Lamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy

The hepatitis B virus (HBV) cytotoxic T lymphocyte (CTL) response in patients with chronic HBV infection is generally weak or totally undetectable. This inability to mount protective CTL responses is believed to be a crucial determinant of viral persistence, and its correction represents an important objective of immune therapies for chronic hepatitis B. However, amplification of CTL responses in vivo may be ineffective if HBV-specific CD8 cells are either absent or nonresponsive to exogenous stimulation. In this study, we asked whether antiviral treatments able to inhibit viral replication and to reduce viral and antigen load can successfully reconstitute CTL responses creating the appropriate conditions for their therapeutic stimulation. For this purpose, the HBV-specific CTL response before and during lamivudine therapy was studied longitudinally in 6 HLA-A2-positive patients with HBeAg+ chronic hepatitis B. Both HBV-specific cytotoxic T cell activity measured by chromium release assay on peptide stimulation in vitro and CD8+ T cell frequency measured ex vivo by HLA-A2/peptide tetramer staining were significantly augmented by lamivudine therapy. This enhancement followed the reconstitution of CD4 reactivity and the decline of viral load induced by therapy. Our study shows that lamivudine treatment in chronic hepatitis B can restore CTL reactivity, making CTL susceptible to exogenous stimulation. This effect may enhance the probability that T cell-based immune therapies delivered after lamivudine treatment can successfully reconstitute a protective CTL response able to cure chronic HBV infection.     

Alpha-interferon (α-IFN) protects B-chronic lymphocytic leukaemia cells from apoptotic cell death in vitro

B-chronic lymphocytic leukaemia cells (CLL) are prone to apoptotic cell death when cultured in vitro . Apoptosis and loss of the bcl-2 protein is prevented in CLL cells cultured in the presence of interleukin-4. In this study we analysed the effects of α-IFN on the DNA fragmentation, bcl-2 protein levels and cell survival in purified B-cells from 16 CLL patients. α-IFN (10³ U/ml) reduced the degree of spontaneous DNA fragmentation of CLL cells after a 30 h culture period (from a mean of 22·2% in control cultures to 10·5%, P <0·01). This inhibition was accompanied by preservation of bcl-2 protein and an increased survival of CLL cells compared to control cultures. In parallel, α-IFN inhibited hydrocortisone induced DNA fragmentation in CLL cells. The effects of α-IFN on DNA synthesis of CLL cells were variable (in two patients a decrease and in seven an increase in ³H-thymidine uptake) and did not correlate with the effect on DNA fragmentation. In conclusion, our data suggest that α-IFN, like IL-4 and γ-IFN, inhibits apoptosis of CLL cells. These in vitro data indicate that the clinical responses of some CLL patients to α-IFN cannot be explained by a direct cytotoxic effect of α-IFN on circulating CLL cells. Alternatively, α-IFN may inhibit the proliferation of the small fraction of clonogenic CLL progenitors, or interfere with cellular interactions necessary for the survival and growth of CLL cells.     

Effects of glycyrrhizin on immune-mediated cytotoxicity

Intravenous administration of glycyrrhizin is known to decrease elevated plasma transaminase levels in patients with chronic viral hepatitis, in which immune-mediated cytotoxicity by cytotoxic T lymphocytes and tumour necrosis factor (TNF)-α is considered to play an important pathogenic role. However, the immunological interpretation of the transaminase-lowering action of glycyrrhizin is not known. Studies were performed to elucidate this action immunologically by assessing the effects of glycyrrhizin on immune-mediated cytotoxicity using an antigen-specific murine CD4⁺ T hybridoma line, which exhibits cytotoxicity against antigen-presenting cells after stimulation with specific antigen, and a murine TNF-α-sensitive fibroblast line. Glycyrrhizin inhibited the cytotoxic activity of the T cells against antigen-presenting cells and also suppressed TNF-α-induced cytotoxicity in the TNF-α-sensitive cell line in vitro. These results suggest that the decrease of elevated transaminase levels by glycyrrhizin in patients with chronic viral hepatitis is mediated in part by inhibition of immune-mediated cytotoxicity against hepatocytes.     

