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.     

T-cell receptor variable region of the β-chain gene use in peripheral blood and multiple synovial membranes during rheumatoid arthritis

In order to look for a site-specific T-cell response in RA SM, PCR analyses using oligonucleotide primers specific for 24 TCRBV (Vβ) families were performed to compare the respective usage of each TCRBV gene by T cells present in PB and SM of 13 patients with RA. In four patients, SM cells from two or three sites of inflammation were subjected to analysis. In one patient, synovial tissue was studied at two different phases of the disease, resulting in a total number of 19 samples of SM cells, which were compared with paired samples of PB cells. The results showed that whereas all 24 TCRBV gene families could be detected in both PB and SM cells, there was some skewing of increased or decreased usage frequencies of particular TCR Vβ genes among SM cells. Three TCRBV families were often overexpressed in SM: Vβ3, Vβl7, and Vβ22. Moreover, Vβ4 was often decreased in SM (7 out of 13). This decrease was statistically significant in the RA population studied. SM from different joints of a given patient showed similar variations of T-cell repertoire compared to PB, even 6 months later in the course of the disease. These results demonstrate a biased TCRBV gene utilization in RA SM. This bias appears to be similar in different joints and at different times in the course of the disease. No correlation was found between the bias of TCR repertoire in SM and the HLA typing of these patients.     

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.     

Analysis of human T-cell receptor α-chain cDNAs isolated from peripheral blood mononuclear cells

T-cell receptor α-chain cDNA were generated from unstimulated peripheral blood mononuclear cells of a DR2,3,52a individual using a modified anchor PCR method. Fifty-six cDNA clones were identified representing 47 distinct T-cell receptor clonotypes and 26 VA loci. This analysis identified a new VA gene family VA30, and aew member of the VA6 gene family.     

T-cell receptor Vα region usage in the cerebrospinal fluid of patients with mumps meningitis

To investigate clonality of local T cells in viral infections of the central nervous system, the T-cell receptor (TCR) repertoire was evaluated in T cells from the cerebrospinal fluid (CSF) of nine patients with mumps meningitis, using the polymerase chain reaction (PCR) assay. The usage of the variable region of TCR α chain gene (Vα gene) in the CSF was widespread, and an average of 13 out of the 18 Vα families were expressed. Quantitative PCR analyses showed that the Vα gene expression was biased toward three or less Vα families in the CSF of each patient. When compared with peripheral blood T cells, the average percentages of Vα 11 and Vα 12 gene expression were significantly higher in the CSF than in the peripheral blood. These results suggested that mumps-specific T lymphocytes with a restricted TCR repertoire are selectively recruited to the central nervous system in mumps meningitis, although polyclonal, probably nonspecific, T-cell populations are present in the CSF.     

A bias in the αβ T cell receptor variable region gene usage in Takayasu's arteritis

Takayasu's arteritis (TA) is a chronic large vessel vasculitis with a predilection for the aortic arch and its branches. T lymphocytes may be important in the pathogenesis, as they have been found to infiltrate the vascular lesions. To elucidate further the role of T cells in the disease, we studied circulating CD4⁺ and CD8⁺ T cells, expression of the activation marker (HLA-DR), marker for naive (CD45RA) and primed (CD45RO) cells and the different variable α/β (AV/BV) gene segments on them. The TCR AV/BV repertoire was studied using a panel of 15 T cell receptor (TCR) V-specific MoAbs by flow cytometry in 18 patients and 23 age- and sex-matched controls. Patients had a higher percentage of AV12S1 (P< 0.05), BV6S7 (P< 0.05) and BV9 (P< 0.001)-bearing CD4⁺ cells. Patients also had a higher frequency of expansions, i.e. of T cell populations with an abnormally high TCR AV/BV gene usage. In patients' CD4⁺ subset of cells, there were 22 expansions out of 231 analyses (9.5%), whereas in controls, four were expanded out of 310 analyses (1%) (P< 0.001). For CD8⁺ cells, the frequency of expansions was 32 in 231 analyses (14%) in patients and nine out of 304 analyses in controls (3%) (P< 0.01). In addition, there was a correlation between CD4⁺ expansions and disease activity; nine out of 10 patients with active disease in comparison with two out of eight patients with inactive disease (P< 0.01) had an expansion. Some of the expanded populations in patients were phenotypically characterized and observed to be HLA-DR^<, CD28^<, CD45RA^< and CD45RO⁺, with a greater proportion being CD45RO⁺. Patients had a higher percentage of expression of HLA-DR on both CD4⁺ and CD8⁺ T cells (P< 0.01). The percentages of naive and primed CD4⁺ and CD8⁺ T cells, γδ⁺ T cells and natural killer cells were comparable to those in the control group.     

Randomized trial of high‐dose interferon‐α‐2b combined with ribavirin in patients with chronic hepatitis C: Correlation between amino acid substitutions in the core/NS5A region and virological response to interferon therapy

The aim of this study was to compare the efficacy of high‐dose interferon (IFN)‐α‐2b with standard dose of IFN‐α‐2b in combination with ribavirin (RBV) for patients with chronic hepatitis C virus (HCV) infection, and to investigate the predictive factors associated with virological response. Two hundred Japanese patients with high HCV viral load (>100 KIU/ml) were randomized to 6 or 10 mega units (MU) of 24‐week IFN‐α‐2b with RBV. Predictive factors were investigated; including pretreatment amino acid (aa) sequences of the core region and the IFN‐sensitive determining region (ISDR). The sustained virological response rate was not different in the two groups (24% vs. 30%) but the incidence of depression was significantly higher in the 10 MU group than 6 MU group (7% vs. 0%, P = 0.02). Younger age (<60) and HCV genotype (2a/b) were significant predictors of sustained virological response. In patients infected with genotype 1b, substitutions of core aa 70 and/or 91 were predictive for non‐virological response (P < 0.001), and substitutions in the ISDR was observed frequently in virological responders. Early viral kinetics study showed that serum HCV core antigen decreased more slowly in both patients with aa 70 and/or 91 substitutions in the core and with absence of substitutions in the ISDR. In conclusion, the use of a higher dose of IFN‐α‐2b in combination with RBV did not improve virological response but resulted in higher incidence of depression. Amino acid substitutions in the core and ISDR are predictive of virological response to the therapy in patients with genotype 1b and high viral load. J. Med. Virol. 81:640–649, 2009 © 2009 Wiley‐Liss, Inc.     

