慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒感染研究

目的 以分子生物学、免疫学有电镜等技术对慢性丙型肝炎患者ＰＢＭＣｓ进行综合检测,以证实其受ＨＣＶ感染,并试图在电镜下发现和证实感染细胞内ＨＣＶ颗粒。方法 对逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和免疫组织化学等技术分别检测患者ＰＢＭＣｓ内的ＨＣＶ ＲＮＡ和ＨＣＶＡｇ,以常规和免疫电镜技术观察研究感染细胞内ＨＣＶ的形态、定位和超微结构。结果 ＨＣＶ ＲＮＡ阳性检出率为７７．２％（１７／２２）,ＨＣＶＡｇ     

Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C

ＨｅｐａｔｉｔｉｓＣｖｉｒｕｓＲＮＡｄｅｔｅｃｔｉｏｎｉｎｓｅｒｕｍａｎｄｐｅｒｉｐｈｅｒａｌｂｌｏｏｄｍｏｎｏｎｕｃｌｅａｒｃｅｌｓｏｆｐａｔｉｅｎｔｓｗｉｔｈｈｅｐａｔｉｔｉｓＣＺＨＯＵＰｉｎｇ，ＣＡＩＱｉｎｇ，ＣＨＥＮＹｏｕＣｈｕｎ，ＺＨＡ...     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒检测及其意义

目的 探讨外周血单个核细胞（ＰＢＭＣｓ）在丙型肝炎病毒（ＨＣＶ）的感染中的作用。方法 对２２例慢性丙型肝炎患者２１例抗－ＨＣＶ（＋）血液管析患者及１２例健康献血员的ＰＢＭＣｓ分别进行ＨＣＶＲＮＡ,ＨＣＶ抗原检测及电镜观察。结果 （１）２２生丙型肝炎肝炎患者ＰＢＭＣｓ中有７７．３％（１７／２２）ＨＣＶＲＮＡ阳性,（２）感染ＨＣＶ的ＰＢＭＣｓ中电镜下发现复制的ＨＣＶ颗粒;（３）ＨＣＶ颗粒阳笥者的血清和     

Testing for hepatitis C virus sequences in peripheral blood mononuclear cells of patients with chronic hepatitis C in the absence of serum hepatitis C virus RNA

Hepatitis C virus (HCV) is able to replicate in peripheral blood mononuclear cells (PBMC) of HCV-infected patients. Few data are available on PBMC testing for HCV RNA in serum HCV RNA negative patients, positive for anti-HCV and with histological evidence of chronic hepatitis. Twenty such patients were studied; of these, 11 were tested during interferon α (IFN) treatment, at the time of serum HCV RNA clearance and ALT normalisation: only one was found to be positive for HCV sequences in PBMC. Within 3 months of IFN withdrawal all 11 patients relapsed with high ALT and recurrence of serum HCV RNA. Of nine serum HCV RNA negative patients with chronic hepatitis C who were not receiving IFN when tested (four untreated patients and five patients who had already completed IFN schedule), PBMC HCV RNA was detected in four. Evidence of active HCV replication (presence of the minus strand genome) in PBMC was also observed in two cases. Thus, five of the 20 patients without detectable serum HCV RNA turned out to be carriers of HCV sequences in PBMC. These data indicate that: 1. PBMC are an extrahepatic replication site of HCV; this is true also in the absence of serum HCV RNA; 2. the role of PBMC as a “viral reservoir” after IFN-induced serum HCV RNA clearance is questioned; 3. the absence of both serum and PBMC HCV RNA in patients under IFN is not predictive of sustained viral loss; 4. testing for PBMC viral sequences might enhance the chances of detecting HCV infection.     

Detection of replicative form of hepatitis C virus RNA in peripheral blood mononuclear cells.