乙型肝炎病毒特异性细胞毒性T淋巴细胞应答与不同临床感染状态的关系

目的 分析人类白细胞抗原(HLA)-A0201限制性的特异性CTL,研究急性肝炎急性期和慢性乙型肝炎活动期患者T淋巴细胞对特异性抗原表位免疫应答的差异.方法 收集HLA-A0201阳性的5例急性肝炎急性期和6例慢性乙型肝炎活动期患者的外周血单个核细胞(PBMC),酶联免疫斑点技术(ELISPOT)测定针对HBV聚合酶区(Pol575-583)、包膜区(Env348-357)和核心区(Core18-27)3个CD8+T淋巴细胞表位肽特异性CTL的数量和功能.数据采用t检验.结果 经Pol575-583、Env348-357和Core18-27三条抗原肽刺激,急性乙型肝炎急性期患者组斑点形成细胞数(SFC)分别为110±13、165±17和185±20;慢性乙型肝炎活动期患者组SFC分别为22±4、23±5和30±5,两组差异有统计学意义(t值分别为10.9、15.2和8.0,均P<0.05).急性乙型肝炎急性期患者各抗原肽特异性CTL的应答能力Pol575-5830.05).非特异性HLA-2402限制性Core117-125刺激也出现SFC增加,但与阴性对照组比较,差异无统计学意义(P>0.05).结论 急性感染者HBV特异性CTL应答水平显著高于慢性HBV感染者,慢性乙型肝炎患者体内的多克隆CTL数量和功能低下.     

负载食管癌抗原的DC诱导特异性CTL对食管癌细胞体外杀伤作用观察

目的观察负载食管癌抗原的树突状细胞（DC）活化的特异性细胞毒性T淋巴细胞（CTLs）对食管癌细胞的体外杀伤作用。方法冻融法获取食管癌细胞抗原,联合应用粒细胞—巨噬细胞集落刺激因子（rhGM-CSF）、白细胞介素4（IL-4）和肿瘤坏死因子α（TNF-α）诱导培养外周血DC并负载肿瘤抗原,激活自体T淋巴细胞,制备特异性CTLs。将其加入食管癌细胞中培养48 h,用MTT法检测食管癌细胞裂解率,ELISA法检测γ干扰素（γ-IFN）水平。结果负载食管癌抗原的DC激活的CTLs表现出对食管癌Eca109细胞的特异性杀伤作用,γ-IFN水平为（1 625±37.55）pg/ml;而对A549细胞仅有微弱的杀伤作用,γ-IFN水平为（169.04±13.81）pg/ml。未负载食管癌抗原DC刺激的CTLs对食管癌细胞几无杀伤作用。结论负载食管癌抗原的DC激活的CTLs在体外对食管癌细胞能产生高效而特异性的杀伤作用。     

Cytotoxic T lymphocyte responses and CTL epitope escape mutation in HBsAg, anti-HBe positive individuals

BACKGROUND/AIMS: Clearance of hepatitis B virus (HBV) is characterised by a strong cytotoxic T cell response. Persistence of HBV in chronic hepatitis B carriers may be related to failure of this response. The aim of this study was to determine whether HLA class I restricted cytotoxic T lymphocyte (CTL) responses persist in anti-hepatitis B e (HBe) positive / HBV DNA negative individuals, and to correlate the presence of viral CTL epitope mutation with clinical outcome. METHODS: An HLA/HBV dual transfectant model was used to demonstrate these CTL responses in individuals chronically infected with HBV. Subsequently, a known hepatitis B core (HBc) CTL epitope was sequenced in a family of five chronically infected individuals all sharing a HLA allele (HLA-A68.1). RESULTS: Low level HLA class I restricted cytotoxic T cell responses were detected in the peripheral blood of five of eight anti-HBe positive individuals. In the family of HLA-A68.1 positive chronically infected individuals, mutation of the HLA-A68.1 restricted hepatitis B core antigen (HBcAg) CTL epitope STLPETTVVRR was found in all four anti-HBe positive individuals but not in the sole hepatitis B e antigen (HBeAg) positive patient. CONCLUSION: These data are consistent with a continued immune selection pressure on HBV in anti-HBe positive chronically infected individuals with low replicating HBV infection and suggest that mutation of a CTL epitope may be a consequence of the immune response, as opposed to the cause of viral persistence.     

