时间较使用拉米夫定的治疗组长。结论 拉米夫定治疗能短暂地增强CHB患者HBV特异性CTL活性。     

慢性乙型肝炎患者抗病毒治疗前后体内T FH细胞的动态变化

 目的:探讨外周血滤泡辅助性T细胞（follicular helper T cells,T FH）及白细胞介素21(interleukin-21, IL-21)的表达在慢性乙型肝炎(chronic hepatitis B,CHB)患者抗病毒治疗前后的变化。方法:抗病毒治疗前后的CHB患者35例（干扰素治疗21 例,替比夫定治疗14例）,取其外周血密度梯度离心法分离外周血单个核细胞（PBMC）,利用流式细胞术动态观察CD4+CXCR5+　T FH细胞及其胞内IL-21的表达水平。结果:外周血T淋巴细胞内IL-21主要来源于T FH细胞的表达,不同抗病毒药物治疗24周对T FH细胞影响无显著差异(P>0.05)。治疗前T FH细胞及其胞内IL-21表达水平在48周病毒学应答（virological response,VR）组显著高于48周病毒学应答不佳（no virological response, NVR）组。NVR组抗病毒治疗后T FH细胞水平呈上升趋势。结论:抗病毒治疗可使应答不佳患者外周血T FH细胞水平升高;治疗前T FH细胞及其胞内IL-21的高表达有助于预测CHB患者更早实现病毒学应答。     

抗病毒治疗对慢性乙型肝炎肝组织HBV DNA及病理的影响

目的 探讨抗病毒治疗对慢乙肝肝组织HBV DNA及病理的影响.方法 75例HBeAg阳性的慢乙肝患者分别接受拉米夫定-干扰素序贯治疗24例、拉米夫定35例或干扰素α2b16例,抗病毒治疗,检测治疗前48周患者肝组织HBV DNA及炎症指数(HAI).结果 抗病毒治疗48周,三组患者血、肝组织HBV DNA及HAI均值明显下降(P＜0.05),序贯治疗组HBeAg血清转换率(38.1%)稍高于其他两组(P=0.1352).HBeAg血清转化患者(17例)和HBV DNA阴转患者(21例)肝组织HBVDNA基线值明显低于其他患者(P＜0.05).结论 抗病毒治疗可以抑制肝组织HBV的复制,改善肝细胞的炎症坏死;肝组织HBV DNA水平低者抗病毒疗效好.序贯治疗可能获得较高的HBeAg血清转化率.     

替比夫定对慢性乙型肝炎患者外周血CD4~+ CD25~+ CD127~(low) T和CD8~+ CD25~+ T细胞频率的影响及临床意义

目的探讨核苷类药物替比夫定对慢性乙肝患者(CHB)外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例的影响,并结合临床指标分析其临床意义。方法替比夫定抗病毒治疗22例CHB患者,在治疗前及治疗后3,6个月时,分别以流式细胞仪检测外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例,实时荧光定量RT-PCR检测Foxp3 mRNA的表达水平,荧光定量PCR检测血清HBV DNA水平,酶联免疫吸附法检测HBV标志物,全自动生化分析仪检测ALT水平。结果 CHB患者外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例显著高于对照组。替比夫定治疗3个月时,这两群细胞比例显著下降,Foxp3 mRNA的表达也显著下降;HBV DNA水平降至检测水平以下的CHB患者,其CD4+CD25+CD127lowT细胞也降至正常水平。治疗3、6个月时,HBeAg阴转率分别为9.1%和18.2%,发生HBeAg血清学转换者的CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例均降至正常水平。结论替比夫定能快速有效抑制CHB患者的病毒复制...     

