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1.
Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology. 总被引:11,自引:1,他引:11
F Negro D Pacchioni Y Shimizu R H Miller G Bussolati R H Purcell F Bonino 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(6):2247-2251
A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or delta, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver cell membrane. 相似文献
2.
Chung CH Yang YH Chang TT Shieh DB Liu SY Shieh TY 《The Kaohsiung journal of medical sciences》2004,20(4):151-159
Oral lichen planus (OLP) is a relatively common skin and oral disease that manifests as a mucous reaction to a variety of etiologic factors, including autoimmune disease, drug reaction, diabetes mellitus (DM), hypertension, hepatitis C virus (HCV), urolithiasis, psychogenic factors, and bacterial infection. The purpose of this study was to investigate the relationship between HCV infection and OLP as there is a high prevalence of HCV infection in Taiwan. A total of 1,075 subjects aged at least 15 years participated in the study. The total prevalence of OLP was 3% (32/1,075). OLP was significantly associated with DM (odds ratio, OR, 3.09) and HCV (OR, 2.05). Atrophic-erosive OLP (13/32) and reticular OLP (21/32) were significantly associated with HCV and DM, respectively. Logistic regression analysis showed that elevation of alanine aminotransferase (ALT) significantly increased the risk of atrophic-erosive OLP. We concluded that OLP is significantly associated with HCV and DM in southern Taiwan, particularly in HCV patients with elevated serum ALT levels and atrophic-erosive OLP. 相似文献
3.
Yumiko Nagao Michio Sata Kazuhiro Abe Kyuichi Tanikawa Tadamitsu Kameyama 《Journal of gastroenterology》1997,32(3):324-329
Oral lichen planus (OLP) is frequently associated with hepatitis C virus (HCV) infection. We have previously reported that
the pathogenesis of OLP arises from host rather than viral factors. In this study, we investigated the role of these factors
in 41 patients with chronic hepatitis C: 22 with OLP (group 1) and 19 without OLP (group 2). All patients had antibodies to
HCV (anti-HCV) and were serum HCV RNA-positive; none were HBsAg-positive. Immune abnormalities in serum were evaluated by
testing for antinuclear antibody (ANA), rheumatoid factor (RF) activity, immunoglobulins (IgG, IgM, and IgA), and cryoglobulin.
The rate for ANA positivity and IgM levels were significantly higher in group 1 than in group 2 (P<0.05). Mean age in group 1 was significantly higher in group 2 (P=0.0001). Of the factors tested, ANA, IgM, and age, logistic regression showed that age correlated independently with OLP
(P=0.003). In 5 patients in group 1, the infiltrating lymphocytic subsets of the OLP lesion were examined histopathologically.
Predominant T cell infiltration was shown in all 5 patients. In addition to host factors, we also examined viral factors in
both groups of patients, measuring serum HCV RNA level and determining HCV genotype. There were no significant differences
between the groups in these viral factors. This study suggested that host factors induced by the HCV infection are more important
than viral factors in the pathogenesis of OLP associated with hepatitis C. 相似文献
4.
Detection of hepatitis C virus RNA in peripheral blood mononuclear cells of infected patients by in situ hybridization 总被引:5,自引:0,他引:5
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. 相似文献
5.
