丙型肝炎患者外周血单个核细胞HCV感染的电镜研究

目的 以常规电镜和免疫电镜技术,发现和证实慢性丙型肝炎患者外周血单个核细胞９ＰＢＭＣｓ）内丙型肝炎病毒（ＨＣＶ）颗粒。试图在病毒形态学和形态发生学上证实ＰＢＭＣｓ的ＨＣＶ感染和复制。方法 以逆转录多聚酶链反应（ＲＴ－ＰＣＲ）和免疫组织化学方法,分别检测２８例患者ＰＢＭＣｓ内ＨＣＶ ＲＮＡ和ＨＣＶＡｇ,对其中阳性标本重点进行电镜研究。结果 ＨＣＶ ＲＮＡ和ＨＣＶ Ａｇ阳性检出率分别为７７．２７％（１     

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 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.     

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.     

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.     

The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team.

BACKGROUND. Chronic liver disease develops in more than half of patients with post-transfusion hepatitis C, but little is known about the natural history of community-acquired hepatitis C. METHODS. In 1985 and 1986 we identified adults with acute non-A, non-B hepatitis in four counties in the United States and followed them prospectively. We used three markers to detect hepatitis C virus (HCV) infection in stored samples of serum: antibody to HCV (anti-HCV) detected by second-generation serologic assays; HCV RNA detected by polymerase-chain-reaction assay; and antibody to HCV antigen (anti-HCVAg) detected by fluorescent-antibody-blocking assay. RESULTS. Of 130 patients with non-A, non-B hepatitis, 106 (82 percent) had HCV infection, 93 were positive for anti-HCV, and 13 were positive only for HCV RNA or anti-HCVAg. Chronic hepatitis developed in 60 (62 percent) of 97 HCV-infected patients followed for 9 to 48 months, with no relation to the risk factors for infection. Ten of the 30 patients who had liver biopsies had chronic active hepatitis. In samples collected 42 to 48 months after the onset of hepatitis, HCV RNA was detected in 12 of 13 tested patients with chronic hepatitis and in all 15 tested patients with hepatitis that had resolved. Anti-HCV persisted in all but two of the initially positive patients, for a rate of antibody loss of 0.6 per 100 person-years. CONCLUSIONS. Patients with community-acquired hepatitis C have a high rate of chronic hepatitis. HCV may be a major cause of chronic liver disease in the United States, and in most patients HCV infection seems to persist for at least several years, even in the absence of active liver disease.     

In situ detection of hepatitis C virus RNA in liver tissue using a digoxigenin-labeled probe created during a polymerase chain reaction

The cellular localization of hepatitis C virus (HCV) RNA in liver tissue was studied by nonisotopic in situ hybridization using a digoxigenin-labeled cDNA probe created during a polymerase chain reaction on samples from 16 patients with chronic HCV infection. Hybridization signals were recognized in the cytoplasm of the hepatocytes, and a few hepatocytes had hybridization signals in the nucleus as well. HCV RNA positive hepatocytes were found in 1 of 9 patients with chronic persistent hepatitis, 2 of 5 patients with chronic active hepatitis, and in each of 2 patients with chronic active hepatitis and cirrhosis. Positive signals were found in many hepatocytes within the lobule in liver sections of patients with advanced chronic active hepatitis. A number of HCV RNA positive hepatocytes were found in nodules, but not in the area of fibrosis. On the other hand, positive signals were found in a few hepatocytes scattered in the lobule in a patient with chronic persistent hepatitis. The mean ALT levels in the patients with positive signal (175.6 ± 44.2 U/L) were significantly higher than in those without a signal (70.27 ± 16.1 U/L) (P < 0.05). The findings suggest that a larger amount of HCV may be present during the advanced than during the early stages of type C hepatitis and nonisotopic in situ hybridization using a digoxigenin-labeled HCV cDNA probe created during a polymerase chain reaction deserves wider application for the detection of HCV replication in specimens. © 1996 Wiley-Liss, Inc.     

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.     