Paired samples of plasma and peripheral blood mononuclear cells (PBMC) from 7 patients with posttransfusion hepatitis C were collected and studied for the presence of replicative forms of hepatitis C virus (HCV). RNA was extracted and tested for both positive and negative strands of HCV RNA by polymerase chain reaction. Positive-strand RNA of HCV was found in both plasma and PBMC of all 7 patients. However, negative-strand RNA was found in PBMC of 3 patients while none was found in the plasma. Levels of negative-strand RNA were about 10-100 times less than those of positive-strand RNA by semiquantification with serial dilution of viral cDNA. The results suggest that active infection and replication of HCV in PBMC is present in patients with posttransfusion hepatitis C and implicate the extrahepatic infection of HCV.     

慢性丙型肝炎患者外周血单个核细胞HCV RNA检测及其与机体免疫功能的相关性研究

目的观察慢性丙型肝炎患者外周血单个核细胞(PBMC)HCVRNA含量及其对T淋巴细胞亚群的影响,以探讨HCV感染者PBMC中HCVRNA水平及其与机体免疫功能的关系。方法采用荧光定量PCR(FQPCR)技术对128例丙型肝炎患者血清、外周血单个核细胞的HCVRNA含量进行了检测,同时检测CD3+、CD4+、CD8+、CD4+/CD8+。结果PBMC内HCVRNA阳性组与HCVRNA阴性组比较,前者CD3+、CD4+水平降低、CD8+水平增高,CD4+/CD8+比值下降大于后者,差异有显著性(P<0.05)。结论丙型肝炎病毒侵染PBMC后可加重患者的细胞免疫功能紊乱。     

原位杂交法检测外周血单个核细胞中HCV RNA

目的比较慢性丙型肝炎患者用干扰素治疗前以及治疗后３个月ＰＢＭＣ中ＨＣＶＲＮＡ。方法应用地高辛素标记ＨＣＶＲＮＡ正链及负链探针，建立原位杂交方法检测外周血单个核细胞（ＰＭＢＣ）中的ＨＣＶＲＮＡ。结果治疗前１９例患者正链ＨＣＶＲＮＡ阳性，８例负链ＨＣＶＲＮＡ阳性，用正链探针杂交在较多的细胞中出现杂交信号，负链探针杂交仅在少数细胞中出现杂交信号，ＨＣＶＲＮＡ在ＰＢＭＣ胞浆中呈均质性分布。用干扰素治疗结束后３个月２０例患者中９例ＨＣＶＲＮＡ转阴性，近期治愈率４５％。结论原位杂交技术的敏感性及特异性较高，且重复性较好，是研究ＨＣＶＲＮＡ在组织中定位分布和病毒复制场所一种切实可行的方法     

Effect of interferon alfa and ribavirin treatment on hepatitis C virus RNA in serum and peripheral blood mononuclear cells in children with chronic hepatitis C

The correlations between the severity of hepatic lesions, age, gender, HBV co-infection and negativisation of HCV-RNA from serum and peripheral blood mononuclear cells (PBMC) after treatment of chronic hepatitis C (CHC) were analysed. 41 children (11 F/ 30 M), aged 5-16 years (mean 10 +/- 2.8), were treated with IFN-alpha and ribavirin for 12 months. Sustained negativisation of HCV-RNA from serum was achieved in 25 patients (61%), in 3 (7%) it reappeared after treatment, and in 13 (32%) it was ineffective. Clearance of HCV did not correlate with age (p = 0.65), sex (p = 0.13), past HBV infection (n = 22 anti-HBc +) (p = 0.24), maximum pre-treatment ALT activity (p = 0.06), grade of inflammation (p = 0.33) or stage of fibrosis (p = 0.9) in liver biopsy. It was achieved in 6/16 children previously resistant to IFN-a monotherapy and in 19/25 naive (p = 0.017). HCV-RNA was detected in PBMC in 9/24 (37%) seronegative children and in 1/21 (5%) in comparative group of seronegative adults; p = 0.004. Persistence of HCV-RNA in PBMC after combined treatment occurred in 5/10 (50%) patients resistant to previous IFN-alpha monotherapy, 6/35 (20%) of them cleared HCV from PBMC (p = 0.04). CONCLUSIONS: Age and gender, infection route, history of HBV infection or severity of histopathologic liver lesions had no influence on the efficacy of treatment with IFN-alpha and ribavirin. Clearance of HCV from serum and from PBMC occurs less frequently in patients previously resistant to IFN-alpha. Children with CHC require longitudinal observation after successful antiviral treatment as in 37% of those considered to be free from the virus by ordinary measures, HCV-RNA was found in PBMC.     