OBJECTIVE: Mucosa-associated lymphoid tissue, which has a follicular structure closely resembling Peyer patches, is absent in the normal gastric mucosa, but it can develop in several chronic conditions. Since we recently detected hepatitis C virus-RNA in gastric mucosa-associated lymphoid tissue of patients with chronic hepatitis C, we tried to treat hepatitis C virus infection to evaluate the effect of antiviral therapy on gastric mucosa-associated lymphoid tissue. METHODS: Eighteen patients (12 men and 6 women; mean age: 52 years, range: 33-71 years) affected by chronic hepatitis C virus and with gastric mucosa-associated lymphoid tissue were enrolled. We enrolled only patients hepatitis C virus-positive, mucosa-associated lymphoid tissue-positive, and Helicobacter pylori-negative (8 patients) or hepatitis C virus-positive patients in whom anti-H. pylori therapy did not obtain disappearance of gastric mucosa-associated lymphoid tissue (10 patients). Hepatitis C virus was evaluated by hepatic biopsy with histologic evaluation and serologic examination; Gastric mucosa-associated lymphoid tissue was scored using Wotherspoon score. All patients were treated with recombinant leukocyte interferon-alpha-2b plus oral ribavirin for 6 months. The hepatitis C virus RNA was assayed at entry and at 3 months after stopping treatment. Virologic response was defined as undetectable levels of serum hepatitis C virus RNA 3 months after stopping treatment; esophagogastroduodenoscopy was repeated at this time to evaluate the effect of anti-hepatitis C virus therapy on acquired gastric mucosa-associated lymphoid tissue. RESULTS: Two (11.11%) patients were withdrawn from the study. Hepatitis C virus cure was obtained in 11/16 patients (68.75%), and in all of them we obtained disappearance of gastric mucosa-associated lymphoid tissue (P < 0.01). Hepatitis C virus infection persisted, but with very lower levels, in 5 of 16 patients (31.25%): in 3 patients gastric mucosa-associated lymphoid tissue persisted (but in 2 it decreased from grade 3 to grade 2), while in 2 it disappeared. CONCLUSIONS: We showed clearly that there is a strict correlation between hepatitis C virus infection and acquired MALT, obtaining the disappearance of this acquired immunologic acquired gastric tissue curing hepatitis C virus infection. However, further studies are needed to clarify there is the same correlation between hepatitis C virus infection and gastric mucosa-associated lymphoid tissue lymphomas in hepatitis C virus-positive patients. 相似文献
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原位杂交法检测外周血单个核细胞中HCV RNA 总被引:4,自引:1,他引:4
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 相似文献
8.
地高辛标记原位杂交技术检测骨髓单个核细胞中HCV RNA 总被引:2,自引:0,他引:2
为探讨丙型肝炎病毒(HCV)肝外感染、复制场所与肝损害的关系,应用地高辛标记HCV RNA 探针原位杂交技术,对20例慢性丙型肝炎患者骨髓单个核细胞中的 HCV RNA(包括正链 RNA 和负链 RNA)进行了检测。结果有18例骨髓单个核细胞用正链 HCV RNA 探针杂交在较多细胞中出现杂交信号(90%),而所有患者骨髓单个核细胞用负链 HCV RNA 探针杂交无杂交信号出现。结果提示:慢性丙肝患者骨髓中存在 HCV RNA,即骨髓可受到丙肝病毒感染.但骨髓单个核细胞负链 RNA 阴性表明骨髓细胞并非 HCV 的复制场所。本研究对阐明 HCV 在人体中的致病作用以及丙肝的慢性化有一定的意义。 相似文献
9.
M Martinot-Peignoux N Boyer D Cazals-Hatem B N Pham A Gervais V Le Breton S Levy C Degott D C Valla P Marcellin 《Hepatology (Baltimore, Md.)》2001,34(5):1000-1005
A significant proportion of patients with detectable antibodies to hepatitis C virus have normal serum alanine transaminase levels. Our aim was to study the outcome of this group. Between 1992 and 1999, 135 consecutive anti-HCV-positive patients with persistently normal ALT were followed for 3.6 +/- 2.3 years (0.5 to 8.5 years), 108 had a liver biopsy at inclusion, and 24 had a second liver biopsy 3.5 +/- 1.0 years later. Serum HCV RNA was detectable with PCR in 94 patients (69%) and not detectable in 41 patients (31%). Patients with and without detectable serum HCV RNA had similar epidemiological characteristics. Serum ALT levels and anti-HCV ratio were lower (P =.001), and histological lesions had lower grade and stage in patients without detectable serum HCV RNA (P =.001). Liver HCV RNA was not detectable with PCR in the 12-serum HCV RNA-negative patients tested. During follow-up, all patients without detectable serum HCV RNA remained HCV RNA-negative and kept normal serum ALT; all patients with detectable serum HCV RNA remained HCV RNA-positive, 20 (21%) had a slight fluctuation of serum ALT above the upper limit of normal. No significant changes were observed in the liver lesions of the 24 patients who underwent a second liver biopsy. In anti-HCV-positive patients with persistently normal serum ALT, histological lesions are significantly lower in HCV RNA-negative than in HCV RNA-positive patients. During follow-up, the HCV RNA status of patients remained unchanged; 21% of the patients with detectable serum HCV RNA had slight increase in serum ALT levels, but histological lesions remained stable. 相似文献
10.
