Quantitation of hepatitis C virus in liver and peripheral blood mononuclear cells from patients with chronic hepatitis C virus infection

Since the natural history of hepatitis C virus-associated liver disease and the therapeutic responsiveness might vary according to liver and blood mononuclear cells viral levels, it may be important to quantitate viral RNA in liver, blood mononuclear cells and serum, and to compare these data with genotype, biochemical and histologic data. A polymerase chain reaction-based assay available for serum hepatitis C virus RNA quantitation has been optimized to quantitate viral genomes in liver and peripheral blood mononuclear cells from 47 chronic hepatitis C patients. The procedure permitted hepatitis C virus RNA quantitation in freshly isolated mononuclear cells and in total RNA extracted from frozen mononuclear cells and liver tissue. The intrahepatic viral amount (median: 2.6 × 10³ copies/μg RNA; range: 0 to 3.6 × 10⁴ copies/μg RNA) correlated significantly with the hepatitis C virus RNA concentration in serum (r = 0.76, P < .001) but not in mononuclear cells. Viral RNA concentrations in liver (P < .001), serum (P < 0.01) and PBMC (P < 0.05) were significantly higher in hepatitis C virus genotype 1 patients (essentially type 1b) than in non-1 type cases, but were unrelated to biochemical or histologic indexes of disease activity. In conclusion, the optimized assay permit HCV RNA quantitation in liver and peripheral blood mononuclear cells, suggesting that serum viral level is an accurate measurement of intrahepatic viral burden. J. Med. Virol. 54:265–270, 1998. © 1998 Wiley-Liss, Inc.     

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.     

慢性乙型肝炎患者外周血单个核细胞Toll样受体3的表达降低

目的 探讨慢性乙型肝炎患者外周血单个核细胞Toll样受体3(TLR3)的表达及其临床意义.方法 分别采集慢性乙型肝炎患者和健康志愿者外周血,荧光定量PCR法检测血清HBV DNA复制水平;使用RT-PCR、流式细胞术以及免疫印迹技术分别检测外周血单个核细胞TLR3的mRNA、蛋白的表达;使用ELISA法检测血清中肿瘤坏死因子α(TNF-α)和干扰素β(IFN-p)水平.结果 慢性乙型肝炎患者外周血单个核细胞中的TLR3表达显著低于健康志愿者,且降低水平与血清HBV DNA复制水平相关;慢性乙型肝炎患者外周血TNF-α、IFN-β浓度显著低于健康志愿者,且降低的水平与血清HBV DNA复制水平相关.结论 慢性乙型肝炎患者外周血单个核细胞TLR3的表达与乙肝病毒的复制水平相关.     

丙型病毒性肝炎患者PBMC mIL-2R的表达

目的　探讨丙型病毒性肝炎患者膜白介素 2受体 (mIL 2R)表达水平及其在丙肝转归中的作用。方法　用PCR和生物素 链霉亲和素法对 78例丙肝患者外周血单个核细胞 (PBMC)分别进行HCV RNA和植物血凝素诱导前后mIL 2R的检测。结果　丙肝患者PBMC静息期和诱导期mIL 2R表达水平分别为 (2 .94± 0 .88) %、(31.5 3± 3.38) % ,与正常对照相比 ,差异有显著性 (P <0 .0 1)。其中 ,急性丙肝患者静息期和诱导期mIL 2R表达水平分别为 (3.2 5± 0 .94 ) %、(32 .82± 3.84 ) % ,慢性丙肝患者静息期和诱导期mIL 2R表达水平分别为 (2 .77± 0 .84 ) %、(30 .97± 3.16 ) % ,两者相比差异有显著性 (P <0 .0 5 )。PBMC内HCV RNA(+)者静息期和诱导期mIL 2R表达水平分别为 (2 .37± 1.16 ) %、(30 .4 1± 4 .0 1) % ,PBMC内HCV RNA(- )者静息期和诱导期mIL 2R表达水平分别为 (3.2 1± 0 .80 ) %、(32 .15± 3.0 9) % ,两者相比差异有显著性 (P <0 .0 5 )。结论　丙肝患者体内mIL 2R水平降低 ,与丙肝的慢性化程度似有一定关系 ;HCV侵入PBMC后可进一步抑制mIL 2R的表达     

Hepatitis C virus infection of mononuclear cells from peripheral blood and liver infiltrates in chronically infected patients