慢性乙型肝炎患者血清HBVDNA水平与滤泡辅助性T淋巴细胞的关系和意义CSCD

目的探讨慢性乙型肝炎（CHB）患者HBVDNA水平与外周血滤泡辅助性T淋巴细胞（Tfh）的关系和意义。方法179例HBVDNA阳性、HBeAg阳性、人白细胞抗原（HLA）一A2阳性的CHB患者,用实时荧光定量PCR检测HBVDNA,流式细胞术检测Tfh、HBV特异性CTL,并作IL一21的检测。将179例CHB患者根据HBVDNA水平分为甲、乙两组,甲组86例,HBVDNA水平为10^4。～10^5拷贝／ml,乙组93例,HBVDNA水平为10^6-10^7拷贝／ml,对两组患者进行以上检测指标的比较。结果甲组HBVDNA水平为（4．85±0．37）logl0拷贝／ml,乙组HBVDNA水平为（6．83±0．31）log。0拷贝／ml,t：27．31,P〈0．001,甲组Tfh（5．96±1．59）％,高于乙组（3．71±2．15）％,t=4．92,P〈0．01,IL．21（42．61±15．11）ng／L,高于乙组（14．91±3．15）ng／L,t=8．62,P〈0．01,HBV特异性CTL（0．36±0．08）％,高于乙组（0．18±0．06）％,t=19．99,P〈0．001。结论CHB患者血清HBVDNA水平与外周血Tfh水平有关：HBVDNA水平低者,Tfh水平高,IL-21水平和HBV特异性CTL水平也高。HBVDNA水平高者,Tfh水平低,IL-21水平和ItBV特异性CTL水平也低。基线HBVDNA水平影响抗病毒疗效的机制可能与Tfh水平有关。     

慢性乙型肝炎患者外周血NKG2A+NK 细胞与Treg 的关系研究

目的:探讨慢性乙型肝炎病毒(CHB)患者外周血NKG2A+NK 细胞与调节性T 细胞(Treg)的关系及临床意义。方法:收集46 例CHB 患者和17 例健康对照者外周血,采用实时荧光定量PCR 法检测血清HBV DNA;采用流式细胞术检测NKG2A+NK 细胞及Treg 的比例。结果:将CHB 患者分为Low HBV DNA 组(300 ~ 104 U/ ml)及High HBV DNA 组(105 ~108 U/ ml)。我们发现High HBV DNA 组ALT 明显高于Low HBV DNA 组(P<0.05)。High HBV DNA 组CD56dim NK 细胞及NKG2A+CD56dim NK 细胞的比例均分别明显高于Low HBV DNA 组(P<0.05)。且High HBV DNA 组Treg 明显高于Low HBVDNA 组和对照组(P<0.05)。此外,NKG2A+CD56dim NK 细胞的比例与High HBV DNA 载量及Treg 水平均呈正相关性(r =0.59,P<0.05;r =0.53,P<0.05)。结论:CHB 患者的NKG2A+CD56dim NK 细胞的水平与HBV 的免疫逃逸及疾病的进展相关。     

经NAs治疗显效慢乙肝患者PBMC中HBX基因检测及临床意义CSCD

目的 检测分析核苷（酸）类似物（Nucleoside analogues,NAs）治疗后血清HBV DNA 阴转的慢乙肝（Chronic hepatitis B,CHB）患者外周血单个核细胞（Peripheral blood mononuclear cell,PBMC）中HBX基因,研究其临床意义.方法 对60例经抗病毒治疗后血清HBV DNA阴转的慢乙肝患者,其中有部分病情进展为乙肝后肝硬化（HBV-related liver cirrhosis）及肝细胞性肝癌（Hepatocellular carcinoma,HCC）,采用实时荧光定量PCR检测其PBMC中HBX基因,并分析其序列特点及临床意义.结果 60例患者中PBMC样品检测出HBX基因片段的有37例,检出率61.67％（37/60）;三组人群中,HCC、乙肝后肝硬化患者PBMC中HBX基因检出率明显高于CHB患者（P=0.000,P=0.010）;通过直接测序法,在2例HCC患者及1例CHB患者中,检测到HBX蛋白突变体蛋白HBX△120、HBX△129、HBX△131截短体;发现A389T/G391A、T380C两个热点突变,且HCC患者中A389T/G391A突变发生率明显高于CHB患者（P=0.021）;对于HBeAg（-）的肝硬化、HCC患者,T380C突变发生率高于HBeAg（＋）患者（P =0.035）.结论 经NAs治疗后血清HBV DNA阴转的患者中,肝硬化、HCC患者的PBMC有更高的HBX检出率;HCC患者A389T/G391A双突变发生率高于CHB患者;HBeAg（-）的肝硬化、HCC患者T380C突变发生率高于HBeAg（＋）患者.     