Yujiro Tanaka Nobuyuki Enomoto Shigeru Kojima Liang Tang Mieko Goto Fumiaki Marumo Chlfumi Sato 《Liver international》1993,13(4):203-208
ABSTRACT— To examine HCV infection histologically, we attempted nonradioactive in situ hybridization of HCV-RNA in the liver. We amplified cDNA probe (360 base pairs) by PCR using the primers deduced from the core region of the HCV genome. The probe was labelled with digoxigenin by PCR and used for in situ hybridization on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline-phosphatase-conjugated anti-digoxigenin and alkaline-phosphatase substrates. HCV-RNA-cDNA hybrids were detected in 21 of 24 patients with positive serum HCV markers, whereas there were no positive signals in the liver of 12 cases without HCV infection. The signal intensity of HCV-RNA-cDNA hybrids was abolished after RNase treatment. Various other specificity experiments also verified specific hybridization of HCV-RNA-cDNA. HCV-RNA was visualized in liver cells and most of them were regarded as hepatocytes from their characteristic features. The infected hepatocytes were frequently associated with mononuclear cell infiltration. Hepatocytes positive for HCV-RNA were sometimes binuclear and distributed in various patterns among cases tested. The present in situ hybridization of HCV RNA is highly sensitive and specific and the results suggest the host immune response to HCV-infected cells. 相似文献
11.
Umemura T Wang RY Schechterly C Shih JW Kiyosawa K Alter HJ 《Hepatology (Baltimore, Md.)》2006,43(1):91-99
To investigate the quantitative characteristics of humoral immunity in patients with hepatitis C, we established an enzyme-linked immunosorbent spot (ELISpot) assay for detection of anti-hepatitis C virus (HCV)-secreting B cells. Receiver operating characteristic curve analysis demonstrated 100% specificity and 58% to 92% sensitivity for detecting B-cell responses to NS5b, NS3, E2, and core antigens. The median sum of anti-HCV-secreting B cells to all HCV antigens tested was significantly higher in 39 patients with chronic hepatitis C (47.3 spot forming cells [SFCs]/10(6) peripheral blood mononuclear cells [PBMCs]) than in 9 recovered subjects (15.3 SFCs/10(6) PBMCs; P = .05) or 11 uninfected controls (5.3 SFCs/10(6) PBMCs; P < .001); the significant difference (P = .018) in chronic versus recovered patients was in reactivity to nonstructural antigens NS3 and NS5b. Anti-HCV immunoglubulin M (IgM)-secreting B cells were also readily detected and persisted decades into HCV infection; there was no difference in IgM-positive cells between chronic and recovered patients. ELISpot reactivity to genotype 1-derived antigens was equivalent in patients of genotypes 1, 2, and 3. There was significant correlation between the numbers of anti-HCV IgG-secreting B cells and serum aminotransferase and to the level of circulating antibody. In conclusion, ELISpot assays can be adapted to study B-cell as well as T-cell responses to HCV. Measurement at the single-cell level suggests that humoral immunity plays a minor role in recovery from HCV infection and that B-cell immunity is strongest in those with persistent infection. 相似文献
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Detection of hepatitis C virus in the bile and bile duct epithelial cells of hepatitis C virus-infected patients 总被引:1,自引:0,他引:1
The pathobiology of hepatitis C virus (HCV) in the biliary system has not been clarified yet, although bile duct damage is a histological finding characteristic of chronic hepatitis C. In this study, we examined whether HCV infects bile ducts and is released into the bile. Twelve patients positive for serum HCV antibody were examined in this study, and eight were seropositive for HCV RNA by polymerase chain reaction (PCR). For those who underwent abdominal surgery, the bile was aspirated from the gall bladders by fine needle puncture. Five underwent wedge liver biopsy. Series of saline-diluted bile were assayed for HCV RNA by PCR to determine the HCV RNA titers. To examine HCV expression in the biliary system, the liver specimens were immunostained using monoclonal antibodies to the HCV proteins. HCV RNA was detected in the bile of 5 patients with high serum HCV RNA load (> or = 2.5 Meq/mL). Comparison of viral loads between serum and bile revealed that the HCV RNA level in the bile was as high as that in serum. Furthermore, immunohistological study showed that bile duct epithelial cells were infected with HCV. In contrast, HCV was not found in either the bile or bile duct of patients seronegative for HCV RNA or with low serum HCV load (< or = 1.1 Meq/mL). These findings suggest that the biliary system is involved in the pathobiology of HCV and that the bile is as highly infectious as the serum. 相似文献
14.