The mechanisms underlying chronicity of hepatitis C virus (HCV) infection are poorly understood, but the importance of impaired viral clearance by the immune system has been suggested. The prevalence of HCV infection of peripheral blood mononuclear cells (PBMC) was in investigated in 34 persistently infected patients with anti-HCV (7 with liver cirrhosis, 10 with chronic active hepatitis, 5 with chronic persistent hepatitis, 4 with chronic lobular hepatitis, and 8 healthy carriers) by polymerase chain reaction (PCR). HCV infection of 116 T cell clones derived from liver infiltrating mononuclear cells obtained from 3 patients with chronic liver disease was examined using the same methods. HCV genomic sequences were found in fresh, unstimulated PBMC from 20 patients with cirrhosis, and chronic active and persistent hepatitis, but in none of the healthy carriers and only in mitogen-activated cells from 1 out of 4 patients with autoresolving chronic lobular hepatitis. Active PBMC infection was confirmed by identification of anti-genomic HCV sequences in the majority of HCV RNA-positive cells (fresh or mitogen-stimulated). A high percentage of T cell clones obtained from liver infiltrates were found to be infected by HCV. These findings suggest that HCV infection of lymphatic cells plays a role in the pathogenesis of chronically evolving liver damage. PBMC may represent a reservoir for latent infection and a site for viral multiplication. © 1995 Wiley-Liss, Inc.     

IL-12对慢性乙型肝炎患者T_(H1)/T_(H2)分化的影响

目的观察 IL- 12对慢性乙肝患者 TH1 / TH2 类细胞分化的影响。方法分离 5 0例慢性乙型肝炎患者 PBMC,分别与植物血凝素 (PHA ,10 0μg/ m L )、HBc Ag(1μg/ m L )、HBe Ag(1μg/ m L )单独或联合 IL - 12 (10 ng/ m L )体外培养 48h,EL ISA法检测培养上清液中 IL - 2、IFN -γ、IL - 4、IL - 10水平。 2 0例健康人群做对照。结果 IL - 12对健康人群 PBMC产生 TH1 /TH2 类细胞因子无显著影响 ,但对慢性乙型肝炎患者则显著增强 PBMC产生 IFN-γ,且慢性中度患者最为突出。 IL - 12与HBe Ag联合诱导 ,不但显著增强慢性乙型肝炎患者 PBMC产生 IL- 2和 IFN- γ,还抑制 IL- 4和 IL- 10的产生。结论 IL- 12可增强慢性乙型肝炎患者 IFN-γ优势表达 ,可促进 HBe Ag诱导的 TH2 型优势表达向 TH1 型优势表达转换     

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     

Quantitation of hepatitis C virus RNA in peripheral blood mononuclear cells in HCV-monoinfection and HIV/HCV-coinfection

Peripheral blood mononuclear cells (PBMCs) represent an extrahepatic hepatitis C virus (HCV) reservoir, the significance of which is unclear due to limited studies and varying test methodologies. In this study, a commercial viral load assay for measuring cell‐associated PBMC HCV RNA was evaluated. HCV RNA was extracted from PBMCs, sorted CD14+, and CD19+ cells and corresponding plasma samples using the Abbott m2000 and Real‐Time HCV assay. Test performance and influence of HIV seropositivity on plasma and PBMC HCV RNA were studied. Among 51 patients, 67 and 62 unique patient samples had detectable plasma and PBMC HCV viral load, respectively. The median PBMC viral load was 535 IU/1 M cells (range 29–5,190). CD19+ cells had significantly higher viral load than CD14+ cells (median log₁₀ HCV viral load 2.63 vs. 1.50 IU/ml; P< 0.001). Stability of PBMC viral load over time was demonstrated in untreated patients; all patients with an undetectable plasma HCV viral load after HCV treatment also demonstrated undetectable PBMC viral load. Repeated testing in nine samples yielded consistent PBMC viral load, differing by only 1.3‐fold (range 1.0–1.7‐fold). Among samples with detectable plasma HCV RNA, the correlation between PBMC and plasma viral load was moderate (r = 0.66) and was greater among HCV mono‐infected compared to HIV/HCV co‐infected subjects (r = 0.80 vs. 0.52). Measurement of cell‐associated PBMC HCV RNA using a commercial assay demonstrated promising test characteristics. Differences in PBMC HCV viral load based on HIV‐coinfection status and the significance of greater copy number in B‐cells requires further study. J. Med. Virol. 84:431–437, 2012. © 2011 Wiley Periodicals, Inc.     