Infection of peripheral mononuclear blood cells by hepatitis C virus.

We investigated the infection of peripheral blood mononuclear cells (PBMNC) by hepatitis C virus (HCV) in 5 patients with HCV-related chronic hepatitis. The presence of HCV-RNA-positive and -negative strands was tested with the polymerase chain reaction (PCR) method. In all subjects, HCV-RNA was shown in PBMNC. In 3 cases, HCV-RNA was shown in the T- and B-cell populations, with viral RNA also present in the monocyte-macrophage fraction of two of these. HCV-RNA-negative stranded molecules, indicative of the viral multiplication, were significantly increased in cells maintained in cultures with PHA/PMA stimulation. The results indicate that HCV infect blood mononuclear cells, thus suggesting that this cellular tropism may play a role in HCV infection.     

HCV RNA在外周血单个核细胞中的复制与丙肝慢性化及复发的关系

通过对外周血单个核细胞（PBMC）中正链及负链HCV RNA的检测来探讨HCV在PBMC中的复制与丙肝慢性化及复发的关系。应用巢式RT—PCR对71例慢性丙型肝炎患者的血清和PBMC进行正负链HCV RNA的检测,其中15例干扰素治疗。并对10例正常人的血清和PBMC进行了HCV RNA检测。71例慢性丙型肝炎患者血清中正链HCV RNA阳性率73．2％,负链HCV RNA阳性率为0,PBMC中正链HCV RNA阳性率为60．5％,负链HCV RNA阳性率为38．0％。15例干扰素治疗患者在治疗后血清正链HCV RNA转阴率为73．3％,PBMC中正链HCV RNA转阴率为45．5％,负链转阴率为71．4％。正常对照组血清及PBMC中HCV RNA阳性率为0。这说明HCV RNA在PBMC中的复制与丙肝慢性化及复发存在一定的关系。干扰素治疗丙肝具有一定疗效,但不能彻底清除病毒。     

多抗原肽免疫血清体外阻断丙型肝炎病毒感染外周血单个核细胞的研究

目的探讨多抗原肽（MAP）免疫兔血清体外阻断丙型肝炎病毒（HCV）感染人外周血单个核细胞。方法用兔抗MAP血清按一定比例混合后接种于HCV感染的人外周血单个核细胞,采用逆转录聚合酶链反应、免疫组化和原位杂交技术分别检测细胞内或上清中HCV RNA、细胞内HCV NS3和HCV NS5抗原的表达。结果兔抗MAP血清按1：1混合后,外周血单个核细胞仍可检出HCV RNA,细胞有HCV NS3抗原表达;而按5：1和10：1混合后,单个核细胞内未检测出HCV正负链RNA,未见特异性抗原表达,原位杂交也未检测出HCV RNA存在。结论兔抗MAP血清在体外能部分阻断HCV感染。     

Detection of hepatitis C virus RNA in peripheral blood mononuclear cells of infected patients by in situ hybridization

We used in situ hybridization to detect hepatitis C virus (HCV) infection of peripheral blood mononuclear cells (PBMNC) from 11 patients with chronic active hepatitis. Using 35S-labeled HCV-RNA probe, HCV-RNA-positive and -negative strands were observed in unstimulated PBMNC from three patients, all of whom were receiving immunosuppressive drugs after orthotopic liver transplantation (OLT). HCV-RNA sequences were also identified in PBMNC from three patients who were not undergoing immunosuppression, after stimulation with either phytohemagglutinin (PHA) or pokeweed mitogen (PWM). In contrast, HCV- RNA was not found in the remaining five patients, who had not undergone OLT and whose cells were not stimulated with mitogens. These results show that mononuclear cells can be infected by HCV and that mitogenic stimulation of infected cells increases HCV-RNA replication.     

