外周血单个核细包中乙型肝炎病毒感染的研究进展

乙型肝炎病毒（ＨＢＶ）感染乙肝（ＨＢ）患者外用血单个核细胞（ＰＢＭＣｓ），ＨＢＶ－ＤＮＡ以游离和整合型两种形式存在于ＰＭＢＣｓ内。在ＰＢＭＣｓ内复制与表达。研究ＰＢＭＣｓ内ＨＢＶ感染对于探讨ＨＢ的临床进程、治愈和指导治疗等均具有重要的意义。最近提出了一种与上述相反的观点，ＰＢＭＣｓ内有关的ＨＢＶ－ＤＮＡ和ＲＮＡ是吸附的结果，而非病毒的复制。     

外周血单个核细胞中乙型肝炎病毒感染的研究进展

乙型肝炎病毒（ＨＢＶ）感染乙肝（ＨＢ）患者外周血单个核细胞（ＰＢＭＣｓ），ＨＢＶ－ＤＮＡ以游离和整合型两种形式存在于ＰＢＭＣｓ内。在ＰＢＭＣｓ内复制与表达。研究ＰＢＭＣｓ内ＨＢＶ感染对于探讨ＨＢ的临床进程、治愈和指导治疗等均具有重要的意义。最近提出了一种与上述相反的观点，ＰＢＭＣｓ内有关的ＨＢＶ－ＤＮＡ和ＲＮＡ是吸附的结果，而非病毒的复制。     

套式PCR和原位杂交技术检测肝病患者单个核细胞？…

目的 了解慢性乙型肝炎（中度）和原发性肝癌（ＨＢｓＡｇ阳性）患者ＰＢＭＣ内ＴＴＶ ＤＮＡ存在情况。方法 采用套式ＰＣＲ以及原位杂交技术检测外周血单个核细胞（ＰＢＭＣ）内ＴＴＶ ＤＮＡ。结果 套式ＰＣＲＳＷＩＭ２６例慢性乙型肝炎（中度）患者ＰＢＭＣ内ＴＴＶ ＤＮＡ阳性７例。阳性率２６．９％,非常显著高于健康对照（Ｘ＾１２＝１４．３,Ｐ〈０．００１）;２１例原发性肝癌（ＨＢｓＡｇ阳性）虱ＰＢＭＣ内ＴＴ     

HBV基因组转梁对小鼠NIH3T3细胞内基质金属蛋白酶组织抑制？…

目的：阐明乙型肝炎病毒（ＨＢＶ）基因组转染对基质金属蛋白酶组织抑制因子的转录调控作用。方法：应用钙磷酸钙转染技术构建表达ＨＢｓＡｇ和ＨＢｅＡｇ的小鼠ＮＩＨ３Ｔ３细胞克隆，以该细胞为模型，应用原位杂交技术测定细胞内特异性ＴＩＭＰ ＲＮＡ含量变化。结果：表达ＨＢｓＡｇ和ＨＢｅＡｇ细胞内ＴＩＭＰ１和ＴＩＭＰ２ ＲＮＡ含量显著升高。     

HCV感染外周血单个核细胞及其对T淋巴细胞亚群的影响

目的：研究丙型肝炎病毒（ＨＣＶ）感染外周血单个核细胞（ＰＢＭＣ）的情况及其对Ｔ淋巴细胞亚群的影响。方法：运用非同位素原位杂交（ＮＩＳＨ）法和链酶亲和素－生物素（ＳＡＢＣ）法分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ－ＲＮＡ和非结构（Ｎｏｎｓｔｒｕｃｔｕｒａｌ,ＮＳ）蛋白ＮＳ５抗原,同时用ＳＡＢＣ法检测其Ｔ淋巴细胞亚群。结果：８例（４０．０％）患者的ＰＢＭＣ中ＨＣＶ－ＲＮＡ呈构（Ｎｏｎｓｔｒｕ     

丙型肝炎患者血清和外周血单个核细胞中HCVRNA检测及其临床意义

丙型肝炎患者血清和外周血单个核细胞中ＨＣＶＲＮＡ检测及其临床意义空军总医院传染科应用套式ＰＣＲ检测了８８例丙型肝炎患者血清和外周血单个核细胞（ＰＢＭＣ）中ＨＣＶＲＮＡ；结果发现：慢性丙肝患者ＰＢＭＣ中ＨＣＶＲＮＡ检出率显著高于急性丙肝患者（Ｐ＜０．０...     