High-density lipoprotein binding rate differs greatly between genotypes 1b and 2a/2b of hepatitis C virus

The hepatitis C virus (HCV) virion is associated with lipoproteins and immunoglobulins in the sera of patients with chronic hepatitis C; however, an accurate binding rate of HCV to lipoproteins or immunoglobulins has not yet been elucidated. Therefore, the accurate binding rate of HCV to low-density lipoproteins (LDL), high-density lipoproteins (HDL), and immunoglobulins was measured quantitatively by a real-time PCR assay. The immunoglobulin binding rate of HCV was found to be greater than 97.5% in most patients, as compared with an LDL binding rate of greater than 80% in most patients. In contrast, the HDL binding rate was greater than 98% in the genotype 2a/2b patients, while it varied in the genotype 1b patients. The genotype 2a/2b HCV not only had a higher LDL binding rate but also had a strikingly higher HDL binding rate than that of the genotype 1b HCV. These lipoprotein binding rates correlated neither to any patient's variables, including the serum apolipoprotein levels, nor to the viral load or the hypervariable region 1 (HVR 1) amino acid sequences. Most of the HCV virions in the sera of such patients have been shown to be associated simultaneously with immunoglobulins and LDL and/or HDL, but not exclusively.     

Detection of hepatitis C virus (HCV) proteins by immunofluorescence and HCV RNA genomic sequences by non-isotopic in situ hybridization in bone marrow and peripheral blood mononuclear cells of chronically HCV-infected patients

Immunofluorescence (IF) to detect HCV antigens and non-isotopic in situ hybridization (NISH) to detect HCV RNA genome were carried out on bone marrow (BM) and peripheral blood (PB) mononuclear cells (MC) of 11 chronically HCV-infected patients. In four patients (36·4%) HCV antigens were detected in monocytes/macrophages as well as in B lymphocytes in both BMMC and PBMC. Positive T lymphocytes in BMMC were found in three of them, but only one patient showed positive T cells in PBMC. NISH invariably demonstrated minus and plus HCV RNA genomic strands either in monocytes/macrophages or B and T lymphocytes in BMMC and PBMC in the four HCV antigen-positive patients and in two further patients not expressing viral proteins in blood MC. IF signals appeared diffusely distributed within the cytoplasm, or as brilliant granules in distinct submembrane areas or else in cytoplasm membrane. Nuclei never stained. Similarly, NISH displayed HCV RNA accumulation restricted to MC cytoplasm only, nuclei being persistently negative. NISH, however, was unable to detect cell membrane signal. Infection of blood MC is a common event in naturally acquired HCV infection, since none of these patients was conditioned by immunomodulating or immunosuppressive therapies. No difference was found in terms of mean age, length of disease, anti-HCV immune response, type and severity of chronic liver damage between patients with HCV-infected MC and patients without cell infection. These results demonstrate that HCV can infect BMMC and PBMC that represent important extrahepatic sites of virus replication, and may help to explain the immunological abnormalities observed in chronic HCV carriers.     

慢性丙型肝炎患者抗病毒治疗前PBMC细胞因子的研究

目的 研究准备抗病毒治疗的慢性丙型肝炎患者的免疫特点.方法 将30例慢性丙型肝炎患者和10例正常对照外周血单个核细胞(PBMC)体外培养72 h后,用ELISA法检测培养上清中细胞因子IL-2、IL-4、IL-10、IL-12、IFN-γ和TNF-α的浓度.结果 (1)慢性丙型肝炎患者PBMC培养上清中IFN-γ、IL-10和TNF-α的水平明显升高(P＜0.05),没有检测到IL-2、IL-4、IL-12的基础分泌.(2)不同病情患者间的细胞因子的分泌水平差异无统计学意义(P＞0.05).结论 慢性丙型肝炎患者体内TH2型细胞因子的分泌占优势.提示通过调整TH1/TH2失衡可能达到抗病毒治疗目的.     

Correlation of AIM2 expression in peripheral blood mononuclear cells from humans with acute and chronic hepatitis B

The AIM2 (absent in melanoma 2) protein promotes host defenses against invading viruses and pathogenic bacteria through corresponding adapter molecules leading to the initiation of innate immune responses. We investigated the expression of AIM2 in peripheral blood mononuclear cells (PBMCs) from patients with acute hepatitis B (AHB) and chronic hepatitis B (CHB) during different clinical phases, and analyzed the correlation between AIM2 and clinical profiles in these groups. This study indicated that there is higher expression of AIM2, IL-1β, and IL-18 in AHB compared with expression in CHB. The expression of AIM2 mRNA was significantly negatively correlated with serum hepatitis B virus (HBV) load, HBeAg, and significantly positively correlated with IL-1β and IL-18 in AHB patients and CHB patients with immune clearance, which suggests that AIM2 expression is correlated with the immune clearance of HBV in the host. We summarized that there is a higher immune status in AHB, and a lower immune response in CHB. This suggests that the down-regulation of AIM2 may be associated with the chronic development of HB.