Infection of peripheral blood mononuclear cells by hepatitis C virus in mixed cryoglobulinemia

A striking association between hepatitis C virus (HCV) infection and mixed cryoglobulinemia (MC) has been shown; thus, HCV seems to play an important etiopathogenetic role in this lymphoproliferative disorder. Because HCV is both a hepatotropic and lymphotropic virus, this study aimed to investigate the prevalence of HCV infection of peripheral blood mononuclear cells (PBMCs) in a series of 16 patients with type II (IgMk) MC. Antibodies against HCV were detected by commercially available kits (Second Generation Chiron enzyme-linked immunosorbent assay [ELISA] and recombinant-based immunoblot assay [RIBA]), and the presence of HCV RNA was evaluated in both sera and isolated PBMCs using the polymerase chain reaction technique. A previous exposure to HCV was shown by ELISA and confirmed by RIBA in all cases (100%). Moreover, HCV RNA was present in the sera of 8 of 16 patients (50%), whereas its frequency markedly increased (13 of 16 [81%]) when genomic sequences were detected in peripheral lymphocytes. HCV RNA was never detected in the PBMCs of 20 control subjects. These findings showed that HCV infection, alone or in combination with other factors, may be responsible for the clonal B-cell expansion underlying the systemic manifestations of MC, and may explain the appearance of a malignant non- Hodgkin's lymphoma in some subjects.     

Hepatitis B virus markers in peripheral blood mononuclear cells]

Recent studies have shown tropism of the hepatitis B virus (HBV) by peripheral blood mononuclear cells (PBMC). The consequences of this phenomenon and their clinical use are not yet clear, however. Seventy-nine patients were studied between March 1989 and October 1990. Sixty-nine patients had chronic liver disease with histological evaluations, and 10 were vaccinated for HBV. The following markers were determined: serum: HBsAg, HBeAg, anti-HBe, antitotal-HBc, anti-HBs, anti-HCV, HBV-DNA; lysated PMBC cells: HBsAg, HBeAg. Hepatic tissue: HBsAg, HBcAg. Four groups were formed according to serology. Group I--positive HBsAg patients (n = 25) HBsAg was observed in the lysated of PBMC in 19 (76%) of the patients. HBeAg in PBMC was detected in 8 (32%), all of them showed evidence of viral replication (presence of HBcAg and/or HBV-DNA in the serum HBcAg in the tissue). Group II--antitotal HBc/anti-HBs positive (n = 14), HBsAg in PBMC was found in 5 (36%) and HBeAg in 1 (7.0%). In this patient replication markers in the serum and in the tissue (HBV-DNA, HBcAg) was also present. Three patients out of 9 anti-HBs positive had HBsAg in PBMC. Group III--seronegative patients for HBV. HBsAg was present in PBMC in 2 (6.6%) of the patients, but was absent in all of them. There was concomitant presence of HBsAg in MN and the hepatic tissue in 1 patient. Replication markers were not observed in the group. Group IV--10 asymptomatic individuals vaccinated for HBV. Except anti-HBs in serum, no other HBV marker could be identified in serum or in PBMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative-strand hepatitis C virus (HCV) RNA in peripheral blood mononuclear cells from anti-HCV-positive/HIV-infected women

BACKGROUND: Hepatitis C virus (HCV) has been reported to replicate in peripheral blood mononuclear cells (PBMCs), particularly in patients coinfected with HCV and human immunodeficiency virus (HIV). However, there are limited data regarding the prevalence of and the factors associated with extrahepatic replication. METHODS: The presence of negative-strand HCV RNA in PBMCs was evaluated by a strand-specific assay for 144 anti-HCV-positive/HIV-infected women enrolled in the Women's Interagency HIV Study. One to 5 PBMC samples obtained from each woman were tested. Multivariate analyses were used to assess for associations with the clinical and demographic characteristics of the women. RESULTS: Negative-strand HCV RNA was detected in 78 (25%) of 315 specimens, and, for 61 women (42%), > or = 1 specimen was found to have positive results. The presence of negative-strand HCV RNA in PBMCs was significantly positively associated with an HCV RNA plasma level of > or = 6.75 log copies/mL (P=.04) and consumption of > or = 7 alcoholic drinks per week (P=.02). It was also negatively associated with injection drug use occurring in the past 6 months (P=.03). A negative association with a CD4+ CD38+ DR+ cell percentage of > 10% and a positive association with acquired immunodeficiency syndrome were borderline significant (P=.05). CONCLUSIONS: HCV replication in PBMCs is common among HIV-coinfected women and appears to be a dynamic process related to lifestyle, virologic, and immunologic factors.     