Evaluation of molecular parameters for routine assessment of viremia in patients with chronic hepatitis C who are undergoing antiviral therapy

OBJECTIVE: To define the usefulness of molecular parameters in patients with chronic hepatitis C who are undergoing antiviral therapy. Anti-hepatitis C virus (HCV) treatment was monitored by determination of serum HCV load and by presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). STUDY DESIGN/METHODS: Fifty-one patients with chronic hepatitis C undergoing antiviral therapy with interferon-alpha plus ribavirin were studied. Serum HCV RNA load was tested with a quantitative assay (Amplicor HCV Monitor Test) before, during, and up to 12 months after end of treatment. If HCV RNA was not detectable, serum samples were subsequently tested with a qualitative assay (Cobas Amplicor HCV Test) and corresponding ethylenediaminetetraacetic acid (EDTA)-treated blood was checked for presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). Sustained virologic response was defined by loss of HCV RNA 12 months after the end of treatment. RESULTS: Four patients (7.8%) were found to be sustained virologic responders, 17 (33.3%) were transient virologic responders, and 30 (58.8%) were virologic nonresponders. No significant difference was found in the median pretreatment serum HCV RNA load between sustained virologic responders, transient virologic responders, and virologic nonresponders. At 1 month after start of therapy, HCV RNA was not detectable with both the serum and the PBMC assay in 12 (23.5%) of 51 patients. Four remained HCV RNA-negative until 12 months after the end of treatment. In 14 of 17 transient virologic responders, reappearance of HCV RNA was detected earlier in PBMCs than in serum. CONCLUSIONS: Based on these results in 51 patients, quantitation of baseline serum HCV RNA does not appear to be a decisive factor to the management of the individual patient. Early assessment of serum HCV RNA level after start of anti-viral treatment seems to be of major importance to identify virologic nonresponders. Reappearance of HCV RNA may be demonstrated earlier in PBMCs than in serum.     

Longitudinal study of hepatitis C viremia in chronic hepatitis C

Serial serum samples from 20 untreated patients with chronic hepatitis C virus (HCV) infection were tested for HCV RNA by a nested polymerase chain reaction assay using primers from the highly conserved 5′ noncoding region to determine the relationship between hepatitis C viremia and the activity of liver disease during the natural course of chronic HCV infection. Semiquantitation of serum HCV RNA level was achieved by testing serial 10-fold dilutions of RNA extracts to determine the end-point titer. All the patients were HCV RNA positive at presentation. There was a poor correlation between the initial HCV RNA titer and serum transaminase levels. All patients except one were persistently HCV RNA positive during a follow-up period of 1.5-15 years, although 17 (85%) had periods of normal or near-normal transaminase levels. There was no correlation between changes in the serum transaminase levels and HCV RNA titer. Patients with chronic HCV infection have persistent viremia despite fluctuations in ALT levels.     

丙型肝炎病毒Ⅱ型阳性PBMC永生化细胞株的生物学特性

目的 观察丙型肝炎病人外 因B细胞长期存活状态下其中是否仍有丙型肝炎病毒（HCV）存在,探讨建立HCV体外复制细胞模型的可能性, 方法 利用EB病毒转化B淋巴细胞技术,建立丙型肝炎患者外周血单个核细胞（PBMC）HCV阳性传代细胞株（LCL）,并应用细胞培养、染色体显示、流式细胞荧光染色、逆转录－聚合酶链反应（RT－PCR）、免疫组化、原位RT－PCR及电镜等技术研究其分子生物学和免疫学特性。结果 （1）LCL细胞核型47,XX,＋mar,细胞表面CD19、CD20抗原阳性,CD21分子消失。（2）传代培养细胞中HCV RNA正链持续12个月阳性,而培养上清中则呈间断性阳性,无明显规律。HCV RNA负链在LCL细胞中也呈间断阳性。HCV基因分型为Ⅱ型株。（3）免疫组化、原位PCR和电镜发现HCV抗原蛋白、HCV RNA和病毒颗粒均在于LCL细胞浆中;电镜发现HCV病毒颗料呈圆球型,双层膜结构,定位于细胞胸质空泡内,直径多为45－70nm,个别为110nm。结论 HCV可以在EB病毒转化病人B细胞株中长期存活、复制和分泌。     

CD81 nucleotide mutation in hepatocellular carcinoma and lack of CD81 polymorphism in patients at stages of hepatitis C virus infection