Hepatitis B surface antigen specific cytotoxic T lymphocyte activity in hepatitis B virus infection

Cytotoxic T lymphocyte (CTL) activity against HBs antigen (HBsAg)-coated autologous mononuclear cells as target cells was examined in hepatitis B virus infection. Target cells were prepared by coincubation of mononuclear cells with purified HBsAg in 0.5 mM CrCl3. The distribution and uniformity amounts of HBsAg on target cells was shown by immune fluorescence and by analyzing the fluorescence intensity with a fluorescent activated cell sorter. CTL activity was detected in 7 of 9 patients with acute hepatitis type B, in 4 of 11 chronic active hepatitis type B, in none of 8 healthy HBsAg carriers, and in none of 22 healthy volunteers. The antigen specificity of the cytotoxicity was confirmed by a blocking experiment with purified HBsAg and by cold target inhibition with HBsAg or bovine serum albumin (irrelevant antigen) coupled cold target cells. CTL lysed HBsAg coupled allogeneic target cells that shared HLA-A or B locus antigens. This finding suggests that HLA restriction may be involved in the killing mechanism. This HBsAg specific CTL clone may participate in the immunopathogenesis of hepatitis B virus infection.     

Intravenous natural β-interferon in white patients with chronic hepatitis C who are nonresponders to α-interferon

Objectives: α-Interferons (α-IFN) have been shown to be effective in the treatment of chronic viral C hepatitis, but their efficacy remains unsatisfactory. Recently natural β-interferon (β-IFN) administered by intravenous infusion has been used successfully.
Methods: To evaluate the efficacy and safety of intravenous β-IFN administration we treated 20 patients with histologically proven chronic hepatitis C who were nonresponders to at least two previous courses of α-IFN treatment. All patients received 6 million units (MU) of natural human fibroblast β-IFN by drip infusion, 6 times per wk for 8 wk and were followed up for 6 months after suspension of treatment.
Results: Five patients (25%) had response at the end of treatment; of these patients only one had sustained response. Patients who responded to therapy had lower, although not significantly, baseline levels of HCV RNA, compared with nonresponders. Whereas mean viral load decreased during therapy, only two patients were HCV RNA negative at the end of treatment, but none were at the end of the follow-up period. Genotype 1 was found in 17 cases, genotype 2 was found in one case, and a combination of genotypes 1b and 2a was found in the remaining two cases. Therapy was well tolerated and β-IFN administration was neither interrupted nor its dosage reduced due to side effects in any of the patients.
Conclusions: Our study shows that intravenous β-IFN is well tolerated and that the modest results obtained may depend on the brevity of treatment. Consequently, further studies are needed to define the optimum dose, schedule, and duration of treatment to eradicate HCV infection.     