乙/丙型肝炎病毒双重感染患者前C区终止变异低频率

目的了解乙型肝炎病毒（ＨＢＶ）与丙型肝炎病毒（ＨＣＶ）双重感染患者前Ｃ区基因变异，及其可能的临床意义。方法用聚合酶链反应（ＰＣＲ）与限制片段长度多态性（ＲＦＬＰ）来分析２５例ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性（Ａ组）和３１例ＨＢｓＡｇ和ＨＢＶＤＮＡ阳性但抗－ＨＣＶ和ＨＣＶＲＮＡ均阴性（Ｂ组）的慢性肝病患者前Ｃ区密码２８终止变异（终２８）。结果ＨＢＶ和ＨＣＶ双重感染患者（Ａ组）血清ＨＢＶＤＮＡ第１次ＰＣＲ阳性率（１６％）明显低于单独ＨＢＶ感染组（６５％）（Ｐ＜０．００１）；前Ｃ终２８检出率（２８％）亦明显低于单独ＨＢＶ感染（６８％）（Ｐ＜０．００１）。结论提示双重感染患者ＨＢＶ前Ｃ终止变异低频率可能与ＨＢＶ低水平复制有关     

在急、慢性HCV感染外周血单核细胞中HCV-RNA比较

在急、慢性ＨＣＶ感染外周血单核细胞中ＨＣＶ－ＲＮＡ比较［英］／ＴｉｎｇＴｓｕｎｇＣｈａｎｇ…／／Ｈｅｐａｔｏｌｏｇｙ，Ｍａｙ．１９９６，２３（５）．－９９７～９８１ＨＣＶ可以感染慢性乙型肝炎患者外周血单核细胞（ＰＢＭＣ）。而没有急性丙型肝炎ＰＢＭＣ存...     

HGV／GBV—C与HCV混合感染者HGV／GBV—C复制中间体的研究

目的 关于ＨＧＶ／ＢＧＶ－Ｃ组织亲和性的研究尚无结论性资料。我们研究了ＨＧＶ／ＧＢＶ－Ｃ与ＨＣＶ混合感染者ＰＢＭＣ和肝脏中ＨＧＶ／ＧＢＶ－Ｃ复制中间体（负链ＲＮＡ）的存在状况。方法 应用逆转录－巢式ＰＣＲ技术,检测了３２例肝炎患者ＨＧＶ／ＧＢＶ－Ｃ和ＨＶＣ正、负链ＲＮＡ。结果 有２６例ＨＧＶ／ＧＢＶ－Ｃ与ＨＣＶ混合感染者ＰＢＭＣ和肝脏中均未检测到ＨＧＶ／ＧＢＶ－Ｃ负链ＲＮＡ;而在９份ＰＢＭＣ和１５     

乙型肝炎病毒感染者癌基因蛋白的表达及与HBeAg的关系

目的为了阐明癌基因蛋白异常表达与乙型肝炎病毒（ＨＢＶ）复制的关系。方法用链霉菌素－生物素（ＳＬＡＢ）免疫组织化学染色和酶联免疫吸附试验（ＥＬＩＳＡ）方法，检测了６４例慢性ＨＢＶ感染者肝细胞中Ｃ－ｅｒｂＢ－２Ｐ１８５、ｒａｓＰ２１和Ｐ５３等癌基因蛋白和血清乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）。结果Ｃ－ｅｒｂＢ－２Ｐ１８５和ｒａｓＰ２１阳性组中，血清ＨＢｅＡｇ的阳性检出率分别为８９４％和８４６％，而阴性组ＨＢｅＡｇ的检出率分别为２０％和４８％，两组差异显著。结论表明癌基因蛋白Ｃ－ｅｒｂＢ－２Ｐ１８５和ｒａｓＰ２１的异常表达与ＨＢＶ复制密切相关。     

Identification and visualization of virus-like particles in peripheral blood mononuclear cells]