慢性乙肝患者抗病毒治疗前后特异性T细胞免疫观察

目的 了解抗病毒治疗前后慢性乙型肝炎患者特异性T淋巴细胞对HBV抗原蛋白免疫应答的变化及其特征.方法 收集17例慢性乙型肝炎患者抗病毒治疗前及治疗后1个月、3个月的外周血单个核细胞,以HBV特异性抗原蛋白HBsAg、HBcAg和HBeAg为刺激物,酶联免疫斑点法检测其分泌IFN-γ产生斑点的情况.同时对血清HBV DNA和HBsAg、HBeAg等病毒学指标及谷丙转氨酶(ALT)等生化学指标进行榆测并分析其相关性.结果 治疗前,所有患者ALT、总胆红素(TBiL)均高于正常上限,17例患者HBV DNA均大于104拷贝/ml;治疗1个月后,ALT复常率为35.3%,9例患者HBV DNA降为检测下限以下;治疗3个月后,ALT复常率为58.8%,有11例患者HBV DNA降为检测下限以下.抗病毒治疗前、治疗1个月、治疗3个月患者针对HBV特异性蛋白总的T细胞反应阳性率分别为64.7%、76.5%和82.4%,其差别无统计学意义.不论治疗前后,患者对HBeAg的特异性T细胞反应频率和平均反应强度最高;治疗后,对3种蛋白的特异性T细胞反应频率和平均反应强度各有不同程度的增加,其中以对HBcAg蛋白的平均反应强度的增强最明显,治疗前和治疗3个月,治疗1个月和治疗3个月之间的差别都有统计学意义.患者对HBcAg蛋白的特异性T细胞反应平均反应强度与病毒载量有明娃负相关,与血清ALT无明显相关性.结论 本研究结果提示抗病毒治疗后,患者对HBV的特异性T细胞免疫应答有所增强,这种改变可能与HBV DNA的下降有关,检测HBV特异性T细胞反应对丁解患者的免疫状态有重要的意义.

Abstract：

Objective To explore the responses of antigen-specific T cells stimulated by hepatitis B virus(HBV)-specific proteins in chronic hepatitis B patients accepting antiviral therapy. Methods Seventeen patients with chronic hepatitis B (CHB) accepting antiviral therapy were included in this study. The peripheral blood monocular cell ( PBMC) were separated from the whole blood collected at the three different time of before and one and three months after accepting antiviral therapy. ELISPOT assay was used to detect the frequency and strength of secreting IFN-γ cells of PBMC stimulated by HBsAg, HBcAg and HBeAg. HBV virus loading, HBsAg, HBeAg, ALT and AST in serum were detected at the same time. Results After three months therapy, ALT, TBiL were improved in all patients, and HBV DNA level were dropped and undetectable in 11 cases. The rates of T cell response in patients to HBV specific proteins were 64. 7% , 76. 5% and 82. 4% at the time of before and one and three months after accepting antiviral therapy, respectively. The frequency of responses of antigen-specific T cells stimulated by HBcAg was higher than that stimulated by HBsAg or HBeAg, and the frequency was enhanced after antiviral therapy. The average response magnitude was expressed as spot forming cells (SFC) per million input cells. SFC of T cell responses to HBcAg was also higher than to HBsAg or HBeAg. There was no significant difference in SFC of T cell responses to HBsAg or HBeAg at the time of before and after antiviral therapy, but there were significant difference in SFC of T cell responses to HBcAg at the time of before and after antiviral therapy. SFC of T cell responses to HBcAg was negatively associated with HBV DNA, and no associated with level of ALT in serum. Conclusion The responses of antigen-specific T cells were improved in CHB patients accepting antiviral therapy which associated with the decrease of HBV DNA. It suggested to investigate HBV specific T cell responses was important.     

Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection

Antibodies directed to the HBs antigen indicate viral clearance and the development of life-long immunity in patients that recovered from HBV infection. In HBs antigen vaccine recipients anti-HBs antibodies provide protective immunity. However, little is known about the regulation of this HBs-specific antibody response. The existence of anti-HBs-secreting B cells was demonstrated using the highly sensitive ELISPOT technique compared with conventional ELISA in serum and cell culture supernatants. In the peripheral blood of patients with acute self-limited hepatitis B, HBs-specific B cells were demonstrated with a high frequency despite undetectable anti-HBs serum antibodies. HBV-immunized patients that had recovered from infection and vaccine recipients had significantly lower frequencies, whereas chronic HBV carriers and negative controls showed no anti-HBs-secreting B cells. Coculture experiments of isolated B and T cells revealed that the anti-HBs antibody response was restricted to the presence of T helper cells, but not to identical HLA class II molecules. Allogeneic T cells derived from vaccine recipients or chronic HBV carriers stimulated the HBs-specific B cell response in HBs vaccine recipients. Otherwise, isolated T helper cells could never provide sufficient help to induce the HBs-specific B cell response in chronic HBV carriers. Furthermore, peripheral blood mononuclear cells (PBMC) of six out of 10 vaccine recipients, one out of five HBV-immunized patients, but of no chronic HBV carrier showed a proliferative response to different HBs antigen preparations. This study demonstrated a high frequency of circulating anti-HBs-producing B cells in the early phase of acute HBV infection, but a lower frequency of HBs-specific B cells years after resolution of HBV infection. In chronic HBV carriers, however, deficient HBs-specific T and B cell responses were observed.     

Flow cytometry CD4+/CD8+ ratio of liver-derived lymphocytes correlates with viral replication in chronic hepatitis B.

T lymphocytes have been assumed to play an essential role in tissue injury in patients with chronic hepatitis B. As hepatitis B virus (HBV) is considered as a major factor controlling liver inflammation, we assessed whether a particular T lymphocyte subset could be preferentially detected in the liver in accordance with viral replication. Liver-derived lymphocytes and peripheral blood lymphocytes were analysed by flow cytometry in 21 patients with histologically confirmed chronic hepatitis B without cirrhosis. Viral replication was quantified by hybridization of serum HBV DNA. Eleven patients exhibited an active viral replication with serum HBV DNA ranging from 10 to 388 pg/ml at the time of the liver biopsy, whereas 10 patients had no detectable serum HBV DNA. In patients exhibiting viral replication, CD4+/CD8+ ratios of liver-derived lymphocytes were significantly higher (P < 0.05) than those obtained in patients without viral replication. In contrast, the percentage of T cells expressing the gamma/delta receptor and that of CD2+/CD57+ cells were similar in both groups of patients. Furthermore, in patients exhibiting viral replication, CD4+CD8+ ratios of liver-derived lymphocytes correlated with serum HBV DNA levels (P < 0.001). No relationship between CD4+/CD8+ ratio of liver-derived and peripheral blood lymphocytes was observed. Our data indicate that, in patients with chronic hepatitis B, the CD4+/CD8+ ratio of liver-derived lymphocytes correlates with viral replication. This suggests that in situ helper/inducer CD4+ T lymphocytes may positively regulate the cytotoxic T cell activity in patients with HBV-related chronic hepatitis.     

Southern hybridisation analysis of HBV DNA in peripheral blood leucocytes and of different cell types: Changes during the natural history and with interferon-α therapy in patients with hepatitis B virus infection

Hepatitis B virus (HBV) DNA was evaluated in peripheral blood mononuclear cells (PBMC) from 50 individuals utilising Southern hybridisation analysis. HBV DNA sequences were detected in PBMC from 16/29 (55 percent) of chronic hepatitis B virus (HBV) carriers with serum HBeAg and HBV DNA, compared with 1/8 (13%) of carriers with anti-HBe and HBV DNA negative (P = NS). Two of 7 patients with previous HBV infection and chronic liver disease had detectable HBV DNA in PBMC. Of the 19 patients with HBV DNA in PBMC, 18 had high molecular weight species. In addition, five of these had free, monomeric HBV DNA and six patients had low molecular weight bands. For nine of the above patients, total peripheral blood leucocytes were separated into PBMC and polymorphonuclear cells. Four had HBV DNA in PBMC only, two only in polymorphonuclear cells and three in both types of cell. Eleven patients with chronic HBV infection were studied at monthly intervals for 6 months. Six were untreated and five received IFN-alpha. Three patients who responded to IFN-a had HBV DNA present in PBMC before therapy, and two became negative. Two of 3 untreated patients had intermittent HBV DNA in PBMC and the other remained persistently negative. Of the patients positive on more than one occasion, the pattern of HBV DNA was similar. Peripheral blood leucocytes often contain multimers of free HBV DNA, more commonly in patients with serum HBeAg and HBV DNA and may occur even in the absence of serum HBsAg. These findings have implications for recurrence of disease after hepatic transplantation. HBV DNA in PBMC is detected intermittently over time and IFN-α therapy may be associated with eradication. © 1994 Wiley-Liss, Inc.     

