EB病毒转化人外周血单个核细胞作为HCV复制模型？…

目的 复制丙型肝炎病毒（ＨＣＶ）感染的细胞模型。方法 用ＥＢ病毒感染其ＨＣＶ阳性患者的外周血单个核细胞并使其转化，获得ＨＣＶ的外周永生化Ｂ细胞株。以后，每隔１个月应用套式逆转录聚合酶链反应（ＲＴ－ＰＣＲ）检测１次培养细胞和上清中的ＨＣＶ ＲＮＡ。结果 ＨＣＶ可以在传代细胞中持续存在１年，培养细胞中的ＨＣＶ负链和培养上清中的ＨＣＶ则呈间断阳性。结中以在该细胞株中较长时间存在、复制和分泌。     

Coordinated increase of miRNA-155 and miRNA-196b expression correlates with the detection of the antigenomic strand of hepatitis C virus in peripheral blood mononuclear cells

A tight relationship has been revealed between cellular microRNAs (miRNAs) and the course of hepatitis C virus (HCV) replication in human hepatoma cells. Although the detection of the antigenomic HCV RNA strand in peripheral blood mononuclear cells (PBMCs) has provided evidence for viral replication in PBMCs, no reports have shown how miRNAs are affected upon HCV RNA synthesis in PBMCs. The aim of the present study was to assess if and how the expression levels of miRNA-155 and miRNA-196b in PBMCs are related to HCV replication in PBMCs of chronic hepatitis C (CHC) patients. Supporting analyses were performed to evaluate the expression of precursor pri-miR-155 (BIC) and Dicer protein. The genomic and antigenomic HCV RNA strands in PBMCs were detected by strand-specific qRT-PCR. The expression levels of miRNAs, BIC RNA and Dicer protein were assayed on PBMCs by qRT-PCR and Western blotting, respectively. miRNA-155 and miRNA-196b were detected in all studied PBMC samples, but their levels varied according to the presence of the antigenomic HCV RNA strand in PBMCs. Increased expression levels of miRNA-155 and miRNA-196b were associated with the presence of the antigenomic HCV RNA strand in PBMCs. In this group of patients higher frequency of BIC RNA and Dicer protein detection was also found. This study demonstrates that HCV RNA replication in PBMCs of CHC patients is connected with the increased and coordinated expression of miRNA-155 and miRNA-196b.     

乙肝病毒相关肝病患者肝移植后外周血单个核细胞及肝组织中乙型肝炎病毒cccDNA的检测

背景：肝移植后受者体内绝大部分病毒负荷被清除,植入新肝后其复发性肝炎病原体从肝外进入肝内的途径及其复制规律目前尚无定论。 目的：检测肝移植前后外周血单个核细胞和肝组织中乙型肝炎病毒cccDNA及血清中乙型肝炎病毒DNA的表达。 方法：采用淋巴细胞分离液从乙肝病毒相关终末期肝病37例患者外周血中分离出单个核细胞,采用荧光定量PCR检测肝移植前后及移植后乙肝复发3个时期外周血单个核细胞和肝组织中cccDNA及血清乙型肝炎病毒DNA表达。 结果与结论：肝移植前,单个核细胞cccDNA阳性12例,肝组织cccDNA阳性6例,检出率分别为32%和16%,单个核细胞、肝组织中cccDNA拷贝范围分别为(3.028~6.508)×10⁴,(4.158~6.234)×10⁴ 拷贝/mL。肝移植后,单个核细胞cccDNA阳性1例,无血清乙型肝炎病毒DNA检测阳性病例。6例肝移植后乙肝复发病例中外周血单个核细胞cccDNA阳性4例,肝组织活检cccDNA阳性1例,6例血清乙型肝炎病毒 DNA均为阳性。提示乙肝病毒相关终末期肝病患者肝移植后乙肝复发途径可能是残留乙肝病毒在外周单个核细胞中以cccDNA为模板复制,然后再迁移到肝脏。     

Lack of hepatitis C virus replication intermediate RNA in diseased skin tissue of chronic hepatitis C patients.

The extent of extrahepatic hepatitis C virus (HCV) replication seems to be low-level and confined to cells of hematopoietic lineage. However, given the spectrum of extrahepatic manifestations associated with HCV, several tissues other than the liver have been suggested as targets of HCV replication and damage. The presence and level of HCV RNA were examined in 19 skin tissue samples from patients chronically infected with HCV and referred for lichen ruber planus (n = 11) or cutaneous vasculitis associated with mixed cryoglobulinemia (n = 8). Serum HCV RNA was quantitated and genotyped by assays that are available commercially. Tissue HCV RNA of genomic- and minus-strand polarity was titrated by a strand-specific semiquantitative RT-PCR. Low titers of genomic-strand HCV RNA were found in three skin specimens from patients with cutaneous vasculitis due to mixed cryoglobulinemia, but in none with lichen ruber planus. The replication intermediate HCV RNA was not detected in any of the skin tissues examined, independent of the serum HCV RNA level or genotype. It is concluded that the occurrence of cutaneous vasculitis and lichen ruber planus in chronic hepatitis C patients is unlikely to be due to HCV replication in the skin.     