Mechanisms determining the chronicity or the pattern of clinical course of hepatitis C virus (HCV) infections have not been clarified. Recently, CD81 was reported to bind the E2 protein of HCV and was suggested to function as a cellular receptor for HCV. Accordingly, the hypothesis was examined that CD81 polymorphism, if it exists, might correlate with certain clinical courses of HCV infection. CD81 cDNA sequences were determined from peripheral blood mononuclear cells (PBMCs). Twenty-four Japanese subjects were enrolled initially as follows: patients with chronic hepatitis C without cirrhosis (n = 3), patients with cirrhosis (n = 3), patients with cirrhosis complicated by hepatocellular carcinoma (HCC) (n = 3), patients with persistent HCV viremia without ALT elevation (n = 3), those with positive anti-HCV antibodies without evidence of HCV viremia (n = 3), and healthy volunteers (n = 9). In all PBMCs samples analyzed, no polymorphism was found in the CD81 cDNA sequence. The sequence was different, however, from the one reported previously at three nucleotide positions: a transversion to thymine instead of cytosine at nt 1130, a deletion at nt 1206, and a guanine insertion at nt 71. Subsequently, CD81 cDNA sequences from PBMCs and HCC tissue were compared among the other 6 patients with chronic hepatitis C bearing HCC. A comparative study of the CD81 sequences from HCC and PBMCs revealed that various nucleotide mutations existed only in the HCC samples in 3 out of 6 patients. Several mutations in the 3' non-coding region of CD81 cDNA were observed exclusively in HCC tissue suggesting its possible role in hepatocarcinogenesis. Because of the absence of polymorphisms, however, CD81 is unlikely to affect the progression of chronic hepatitis C in terms of chronicity, hepatitis activity, or disease stage.     

Clinical significance of hepatitis C viral RNA status and its correlation to antibodies to structural HCV antigens in anti-HCV reactive patients with normal liver tests

Extensive serological testing and HCV RNA determination by RT-PCR was performed in serum, PBMCs, and liver tissue in thirteen anti-HCV reactive patients with persistently normal liver tests. Absolute concordance in the status of HCV RNA between serum, PBMCs, and liver was noted. Five patients were HCV RNA positive but only three had mild histological changes. Eight patients were HCV RNA negative in all three sites and had virtually normal liver histology. Patterns of reactivity in RIBA™ 2.0 strip immunoblot assay did not differentiate viremic from nonviremic patients. ELISA testing using multiple individual HCV recombinant antigens from the structural and non-structural regions of HCV demonstrated mean antibody titers to the structural antigens, in particular HCV E2 antibodies, to be significantly lower in HCV RNA negative patients. The status of HCV RNA in the serum appears to infer the status of HCV RNA in the liver and PBMCs in patients with persistently normal liver tests. Patients with persistently normal liver tests and undetectable HCV RNA have probably spontaneously cleared HCV infection. © 1996 Wiley-Liss, Inc.     

Decreased level of intracellular cholesterol in peripheral blood mononuclear cells is associated with chronic hepatitis C virus infection

Peripheral blood mononuclear cells (PBMCs) constitute the main extrahepatic place of, hepatitis C virus (HCV) replication. We aimed to determine the impact of CHC infection and microRNA-, 122 expression on cholesterol expression in PBMCs. HCV RNA strand, intracellular cholesterol, HMGCoA, reductase and miR-122 expression in PBMC were determined in 54 CHC patients. The study shows that significant decrease of intracellular cholesterol level in PBMC (p = 0.000000), accompanied by serum hypocholesterolemia is the characteristic feature of chronic hepatitis C infection. Although, microRNA-122 expression was detectable in PBMCs of CHC patients (52.5%), the alteration of intracellular cholesterol level was independent of miR-122 expression.     

Hepatitis C virus infection in the family setting of patients with occult hepatitis C

Family members of patients with chronic hepatitis C virus (HCV) infection are at increased risk of HCV infection but the prevalence of HCV among family members of patients with occult HCV infection is not known. Anti‐HCV, serum HCV RNA and levels of liver enzymes were determined in 102 family members of 50 index patients with occult HCV infection and in 118 family members of 59 chronic hepatitis C index patients. HCV RNA and/or anti‐HCV were detected in 10/102 (9.8%) relatives of patients with occult HCV infection and in 4/118 (3.4%) of patients with chronic hepatitis C. Fourteen additional family members (seven were relatives of index patients with occult HCV infection) had abnormal values of liver enzymes without serological markers of HCV infection. Two of these patients (who were relatives of two index patients with occult HCV infection) underwent a liver biopsy and were diagnosed with an occult HCV infection because HCV RNA was detected in the liver cells in the absence of serological HCV markers. In conclusion, the prevalence of HCV infection among family members of patients with occult HCV infection was similar to that found among family members of patients with chronic hepatitis C. This stresses the need to adopt strategies to prevent the transmission of HCV in the family setting of patients with occult HCV infection. J. Med. Virol. 81:1198–1203, 2009. © 2009 Wiley‐Liss, Inc.