Different forms of hepatitis B virus DNA and expression of HBV antigens in peripheral blood mononuclear cells in chronic hepatitis B

The presence of both hepatitis B virus (HBV) DNA and HBV antigens (HBsAg, HBeAg) was assessed in peripheral blood mononuclear cells (PBMC) from 32 patients chronically infected with HBV. Three different molecular forms of HBV DNA were observed: free monomers (5), high-molecular-weight free concatemers (11), and integrated HBV DNA (9). The HBV DNA patterns in the PBMC were different from those found in liver and did not correlate with any specific profile of serum HBV markers. When the same PBMC were assayed for HBsAg, 22 of the 25 HBV DNA positive samples, but only three of the seven HBV DNA negative samples, were positive. By contrast, none of the PBMC samples from five healthy HBV vaccine recipients gave any positive signal in the HBV DNA or HBsAg assays. In some patients, T and B cells, monocytes, and polymorphonuclear (PMN) cells were assayed separately, showing that the DNA pattern was similar for these different leucocytes subsets and ruling out the possibility that these patterns might reflect PMN cell contamination. Thus, in chronic HBV infection, 87.5% (28/32) of patients were found to contain at least one HBV marker in their PBMC, and a strong correlation was found between the presence of HBV DNA and viral antigens, suggesting a specific expression of HBV encoded proteins.     

Th17 / Treg 比率在慢性乙型肝炎患者外周血的动态变化及意义

目的: 通过比较不同治疗方法对慢性乙型肝炎(Chronic hepatitis B ,CHB) 患者外周血Th17(T helper lymphocyte 17)、Treg(regular T cell)细胞频率及Th17/ Treg 比率的变化,探讨其在CHB 患者疾病转归中的意义。方法:CHB 患者40 例,根据保肝治疗基础上是否加用核苷酸抗病毒药物分为未抗病毒治疗组(Chronic hepatitis B without antiviral,CHBWA)20 例、抗病毒治疗组(Chronic hepatitis B antiviral,CHBA)20 例、乙肝病毒携带者(Asymptomatic hepatitis B carriers ,AsC)10 例、健康对照组(Health control,HC)10 例;采用流式细胞术检测CHBWA 组0 月、1 月及CHBA 组0 月、1 月、3 月,AsC 患者和HC外周血Th17、Treg 细胞频率,计算Th17/ Treg 比率,观察其在疾病不同阶段的动态变化。结果:CHBWA 组保肝治疗1 月后,Th17/ Treg 比率(0.39±0.11)比0 月(1.20±0.26)明显降低(P<0.01),已接近AsC 组(0.39±0.14)及HC 组(0.42±0.20)Th17/Treg 比率水平(P>0.05),且Th17/ Treg 比率与ALT 有良好的正相关关系(r =0.709,P =0.000);在CHBA 组,Th17/ Treg 比率在1 月(0.73依0.32)、3 月(0.76±0.44)均较0 月(1.18±0.27)明显下降,与0 月比较差异有统计学意义(P<0.01),但继续治疗到3 月时Th17/ Treg 比率较1 月时变化不明显,Th17/ Treg 比率在0、1、3 月均高于AsC 组及HC 组(P<0.01,0.05);Th17/ Treg 比率与ALT、HBV DNA 有良好的正相关关系(r =0.500,P =0.000;r =0.345,P =0.007);两组间比较,0 月时两组间Th17/ Treg 比率差异无统计学意义(P>0.05),但经治疗1 月后CHBA 组Th17/ Treg 比率高于CHBWA (t=4.471,P<0.01)。结论:CHB 患者中,抗病毒治疗将迅速影响Th17、Treg 细胞频率,导致Th17/ Treg 比率的变化,其变化可能与清除病毒有关。     