OBJECTIVE: PBMCs from patients with chronic hepatitis C were examined by electron microscopy (EM), immune EM (IEM) combined with immunohistochemistry to trace the infection and morphology of HCV in the PBMCs. METHODS: The PBMCs from 28 patients with chronic hepatitis C were analyzed firstly for HCV RNA and HCV antigens by RT-PCR and immunohistochemistry respectively. The PBMCs with positive HCV RNA and HCV antigens were then observed by electron microscopy. RESULTS: The positive rate of HCV RNA and HCV antigens were 77.27% (17/22) and 75.00% (21/28), respectively. Two types of polymorphic HCV-like particles with diameter of approximately 65 nm and 110 nm were observed in cytoplasmic vesicles of the PBMCs from HCV Ag positive patients. 10 patients with HCV Ag over expression were studied by electron microscopy and immune election microscopy. The budding phenomenon of the particles could be also visualized in the cytoplasmic vesicles of the PBMCs. Some ultrastructural changes showed an increased ratio of cytoplasm to nucleus and pyknosis of nucleus was also suggestive of the virus infection. CONCLUSIONS: The EM study demonstrated the infection and replication of HCV in the PBMCs of chronic hepatitis C patients by morphology and morphogenesis.     

HCV replication in mononuclear cells stimulates anti-HCV-secreting B cells and reflects nonresponsiveness to interferon-α

Recently, it was demonstrated in chronic hepatitis C that the release of IgG and IgM anti-HCV antibodies by mononuclear cells (PBMCs) correlated with inflammatory activity, HCV persistence in serum, and negative outcome from antiviral therapy. Thus, persistent antigenic stimulation of the antibody-secreting B cells has been suggested. In this study, PBMCs were derived from 13 patients with chronic hepatitis C. Nucleic acids were extracted by the guanidine-thiocy-anate-method, and plus- and minus-stranded HCV-RNAs were determined using primers from the 5′-untranslated region of HCV. Simultaneously, unstimulated PBMCs were cultured for 8 days and anti-HCV antibodies were detected in the supernatants by EIA and RIBA. Seven patients (53.8%) had both plus- and minus-stranded HCV-RNA in PBMCs, while anti-HCV antibodies were secreted in vitro. One of 2 patients with plus- but not minus-stranded HCV- RNA in PBMCs was anti-HCV positive in vitro, whereas 4 patients without HCV-infected PBMCs were anti-HCV negative in vitro. Eight patients received antiviral therapy with interferon-α2b. Four nonresponders and 1 partial responder had plus- and minus-stranded HCV- RNA in PBMCs and anti-HCV secretion in vitro. On the other hand, 2 complete responders and another partial responder showed neither HCV infection of PBMCs nor anti-HCV secretion in vitro. In conclusion, infection of PBMCs by HCV is observed frequently in patients with chronic hepatitis C, and may be related to the high rate of nonresponders to antiviral treatment. The close correlation of anti-HCV secretion in vitro and viral replication suggests that the humoral response to HCV is triggered by viral antigens that are expressed on infected mononuclear cells. © 1995 Wiley-Liss, Inc.     

Fluorescent "in situ" hybridization of hepatitis C virus RNA in peripheral blood mononuclear cells from patients with chronic hepatitis C

Although the liver is the main target for hepatitis C virus (HCV) infection, HCV RNA of positive and negative polarity has also been detected in peripheral blood mononuclear cells (PBMCs) by polymerase chain reaction. However, no data have been published on the relationship between the number of HCV-infected PBMCs and serum viremia levels. To address this issue, PBMC samples from 20 patients with chronic hepatitis C were examined by fluorescent "in situ" hybridization. Serum viremia levels and viral load in infected PBMC were measured using the Amplicor Monitor test. HCV was detected in all PBMC samples corresponding to the HCV-positive patients. Fluorescent signals were found mainly in the cytoplasm of the cell. The percentage of positive cells ranged from 0.08% to 4%, with a statistical correlation with the viral load in PBMC (r = 0.69; p =. 001) but not with the serum viremia levels (r = 0.23). It was demonstrated that HCV infection of PBMCs is a common feature of HCV chronic carriers. The results suggest that HCV infection of PBMCs does not contribute significantly to HCV viremia.     