结核杆菌热休克蛋白质70作为乙型肝炎核心抗原细胞毒性T淋巴细胞表位肽载体的研究

目的探讨结核杆菌热休克蛋白质70（TB．HSP70）作为乙型肝炎病毒（HBV）核心抗原细胞毒性T淋巴细胞（CTL）表位肽载体，诱导HBV特异性免疫应答的可能性。方法体外观察重组TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物诱导慢性乙型肝炎患者外周血淋巴细胞增殖以及HBV特异性细胞毒活性；以Balb／c小鼠为体内研究对象，行流式细胞术分析免疫后小鼠外周血和脾细胞中CD4^＋与CD8^＋T淋巴细胞及自然杀伤（NK）细胞的比率，并观察能否诱导HBV特异性免疫保护作用。结果体内外研究表明TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物能有效地诱导HBV特异性细胞毒活性，体内能激活CD4^＋与CD8^＋T淋巴细胞及NK细胞增殖。在体内，TB．HSP70-CTL融合蛋白质较复合物能更有效地激活免疫应答，其杀伤率为28．9％。CD8^＋T淋巴细胞在脾细胞中比率为43．9％，NK细胞为13．6％。而单纯的TB．HSP70和CTL表位肽并不能有效地引起机体的免疫应答。结论TB．HSP70能够作为乙型肝炎核心抗原CTL表位肽的载体，提高小分子表位肽的免疫原性。     

慢性乙型肝炎患者特异性细胞毒性T淋巴细胞的变化

目的探讨慢性乙型肝炎急性发作及病情严重程度与特异性细胞毒性T淋巴细胞(CTL)水平的关系。方法从29例人白细胞抗原(HLA)-A2阳性慢性乙型肝炎急性发作及免疫耐受慢性乙型肝炎患者外周血中分离外周血单个核细胞,用HLA-A2*HBcAg抗原表位肽-五聚体复合体及CD8单克隆抗体染色后,用流式细胞仪检测乙型肝炎病毒(HBV)特异性的CTL细胞。并对6例急性发作患者进行动态检测HBV特异性的CTL、肝功能和病毒载量。结果19例急性发作慢性乙型肝炎患者五聚体阳性细胞占CD8阳性细胞的比例平均为1．4%±0．8%,而10例免疫耐受患者为0．6%±0．4%,两组间比较,t=2．180,P＜0．05,差异有统计学意义。急性发作导致重型肝炎的患者中,乙型肝炎核心抗原特异性CTL细胞阳性率平均为1．3%±1．0%,而非重型肝炎患者为1．4%±0．8%,两组间比较,差异无统计学意义。在12周的随访期内,患者丙氨酸氨基转移酶和病毒载量逐渐下降,但乙型肝炎核心抗原特异性CTL维持在较高的水平(＞0．7%)。结论慢性乙型肝炎患者急性发作与HBV特异性CTL有关,但主要的肝细胞损害可能并非直接由HBV特异性CTL引起的。     

阻断Notch1信号通路对慢性乙肝患者外周血Foxp3 mRNA表达的影响

目的观察阻断Notch1信号通路对慢性乙肝患者外周血单个核细胞（PBMC）Foxp3 mRNA表达以及细胞毒性T淋巴细胞（CTL）细胞毒活性的影响,探讨Notch1信号通路在乙型肝炎慢性化过程中可能的作用机制。方法抗Notch1抗体阻断Notch1信号通路,利用实时荧光定量PCR（real-tim e PCR）检测其外周单个核细胞Foxp3 mRNA的表达情况,并用乳酸脱氢酶释放法检测CTL对靶细胞HepG2.2.15细胞毒活性的影响。结果用抗体阻断后,慢性乙型肝炎患者外周单个核细胞Foxp3 mRNA表达显著减弱,且CTL细胞毒活性明显增强,与阴性对照组和空白对照组相比差异有统计学意义（P均〈0.01）。结论阻断Notch1信号通路可减少慢性乙型肝炎患者外周单个核细胞Foxp3 mRNA的表达,增强CTL细胞毒活性,说明Notch1信号通路可能是HBV持续感染的一个因素,且可能与Foxp3有关。     