Detection of hepatitis-C virus and hepatitis-C virus replication in hepatocellular carcinoma by in situ hybridization. 总被引:3,自引:0,他引:3
M Ohishi S Sakisaka M Harada H Koga E Taniguchi T Kawaguchi K Sasatomi M Sata T Kurohiji K Tanikawa 《Scandinavian journal of gastroenterology》1999,34(4):432-438
BACKGROUND: Although hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma (HCC), the role of this virus in carcinogenesis is not fully understood. METHODS: We studied HCV infection and replication by detecting plus- and minus-strand HCV RNA by in situ hybridization (ISH) in surgically resected HCCs and adjacent non-cancerous tissue obtained from 15 anti-HCV antibody-positive patients with HCC. RESULTS: Plus-strand HCV RNA was found in 9 of 15 HCCs. Both plus- and minus-strand HCV RNA were detected in four of these nine patients. In non-cancerous tissues obtained adjacent to the HCC, plus-strand HCV RNA was found in 10 of 15 patients, whereas both plus- and minus-strand HCV RNA were detected in 7 of these 10 patients. The degree of staining by ISH did not correlate with the differentiation of the HCC, histologic classification of the non-cancerous tissue, serum HCV RNA levels, or serum transaminase levels. CONCLUSIONS: HCV can be found in and replicates in both hepatoma cells and in non-cancerous hepatocytes in anti-HCV antibody-positive patients with HCC. The direct effects of HCV viral gene products on normal hepatocytes in the development of HCC require additional study. 相似文献
15.
Abstract: Persisient infection with hepatitis C Virus (HCV is associated with chronic hepatitis and cirrhosis which may eventually develop into primary hepatocellular carcinoma. The mechanism of pathogenesis is illdefined and nothing is known of the distribution, frequency or type of infected cell in the liver of HCV-infected individuals. In this study we have examined liver tissue taken at autopsy from 2 anti-HCV-positive patients by in situ hybridization for the presence of HCV RNA. Viral RNA was detected by autoradiography after hybridizaiton with 125I-labelled riboprobes. reprsenting approximately 35% of the HCV genome. Only a few positive cells were indentified in the HCV-infected liver samples. but not a normal liver sample. Hybridizaiton with an unrelated probe was negative in all samples. The HCV RNA-positive cells were detected with anti-sense but not sense RNA probes, suggesting that they contained a high ratio of genomic:antigenomic RNA. The appearance and distribution of the HCV RNA-positive cells suggested that they were not hepatocytes and were more likely to be lymphocytes or macrophages. 相似文献
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重型丙型肝炎患者肝外脏器病毒感染和复制状态 总被引:2,自引:0,他引:2
目的 探讨丙型肝炎患者肝外脏器丙型肝炎病毒(HCV)感染和复制状态。方法 采用逆转录-聚合酶链反应(RT-PCR)、原位杂交(ISH)和免疫组织化学法,对9例重型丙型肝炎患者肝外8种脏器内HCV基因、HCV复制中间体和HCV抗原表达进行了研究。结果 采用RT-PCR,9例患者肝外脏器均可检出HCV基因,6例(66.7%)检出HCV复制中间体及HCV抗原;采用ISH和免疫组化法,5例(55.6%)检出HCV基因。除脾脏外,心脏、肾脏、胰腺、肾上腺、肠道、胆囊和淋巴结等脏器细胞内有HCV基因和HCV抗原表达。结论 肝外多种脏器细胞可能支持HCV复制;肝外HCV感染程度低,复制水平低。 相似文献
18.