丙型肝炎病毒核心蛋白在患者外周血单个核细胞内的核表达检测

目的　检测和研究丙型肝炎病毒 (hepatitisCvirus,HCV)核心蛋白在患者外周血单个核细胞 (peripheralbloodmononuclearcells ,PBMC)内核表达的意义 ,并探讨其与临床的关系。方法　对 6 6例慢性丙型肝炎患者PBMC标本进行免疫组化检测 ,并将HCV蛋白抗原定位分布情况与患者临床状况进行比较分析 ,对其中 2 7例患者PBMC进行HCVRNA和HCVAg的平行检测和分析。结果　免疫组化结果显示 ,慢性丙型肝炎患者PBMCHCVAg(core +NS3)阳性检出率为 77 2 7% (5 1 6 6 )。结果还证实 ,HCV核心蛋白均定位于胞核内 ,且呈强表达 ;NS3蛋白主要定位于胞质内 ,呈弱表达。当进行HCVAg在PBMC内定位情况与患者临床状况比较分析时显示 ,病情较重患者PBMC内核心蛋白表达阳性率 (35 2 9% )明显高于病情较轻者 (5 88% ) (P <0 0 0 1)。结论　HCV核心蛋白在PBMC内核表达与患者临床状况相关 ,提示其可能是丙型肝炎慢性化的一个指标 ,并可能在肝硬化和肝癌发生上起一定作用     

Detection of hepatitis C virus RNA in peripheral blood mononuclear cells and in saliva

The nested polymerase chain reaction (PCR) technique was applied to investigate hepatitis C virus (HCV) RNA in the peripheral blood mononuclear cells (PBMCs), saliva, and serum of patients with chronic type C hepatitis. The specificity of the amplified products was analyzed and confirmed by agarose gel electrophoresis, Southern blot hybridization, and restriction endonuclease pattern analysis. HCV RNA was detectable in the PBMCs of 24% (12/50) of the patients. The HCV RNA detected in PBMCs was not due to the contamination from plasma, since no viral sequences could be detected in the third washing of PBMCs. Of the 12 patients with HCV RNA in PBMCs, five were negative for HCV RNA sequences in the serum. Thus the presence of HCV RNA in PBMCs was not strictly correlated to the results for sera. Among 25 patients with HCV RNA in their saliva, 18 were negative for PBMCs. Among 25 patients without HCV RNA in their saliva, five had HCV RNA in PBMCs. In conclusion, PBMCs are an extrahepatic target for chronic HCV infection. However, we do not suggest that PBMCs act as a vehicle for carrying HCV to saliva, since the presence of HCV RNA in PBMCs and in saliva was not closely correlated.     

Prominent proliferative response of peripheral blood mononuclear cells to a recombinant non-structural (NS3) protein of hepatitis C virus in patients with chronic hepatitis C.

The proliferative response of peripheral blood mononuclear cells (PBMC) to a recombinant non-structural (NS3) protein of hepatitis C virus (HCV) was studied in 41 patients with chronic hepatitis C. Of them, 28 had chronic persistent hepatitis (CPH) and 13 chronic active hepatitis (CAH). The positive proliferation rate of PBMC to the recombinant NS3 protein, T9Ag, was 66% in the 41 patients (77% in CAH versus 61% in CPH; P > 0.05) when stimulation index (SI) = 4 was set as the cut-off value. However, mean SI of CAH patients was significantly higher than that of CPH patients (8.3 +/- 5.2 versus 5.1 +/- 3.6; P < 0.05). Six other chronic hepatitis patients who were repeatedly negative for anti-HCV antibody but positive for serum HCV RNA also had an SI of > or = 4.0. The frequency of cellular immune response to the T9Ag is among the highest results obtained by using HCV antigens tested so far. Our studies thus indicate that NS3 is an immunologically important region of HCV for T cells. Moreover, the proliferative response to T9Ag may help to establish hepatitis C etiology in chronic hepatitis patients who are seronegative with currently available anti-HCV assays.     