HBsAg及B_(7-2)抗原重组腺病毒载体感染免疫研究

目的　为激发机体对ＨＢｓＡｇ的ＣＴＬ反应，寻求对慢性乙型肝炎更有效的治疗方法。方法　构建了共表达ＨＢＳＡｇ及Ｂ７－２抗原的Ｅ１区插入重组腺病毒载体，用脂质体法转染２９３细胞，经空斑筛选表达目的抗原的重组腺病毒，用ＥＬＩＳＡ法及Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ法分别检测ＨＢｓＡｇ及Ｂ７－２抗原表达。在２９３细胞中扩增，再纯化、浓缩后，体内感染Ｃ５７小鼠，检测体液免疫反应和细胞免疫反应。结果　重组腺病毒在体外培养的２９３细胞及ＨｅｐＧ２细胞中高效表达ＨＢＳＡｇ及Ｂ７－２抗原。感染小鼠体液免疫较弱，但可通过注射乙型肝炎疫苗而加强；重组腺病毒载体能诱导强而有效的细胞免疫反应；未观察到明显毒副作用。结论　ＨＢｓＡｇ及Ｂ７－２抗原重组腺病毒载体体外感染２９３、ＨｅｐＧ２细胞后高效表达目的抗原，感染免疫局诱导有效的细胞免疫反应。初步结果显示腺病毒载体是一种高效、安全的载体系统。     

慢性乙型肝炎病毒感染者外周血单个核细胞表达程序性死亡分子-1及其配体与血清乙型肝炎病毒DNA水平的相关性

目的 研究程序性死亡分子-1(PD-1)及其配体(PD-L1)表达水平与慢性HBV感染者HBV DNA水平的相关性及抗病毒治疗对其表达的影响.方法 检测137例慢性HBV感染者的外周血单个核细胞(PBMC)表面PD-1和PD-L1,并检测其中64例人类白细胞抗原(HLA)-A2阳性者HBV特异性CTL数量.ELlSA法检测PBMC体外培养上清液中IFN-γ浓度.比较10例HBeAg阳性慢性乙型肝炎(CHB)患者予替比夫定抗病毒治疗24周前后上述指标的变化.两组间均数比较采用两独立样本的t检验,多组间的差异采用单因素方差分析,相关分析采用Pearson相关分析.结果 HBV DNA＜3 lg、3～6 lg和＞6 lg拷贝/mL组问PBMC表面PD-1和PD-L1表达均明显高于健康对照组,但差异无统计学意义;3组HBV特异性CTL表面PD-1表达分别为(69.3±11.2)%、(76.5±9.1)%和(78.0±11.7)%,HBV DNA＞6 lg拷贝/mL 组PD-1表达明显高于＜3 lg拷贝/mL组,而HBV特异性CTL数量明显低于＜3 lg拷贝/mL组;3组PBMC体外培养上清液中IFN-γ水平差异无统计学意义.HBeAg阳性组和阴性组间上述指标差异无统计学意义.替比夫定抗病毒治疗12周和24周时,PD-1、PD-L1表达较治疗前明显下降,伴有HBV特异性CTL数量逐渐增加和IFN-γ水平升高.结论 慢性HBV感染者PBMC表面PD-1的表达较健康者明显上调,且HBV特异性CTL表面表达PD-1水平与血清HBV DNA水平相关,但与HBeAg状态无关.抑制HBV复制能降低PD-1、PD-L1表达,并增加HBV特异性CTL的数量和功能.