Late disappearance of hepatitis C virus RNA from peripheral blood mononuclear cells in patients with chronic hepatitis C in sustained response after α-interferon therapy

OBJECTIVE: We aimed to investigate the modifications of HCV RNA (genomic and antigenomic strands) in peripheral blood mononuclear cells (PBMCs) of long-term responder patients to alpha-interferon therapy, and their usefulness as criteria of definitive HCV eradication. METHODS: We studied 10 patients with chronic hepatitis C with > 1 yr of sustained response after alpha-interferon therapy (normal alanine aminotransferase [ALT] and negative serum HCV RNA). Serum HCV RNA and genotyping were determined. Approximately 2 and 4 yr after completion of treatment we investigated the presence of HCV RNA (genomic and antigenomic strands) in PBMCs. Eight of 10 patients were rebiopsed 2 yr after discontinuation of treatment. RESULTS: The mean follow-up was 46.6 +/- 4.6 months (range, 39-51 months). In this period, all patients remained in sustained response. In the first determination, all patients had HCV RNA genomic strands and two patients had antigenomic strands detectable in PBMCs. Two years later only two patients had genomic and none had antigenomic strands detectable. After 4 yr of sustained response, eight of 10 patients lost HCV RNA from PBMCs. CONCLUSIONS: In the long-term follow-up, the majority of patients with chronic hepatitis C with sustained response after alpha-interferon therapy progressively lost HCV RNA from PBMCs. This determination in PBMCs is not a predictor of response.     

Plasmacytoid dendritic cells in acute and chronic hepatitis C virus infection

Chronic evolution of acute hepatitis C (aHC) occurs in more than 80% of patients but can frequently be prevented by early treatment with interferon (IFN)-alpha. Plasmacytoid dendritic cells (pDCs) are the major endogenous IFN-alpha producers, but their role in aHC is unknown. In this study, frequency, phenotype, and pDC function were analyzed in 13 patients with aHC and 32 patients with chronic hepatitis C (cHC) compared with 20 healthy controls, 33 sustained responders to antiviral treatment, 14 patients with acute hepatitis B (aHB), and 21 patients with nonviral inflammatory disease. In aHC, pDCs in the peripheral blood were significantly reduced compared with healthy controls (median, 0.1% vs. 0.36%, P < .0005) and were inversely correlated to alanine aminotransferase levels (r = -0.823; P < .005). Circulating pDCs in aHC were immature, as determined via reduced expression of HLA-DR and CCR7, and produced little amounts of IFN-alpha (median, 3.5 pg/50,000 peripheral blood mononuclear cells [PBMCs] vs. 498.4 pg/50,000 PBMCs in healthy controls; P < .0005). Less pronounced changes were present in cHC (median, 0.17%, 28.0 pg/50,000 PBMCs IFN-alpha, respectively). However, a significantly reduced frequency and IFN-alpha production was also found in self-limited aHB (median 0.1%, 8.6 pg/50,000 PBMCs) and in patients with nonviral inflammatory disease (median 0.19%, 7.5 pg/50,000 PBMCs). In conclusion, in aHC frequency and IFN-alpha-producing capacity of peripheral blood pDCs are dramatically reduced and inversely correlated with the degree of liver inflammation. In cHC there is incomplete recovery of pDC function, which, however, could be solely due to the chronic inflammatory state.