A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells

Hepatitis C virus (HCV) is reported to be lymphotropic under certain circumstances. In order to evaluate viral replication in peripheral blood mononuclear cells (PBMCs), a novel strand-specific RT-PCR was developed for the determination of HCV negative-strand RNA. The detection limit of this strand-specific RT-PCR was 100 copies of HCV negative-strand RNA in the presence of 1 μg liver RNA and 10⁷–10⁸ copies of positive-strand RNA. False positive PCR signals occurred only when HCV positive-strand RNA exceeded 10⁹ copies. With this RT-PCR, the replicative-intermediates could be detected specifically in eight of ten liver tissues, but not in any samples of serum or plasma (0/65) of patients with chronic hepatitis C. When examining the PBMCs of 46 hepatitis C patients, including 12 patients who had undergone orthotopic liver transplantation, HCV negative-strand RNA was detected in only one patient (1/46). In addition, HCV replicative intermediates were not detected in PBMCs of patients using immunosuppressive agents. It is concluded that the replication of HCV in PBMCs is very unusual.     

Detection of the hepatitis C virus genome in acute and chronic experimental infection in chimpanzees.

In order to gain an understanding of the relationship of various markers of hepatitis C virus (HCV) infection in acute and chronic cases of the disease, serial blood samples obtained from chimpanzees before and after infection with HCV were analyzed for the presence of the HCV genome by using polymerase chain reaction (PCR) amplification of cDNA (cDNA PCR) synthesized from plasma- and serum-derived RNA. In a chimpanzee with acute hepatitis C, signals detectable by cDNA PCR appeared 1 week before characteristic ultrastructural changes visualized by electron microscopy, persisted throughout the peak alanine aminotransferase levels, and diminished with the disappearance of alterations visualized by electron microscopy. This was in contrast to the results obtained from chimpanzees with chronic HCV infection, in which the HCV genome was consistently detectable for up to 10 years after infection. The results indicate the usefulness of detection of HCV RNA by cDNA PCR as a sensitive and semiquantitative method for monitoring the course of HCV infection and as a potential marker for differentiating between chronic and acute cases of disease.     

Detection of Occult Hepatitis C and Hepatitis B Virus Infections from Peripheral Blood Mononuclear Cells

Purpose: To investigate the problem of occult HCV & HBV infections in patients with persistently longstanding abnormal liver function test results of unknown etiology and to investigate occult HCV in patients with sustained virological response (SVR). Methods: The study included two groups; first group included 40 patients with persistently longstanding abnormal liver function test, in addition to 62 patients with history of hepatitis C who developed SVR. HCV RNA status was tested in serum by conventional RT-PCR and by real-time PCR in Peripheral Blood Mononuclear Cells (PBMCs). HBV DNA in PBMCs was done in first group only. Results: In first group, PCR in PBMCs was positive for HCV RNA in 4 patients with elevated liver enzymes and HBV DNA was positive in PBMCs in 3 patients. In patients with SVR, 7 patients were positive for HCV RNA in PBMCs. Conclusions: Patients with long-standing abnormal results of liver-function tests with unknown etiology may have HCV RNA or HBV DNA in their PBMCs in the absence of anti-HCV antibodies, HBV markers, serum HBV DNA and serum HCV RNA.In Patients with SVR, HCV RNA in PBMCs is recommended to detect residual infection especially in those with high serum HCV RNA levels before treatment.     

Intrahepatic expression of hepatitis B virus antigens: effect of hepatitis C virus infection.

Coinfection with hepatitis B and C viruses (HBV, HCV) is not uncommon, but the expression of HBV antigens in the liver of patients with concomitant HCV infection has not been investigated. This study aimed to evaluate the effects of concomitant HCV infection on the intrahepatic expression of HBV antigens in chronic hepatitis. HBV surface and core antigens (HBsAg, HBcAg) were immunohistochemically evaluated and semiquantitatively scored in liver biopsy specimens from patients with chronic hepatitis, comprising 17 cases with dual HBV/HCV infection and 25 with HBV infection alone. The prevalence of HBV Ag expression proved significantly lower in the group with dual infection. In the presence of active HBV replication (HBV DNA-positive serum) the prevalence of HBsAg and HBcAg immunoreaction was similar in the two groups, though a significantly lower percentage of cells expressed HBcAg in the group of coinfected patients. HBV Ag was not detected at all among HBV DNA-negative/HCV RNA-positive cases. In conclusion, these observations suggest that HCV might influence HBV antigen expression in the liver and that either partial or complete suppression might occur.