Abstract：

Objective To study the relationship between programmed death-1 (PD-1)/programmed death-1 ligand (PD-L1) expressions and serum hepatitis B virus (HBV) DNA levels in chronic hepatitis B (CHB) patients. Methods A total of 137 CHB patients and 10 healthy controls were enrolled in the study. The peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples. HBV-specific cytotoxic T lymphocyte (CTL) was expanded in vitro in 64 human leucocyte antigen (HLA)-A2 positive patients. Flow cytometry was used to detect HLA-A2 type,expressions of PD-1/PD-L1 on PBMCs and PD-1 on HBV specific CTL. Interferon gamma (IFN-γ)was measured by commercial enzyme-linked immunosorbent assay (ELISA) kits. PD-1/PD-L1expressions on PBMCs, HBV-specific CTL and IFN-γ level in PBMC culture medium were compared among patients with different baseline HBV DNA levels. Ten hepatitis B e antigen (HBeAg) positive patients were treated with telbivudine for 24 weeks. The above mentioned parameters were determined and compared before and after the antiviral treatment. Independent-samples t test were used to compare means between two groups and one-way A NOVA were used to compare means among multigroups. We used the pearson corretation test to assess corretation significance. Results The PD-1 and PD-L1 expressions on PBMCs in patients with baseline HBV DNA＜3 lg copy/mL, 3-6 lg copy/mL and ＞6 lg copy/mL were all significant higher than those in healthy control group, but no statistical differences were found. PD-1 expressions on HBV-specific CTL in the three CHB patient groups were (69.3±11.2)%, (76.5±9. 1)% and (78.0±11.7)%, respectively. However, PD-1 expression on HBV-specific CTL was higher, while the frequency of HBV-specific CTL cells was lower in HBV DNA ＞6 lg copy/mL group compared to HBV DNA＜3 lg copy/mL group. The above parameters, including expressions of PD-1 and PD-L1, the frequency of HBV-specific CTL and its PD-1 expression were not significantly different between HBeAg-positive group and HBeAg-negative group. Compared with baseline, PD-1 and PD-L1 expression decreased obviously accompanying with increase of HBV-specific CTL cells frequency and IFN-γ level after 12 weeks and 24 weeks of telbivudine treatment. Conclusions PD-1 expression on HBV-specific CTL correlates with serum HBV DNA level, but not HBeAg status in CHB patients. Suppression of HBV replication can reduce PD-1/PD-L1 expressions and partially restore HBV specific CTL function.     

Efficacy of an analysis of lymphocyte subsets in predicting the clinical response to α-interferon therapy in thalassaemia patients with chronic infection by hepatitis C virus: a pilot study

Summary. α-interferon (α-IFN) has been used to treat chronic non-A non-B hepatitis in thalassaemic patients with response rates from 45% to 83%. Unfortunately, treatment with α-IFN is associated with side-effects which have a negative effect on the quality of life of the patient. Therefore it would be useful if we could distinguish in advance those patients who would benefit from such therapy from those who would not. In the present study we found that the modification of lymphocyte subsets 20 h after the administration of the first dose of α-IFN revealed that relative numbers of T helper lymphocytes (CD4⁺) increased in three non-responding patients and decreased in five responding patients, whereas those of T suppressor lymphocytes (CD8⁺), and natural killer cells (CD57⁺. CD16⁺) decreased in non-responding patients and increased in responding patients. Therefore analysis of the lymphocyte subsets CD4, CD8, CD57 and CD16 before and 20 h after the administration of α-IFN can be used to predict the clinical response to treatment with α-IFN.     

重型肝炎患者乙型肝炎病毒特异性细胞毒性T淋巴细胞应答功能研究

目的 用主要组织相容性复合物（MHC）抗原肽四聚体（Tetramer）流式细胞技术，分析乙型重型肝炎患者外周血中特异性细胞毒性T淋巴细胞（CTL）应答状况，并探讨其临床意义。方法 采用Tetramer流式细胞技术检测乙型重型肝炎患者外周血中受人类白细胞抗原Ⅰ类分子限制的三类特异性CD8^＋CTL细胞数量；采用酶联免疫吸附斑点试验技术，测定经特异性乙型肝炎病毒（HBV）肽段诱导培养的特异性CTL表达膜内细胞因子IFNγ、TNFα、IL-4和IL—10等的水平；采用Promega CytoTox96非放射性细胞毒试验技术，测定经特异性HBV肽段诱导培养的特异性CTL杀伤靶细胞能力。结果 急性乙型重型肝炎组外周血中针对HBVcore18-27表位的特异性CTL数量高于慢性乙型重型肝炎组（P〈0．05），而低于急性乙型肝炎组（P〈0．05）；急性乙型重型肝炎组表达干扰素γ和肿瘤坏死因子α较慢性乙型重型肝炎组高（P值均〈0．05）；急性乙型重型肝炎组HBVcore18—27特异性CTL裂解靶细胞能力高于慢性乙型重型肝炎组（P〈0．05）。结论 急性乙型重型肝炎患者特异性CTL应答作用增强，而慢性乙型重型肝炎患者特异性CTI。应答缺乏。急性乙型重型肝炎患者外周血特异性CTL持续存在，可能与促进病毒清除等相关。     