G. Li K. Li A. S. Lea N. L. Li N. E. Abdulla M. A. Eltorky M. R. Ferguson 《Journal of viral hepatitis》2013,20(3):183-192
In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. Detection of the HCV genomes in clinical specimens is useful for differential diagnosis, particularly between recurrent HCV infection and acute cellular rejection in transplant specimens. We optimized an ISH protocol that demonstrated sensitivity and specificity for detecting genomic and replicative form of HCV RNA in tissue biopsies. Digoxigenin (Dig)‐labelled sense and anti‐sense riboprobes were synthesized using a plasmid containing a fragment of the highly conserved HCV noncoding region as a template. The efficiency of the Dig‐labelled riboprobes in detecting genomic and replicative‐intermediate HCV RNA was analysed in 30 liver biopsies from patients infected or uninfected with HCV in a blinded study. A Huh7 cell line that stably replicates genome‐length HCV RNA was developed to be used as a positive control. Negative control riboprobes were used in parallel to evaluate and control for background staining. The anti‐sense probe detected HCV RNA in 20/21 specimens from HCV‐infected liver tissues obtained from patients and in 0/9 samples from patients with non‐HCV‐related liver diseases, resulting in a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA was variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes. Detection of HCV RNA by our optimized ISH protocol appears to be a sensitive and specific method when processing clinical specimens. It may also be revealing when exploring the pathophysiology of HCV infection by verifying the presence of viral genetic material within heptocytes and other cellular elements of diseased liver tissue. This methodology might also evaluate the response to antiviral therapies by demonstrating the absence or alteration of genetic material in clinical specimens from successfully treated patients. 相似文献
19.
In situ hybridization was used to identify the cell types infected by hepatitis C virus (HCV) in the liver. Using an antisense HCV-RNA probe from the 5' non-coding region, HCV-RNAs molecules were detected in liver sections of 4/11 patients with chronic hepatitis C. These 4 positive subjects were also infected by HIV. HCV-RNA-positive strands were detected in scattered hepatocytes as well as in cells identified as mononuclear cells within the inflammatory infiltrates. HCV-RNA negative strands, likely replicative intermediates, were also detected in these cells. This study therefore indicates that replication of HCV may occur in both hepatocytes and mononuclear liver cells. 相似文献
20.
RNA interference (RNAi) is being evaluated as an alternative therapeutic strategy for hepatitis C virus (HCV) infection. The use of viral vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors. However, overexpression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity. The use of endogenous microRNAs (miRNAs) as scaffolds for short interfering (siRNAs) may avoid these problems, and miRNA clusters can be engineered to express multiple RNAi effectors, a feature that may prevent RNAi-resistant HCV mutant generation. We exploited the endogenous miRNA-17-92 cluster to generate a polycistronic primary miRNA that is processed into five mature miRNAs that target different regions of the HCV genome. All five anti-HCV miRNAs were active, achieving up to 97% inhibition of Renilla luciferase (RLuc) HCV reporter plasmids. Self-complementary recombinant adeno-associated virus (scAAV) vectors were chosen for therapeutic delivery of the miRNA cluster. Expression of the miRNAs from scAAV inhibited the replication of cell culture-propagated HCV (HCVcc) by 98%, and resulted in up to 93% gene silencing of RLuc-HCV reporter plasmids in mouse liver. No hepatocellular toxicity was observed at scAAV doses as high as 5 × 10(11) vector genomes per mouse, a dose that is approximately five-fold higher than doses of scAAV-shRNA vectors that others have shown previously to be toxic in mouse liver. Conclusion: We have demonstrated that exogenous anti-HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity. The combination of an AAV vector delivery system and exploitation of the endogenous RNAi pathway is a potentially viable alternative to current HCV treatment regimens. 相似文献