Cytokine-dependent anti-viral role of CD4-positive T cells in therapeutic vaccination against chronic hepatitis B viral infection

There are several lines of evidence suggesting that specific vaccine therapy with a standard hepatitis B virus (HBV) vaccination reduces HBV replication. The aim of this study was to investigate the anti-viral mechanism of vaccine therapy in chronic hepatitis B patients. Nineteen patients were assigned to receive either vaccine therapy (n = 13) or no treatment as a control (n = 6). Vaccinated patients were analyzed for T cell proliferative responses specific for envelope antigen and cytokine production by antigen-specific T cells. ELISPOT and cytotoxicity assays also were carried out for limited blood samples. Serum HBV DNA levels decreased significantly at 3 months after completion of therapy and thereafter as compared to the baseline ones, and were significantly lower in vaccinated patients than in controls at 12 and 18 months after completion of therapy. Vaccination induced antigen-specific CD4+ T cell proliferative responses in four patients (30.8%). The production of high levels of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) by antigen-specific T cells was found in six patients (46.0%) who showed significantly lower HBV DNA levels in serum at 6 (P = 0.04) and 18 months (P = 0.005) after completion of therapy than those without high levels of cytokine production. Vaccination did not induce antigen-specific CD8+ T cells or cytotoxic T cells. These results suggest that envelope-specific CD4+ T cells may control directly HBV replication by producing anti-viral cytokines rather than providing help for cytotoxic T cells in therapeutic vaccination against chronic HBV infection.     

慢性乙型肝炎急性发作与血清HBN DNA含量关系

目的 研究慢性乙型肝炎急性发作与血清ＨＢＶ ＤＮＡ含量的关系。方法 采用荧光ｍｐｌｉＳｅｎｓｏｒ）定量方法。测定一组自发性反复发作的慢性乙型肝炎患者发作前,中和后的血清ＨＢＶ ＤＮＡ含量的变化。结果 （１）１１例患者中有９例（８２％）血清ＨＢＶ ＤＮＡ含量的高峰值是在最大肝损害之前出现或与丙氨酸转氨酶（ＡＬＴ０同时达到高峰。     

Immunoregulation of the in vitro anti-HBs antibody synthesis in chronic HBsAg carriers and in recently boosted anti-hepatitis B vaccine recipients.

We report a study on immunoregulation of in vitro antibody to hepatitis B surface antigen (anti-HBs) synthesis induced by pokeweed mitogen (PWM) from peripheral blood mononuclear cells (PBMC) in chronic hepatitis B surface antigen (HBsAg) carriers and in 'high responders', (anti-HBs RIA ratio greater than or equal to 20 in serum), recently boosted with anti-hepatitis B vaccine. Anti-HBs was detected in 11 days PBMC supernatants (SN) from 24 out of 36 'high responders', but in none from 31 chronic HBsAg carriers, despite detectable amounts of polyclonal IgG and antibody to hepatitis B core antigen (anti-HBc) were produced. The lack of anti-HBs production by chronic HBsAg carriers did not seem to be determined by suppressor influences because T lymphocytes from the majority of chronic HBsAg carriers, co-cultured with 'high responders' PBMC did not suppress anti-HBs production. Co-cultures between HBsAg carriers T4 positive (helper/inducer) cells and allogenic 'high responder' non-T cells produced anti-HBs antibody, indicating that HBsAg carrier T cells are not deficient in this allogenic helper function under PWM stimulation. Allogenic cocultures between HBsAg carrier non-T cells and 'high responder' T4 positive cells failed in anti-HBs production: a specific B lymphocyte defect might be involved in the lacking anti-HBs synthesis in chronic HBV patients. Antigen-induced specific anti-HBs synthesis experiments indicate that B cells themselves seem to be the target for HBsAg-induced suppression of anti-HBs antibody response.