Prevalence and significance of anticardiolipin, anti-β2 glycoprotein I and anti-prothrombin antibodies in chronic hepatitis C

Antiphospholipid antibodies have been demonstrated in chronic hepatitis C, but their clinical and pathogenetic significance remains elusive. We prospectively studied 115 patients (85 men, mean age 36.9 years) with chronic hepatitis C without cirrhosis and treated by α-interferon (α-IFN). Antiphospholipid determinations comprised anticardiolipin (ACA), anti-β₂-glycoprotein I and anti-prothrombin antibodies of the IgG and IgM classes. At entry, 24 patients (21%) were found to possess low to moderate ACA levels (18 IgG, two IgM and four both isotypes) compared with only 4/115 age- and sex-matched control subjects (3.5% P  = 0.001). ACA positivity rate increased to 31% ( P  = 0.01) after a 6-month course of α-IFN treatment. In contrast, the prevalence of anti-β₂-glycoprotein I and anti-prothrombin antibodies was not significantly different from controls at either time point. The presence of ACA correlated with that of antinuclear antibodies ( P  = 0.0002), but was not associated with parameters such as histological activity, viral burden and response to α-IFN, nor with a history of thrombosis or pregnancy loss. However, a non-significant trend of higher incidence of mild thrombocytopenia among ACA-positive patients was observed. We conclude that low-titre ACA positivity is a common finding in patients with chronic hepatitis C, especially following α-IFN treatment, but does not select a category with different clinical features. These data are in keeping with the absence of associated anti-β₂GPI and anti-prothrombin antibodies, and do not support a role for HCV infection in the pathogenesis of the antiphospholipid syndrome.     

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia

BACKGROUND/AIMS: The aim of this study was to identify and characterize hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) epitopes presented by human leukocyte antigen (HLA)-A*2402, most common HLA class I allele in East Asia. METHODS: HLA-A*2402-restricted CTL epitopes were identified by reverse immunogenetics. Immunogenecity of these epitopes was investigated using peripheral blood mononuclear cell (PBMC) from HLA-A24+ patients with acute hepatitis B. RESULTS: An HLA-A*2402 stabilization assay demonstrated that 36 of 63 HBV peptides carrying HLA-A*2402 anchor residues have high- and medium-HLA-A*2402 binding affinity. Two (C117-125 and P756-764) of the 36 peptides induced peptide-specific CTLs. CTL clones and lines specific for these peptides killed HBV recombinant vaccinia virus-infected target cells expressing HLA-A*2402, indicating that these two peptides are CTL epitopes presented by HLA-A*2402. These two peptides were able to induce specific CTLs in 7 and 11 of 12 HLA-A24+ patients with acute hepatitis B, respectively. CONCLUSIONS: We identified two immunodominant CTL epitopes restricted by HLA-A*2402. Because HLA-A*2402 is the most common allele in East Asia, a region in which there are approximately 200 million HBV carriers, these epitopes will be useful for analysis of CTL responses in patients from East Asia.     