Intrahepatic cytokine expression is downregulated during HCV/HIV co-infection

HIV co-infection is associated with reduced HCV treatment response rates and accelerated HCV-related liver disease. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the roles of HIV and/or its therapies on cytokine expression are unknown. Total RNA was extracted from liver biopsies of 12 HCV mono-infected and 14 HCV/HIV co-infected persons. We used real-time PCR to quantify cytokines that contribute to innate and adaptive immune responses, including IFNalpha, IFNgamma, TNFalpha, TGFbeta(1), IL-2, IL-4, IL-8, IL-10, and IL-12p40. Positive- and negative-strand HCV RNA levels were quantified using a molecular beacon approach. Detection of positive-strand HCV RNA was 100% in both groups; negative-strand HCV RNA was detected in four (33%) HCV mono-infected persons and in nine (64%) HCV/HIV co-infected persons. Median strand-specific HCV RNA levels were not significantly different between the two groups. Detection rates of cytokine mRNAs were lower for the HCV/HIV co-infected group compared to the HCV mono-infected group; the detection rates for TNFalpha, IL-8, and IL-10 were statistically significant. Overall, cytokine mRNA quantities were lower for HCV/HIV co-infected compared to HCV mono-infected persons, with the exception of TGFbeta1. These data suggest that a defect in cytokine activation may occur in HCV/HIV co-infected persons that limits efficient clearance of HCV from the liver.     

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.     

环孢素治疗丙型肝炎病毒RNA阳性肾移植患者的抗病毒复制效应

背景：丙型肝炎病毒阳性患者接受肾脏移植后,免疫抑制剂的选择及抗丙型肝炎病毒药物的选用是目前关注的重点。 目的：探讨环孢素在丙型肝炎病毒RNA阳性肾移植患者中除抗排斥作用以外的抗病毒复制作用。 方法：纳入11例丙型肝炎病毒RNA阳性肾移植患者,于采用环孢素+咪唑立宾+泼 尼松治疗方案时记为入组,分别对入组前、入组后6,12个月时患者丙型肝炎病毒RNA、血红蛋白、肝肾功能等指标的变化进行检测。 结果与结论：入组前、入组6个月、入组12个月11例患者丙型肝炎病毒RNA中位数(copies/mL)分别为1.22×10⁷,1.11×10⁴,4.19×10⁶;入组6个月时,有8例患者丙型肝炎病毒RNA转阴(丙型肝炎病毒RNA<500 copies/mL),总应答率为73%(8/11);至随访结束,持续病毒学应答率为55%(6/11)。且入组治疗前后患者谷丙转氨酶、血清肌酐、血尿酸水平差异均无显著性意义(P > 0.05),患者血红蛋白水平在入组后升高。随访过程中,仅1例发生排斥反应,甲基强的松龙冲击治疗 3 d后好转。提示对于合并丙型肝炎的肾移植患者,选用环孢素为主的治疗方案,在达到抗排斥治疗作用的同时,可发挥抑制丙型肝炎病毒复制的作用。     

Study of the infection of human blood derived monocyte/macrophages with hepatitis C virus in vitro

Hepatitis C virus (HCV) is essentially hepatotropic, but clinical observations based on quasispecies composition in different compartments or on viral RNA detection in cells suggest that the virus is able to infect and persist in cells other than liver cells. It was shown previously that peripheral blood mononuclear cells (PBMCs) are permissive to HCV replication in vitro but at a very low rate. Since different viruses associated with chronic infections are known to persist in monocyte/macrophages, it is important to determine whether these mononuclear blood cells are susceptible preferentially to HCV. In order to study HCV interaction with monocytes/macrophages, these cells were isolated from the blood of healthy donors and incubated with HCV genotype 1b positive sera. The detection by RT-PCR of the positive- and negative-strand RNA in the cells at different times and the increase in the amount of intracellular viral RNA measured by the branched DNA assay suggest that monocyte/macrophages can support HCV RNA replication. The rate, however, is very low. The analysis of hypervariable region 1 (HVR-1) nucleotide sequences indicated that some minor variant present in the inoculum might display a specific tropism for the monocytes/macrophages. These results provide evidence that human monocytes/macrophages might represent a reservoir for HCV. This cell tropism may be a crucial factor in the pathogenesis of hepatitis C.     

Positive and negative strand of hepatitis C virus RNA sequences in peripheral blood mononuclear cells in patients with chronic hepatitis C: No correlation with viral genotypes 1b, 2a,and 2b

Hepatitis C virus (HCV) has many genotypes which are closely associated with the severity of chronic hepatitis and the response to antiviral therapy. Although HCV is essentially hepatotropic, several lines of evidence suggest that this virus can infect peripheral blood mononuclear cells (PBMC) in most patients with chronic HCV infection. However, the methods used previously to detect negative-strand HCV RNA have been questioned, and the PBMC tropism of different HCV genotypes remains unknown. A stringent method was used to investigate the prevalence of positive- and negative-strand HCV RNA in the PBMC of 106 patients with chronic hepatitis C and to analyze the influence of HCV genotype on the tropism of PBMC. HCV type 1b was the predominant strain in the patients. Positive-strand RNA in PBMC was detected in 83 (78%) and 40% had negative-strand RNA. The demographic and clinical features were comparable among different patients grouped by the replication status of HCV in the plasma and PBMC samples. In addition, there was no significant difference of PBMC tropism between type 1b and non-1b HCV. In summary, HCV does indeed infect actively the PBMC of chronic hepatitis C patients and such infection is not correlated to the pathogenesis of liver cell damage. Moreover, the genotype is not associated specifically with PBMC tropism of HCV. J. Med. Virol. 52:270–274, 1997. © 1997 Wiley-Liss, Inc.     

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     

Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire

Background

Co-infection with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown.

Results

HIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10) mRNA in peripheral blood mononuclear cells (PBMCs). HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production.

Conclusions

HIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.     

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