HBsAg及B7—2抗原重组腺病毒载体感染免疫研究

目的 为激发机体对HBsAg的CTL反应，寻求对慢性乙型肝炎更有效的治疗方法。方法 构建了共表达HBsAg及B7－2抗原的E1区插入重组腺病毒载体，用脂质体法转染293细胞，经空斑筛选表达目的抗原的重组腺病毒，用ELISA法及Western blotting法分别检测HBsAg及B7－2抗原表达，在293细胞中扩增，再纯化、浓缩后，体内感染C57小鼠，检测体液免疫反应和细胞免疫反应。结果 重线腺病毒在体外培养的293细胞及HepG2细胞中高效表达HBsAg及B7－2抗原。感染小鼠体液免疫较弱，但可通过注射乙型肝炎疫苗而加强；重组腺病毒载体能诱导强而有效的细胞免疫反应；未观察到明显毒副作用。结论 HBsAg及B7－2抗原重组腺病毒载体体外感染293、HepG2细胞后高效表达目的抗原，感染免疫后诱导有效的细胞免疫反应。初步结果显示腺病毒载体是一种高效、安全的载体系统。     

前列腺素E1抑制慢性乙型重型肝炎树突状细胞成熟及CD4~+/CD8~+T细胞活性的观察

目的研究前列腺素E1对慢性乙型重型肝炎树突状细胞成熟及CD4+/CD8+T细胞活性的影响。方法抽取前列腺素E1治疗10 d前后的慢性乙型重型肝炎患者外周静脉血,密度梯度离心法分离淋巴细胞,贴壁培养获得PBMC,经重组人白细胞介素4(rhIL-4)、重组人粒-巨噬细胞集落刺激因子(rhGM-CSF)、加或不加前列腺素E1刺激培养8 d获得树突细胞(DC)。经流式细胞仪检测DC表达的HLA-DR、CD83、CD86,ELISA测定分泌的IFNγ、IL-12p70。悬浮T细胞部分检测CD4+/CD8+T细胞比例及其细胞表面CD69、HLA-DR表达;部分经IL-2培养,用于检测DC体外诱导的T淋巴细胞毒活性。结果前列腺素E1治疗慢性乙型重型肝炎,对患者CD4+/CD8+T细胞比例无明显影响,可抑制CD4+/CD8+T细胞活化分子CD69、HLA-DR的表达;前列腺素E1体外培养的DC低表达HLA-DR、CD83、CD86,分泌IFNγ、IL-12p70均下降,体外诱导的自体淋巴细胞毒活性减弱,与未加用前列腺素E1比较差异有统计学意义(P<0.01)。结论前列腺素E1治疗慢性乙型重型肝炎,可抑制CD4+/CD8+T细胞活化;体外培养可抑制DC成熟,诱导减弱自体淋巴细胞毒活性。     

A pilot study of the CY-1899 T-cell vaccine in subjects chronically infected with hepatitis B virus. The CY1899 T Cell Vaccine Study Group.

Clinical observations suggest that eradication of the hepatitis B virus (HBV) is immune-mediated. Vigorous cytotoxic T lymphocyte (CTL) activity directed at HLA class I-bound viral epitopes are detected during acute hepatitis B, but not in chronic hepatitis B carriers. A CTL epitope derived from the hepatitis B core protein amino acids 18-27 has been incorporated into a vaccine also comprised of a T-helper cell epitope and 2 palmitic acid residues (CY-1899). The aim of this study was to determine whether repeated doses of CY-1899 given to patients with chronic hepatitis B could initiate in vivo CTL activity and viral clearance. Patients with chronic hepatitis B received up to 4 doses (ranging from 0.05 mg to 15 mg) 6 weeks apart. Following vaccination, patients were monitored for hepatitis B surface antigen and "e" status, HBV-DNA levels, liver biochemistry, CTL activity, and any adverse events. Ninety patients with chronic hepatitis B infection received CY-1899. Mean CTL responses were all low but were maximal following vaccination with 5 mg CY-1899. Peak CTL responses never exceeded 10 lytic units (LU) regardless of vaccine dose, this value being well below that seen following resolution of acute hepatitis B. No significant changes in liver biochemistry or viral serology were observed during follow-up. No serious adverse events were noted. Administration of the single-epitope vaccine, CY-1899, initiated CTL activity, but of a magnitude lower than that observed during spontaneous HBV clearance. This low-level CTL activity was not associated with viral clearance.