丙型肝炎高变区1合成肽体外刺激HCV感染PBMC细胞因子分泌的研究

目的检测丙型肝炎病毒(HCV)高变区1(HVR1)模拟表位刺激自然感染患者外周血淋巴细胞后的细胞因子水平变化。方法细胞因子检测运用ELISA、流式细胞仪(FACS)淋巴细胞分类以及计算机模拟多肽抗原性。结果(1)1号和5号合成肽刺激HCV患者外周血单核细胞(PBMC)后,患者PBMC出现了明显的增殖;(2)培养上清中干扰素γ(IFNγ)、白介素4(IL-4)、白介素10(IL-10)都有不同程度的提高,白介素2(IL-2)和肿瘤坏死因子α(TNF-α)的水平并没有提高;(3)增殖细胞主要为CD4阳性淋巴细胞。结论我们设计的HCV高变区1合成肽引发的免疫因子的释放倾向于TH2类因子。     

Cytotoxic protein expression in natural killer cell lymphomas and in αβ and γδ peripheral T-cell lymphomas

Lymphomas with T-cell phenotype represent a heterogeneous group of diseases differing in histopathology, tumour site, and cell origin. They include peripheral T-cell lymphomas (PTCLs) derived from αβ cells, but also some recently recognized entities such as γδ hepatosplenic lymphomas and natural killer (NK) cell lymphomas. Only a few studies have investigated the possibility that at least some PTCLs could be derived from lymphocytes with cytotoxic potential. In order to investigate this possibility, 60 cases of PTCL, including 27 cases expressing the αβ T-cell receptor (TCRαβ), 15 TCRγδ cases and 18 cases expressing neither TCR (TCR silent), as well as 14 cases of NK-cell lymphomas, were studied by immunohistochemistry for the expression of TIA-1, perforin, and granzyme B proteins. Expression of TIA-1 is characteristic of cytotoxic cells regardless of their activation status, whereas expression of perforin and granzymes is highly increased in activated cytotoxic cells and correlates with the induction of cytolytic activity. All NK-cell lymphomas (11 sinonasal, three systemic cases) expressed TIA-1, perforin, and granzyme B in most tumour cells. All γδ PTCLs (15 cases) expressed TIA-1 protein in most tumour cells, with a different cytotoxic antigen profile in hepatosplenic γδ PTCL (TIA-1+, perforin−, granzyme B−) and in non-hepatosplenic γδ PTCLs (three nasal, one skin, one lung), the latter expressing the three cytotoxic proteins. Of the 45 cases of αβ and TCR silent PTCL, 15 (33 per cent) were considered to be derived from cytotoxic lymphocytes with expression of at least one cytotoxic protein (TIA-1, 15/45; perforin, 10/41; granzyme B, 14/38) in tumour cells. This cytotoxic protein expression appeared to be related to the site of localization, since 7/13 (54 per cent) extranodal and only 8/32 (25 per cent) nodal αβ and TCR silent PTCLs expressed TIA-1, and to histology, since this pattern was observed in a proportion of anaplastic (6/8, 75 per cent) and pleomorphic (8/17, 47 per cent) lymphomas, but not in AILD-type NHL (0/16). Taken together, our data suggest that NK-cell lymphomas and non-hepatosplenic γδ PTCLs represent tumours of activated cytotoxic NK cells and γδ T cells, respectively; that hepatosplenic γδ PTCLs represent tumours of non-activated cytotoxic γδ T cells; and that a small proportion of αβ and TCR silent PTCLs, mostly extranodal cases, or nodal anaplastic lymphomas, represent tumours of cytotoxic T cells. © 1997 John Wiley & Sons, Ltd.     

The T cell receptor β genes of Xenopus

cDNA of the T cell receptor β (TCRB) have been isolated from the anuran amphibian Xenopus and they show strong structural homology to TCRB sequences of other vertebrates. Ten BV families, two D segments, ten J segments, and a single C region have been defined so far. Each V family consists of one to two members per haploid genome. A unique feature of the Xenopus TCRB constant region is the lack of N-linked carbohydrate glycosylation sites. The recombination signal sequences suggest that the mechanism of rearrangements are identical to those of mammals. The locus is inherited in a diploid manner despite the pseudotetraploidy of the Xenopus laevis and X. gilli used in this study.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

Serum hepatitis C virus RNA level as a predictor of subsequent response to interferon-α therapy in Japanese patients with chronic hepatitis C

Serum hepatitis C virus (HCV) RNA level has been shown to be a good predictor of subsequent response to interferon-α (IFN) therapy in US patients in whom genotype 1a/1b are both predominant. To determine whether serum HCV RNA level is a predictor of subsequent response to IFN in Japanese patients or not, appropriately collected pre-IFN therapy serum samples from 35 Japanese patients with chronic HCV infection were studied. Serum HCV RNA level and HCV genotype were determined and correlated with the subsequent response to IFN. Response to IFN was defined by serum alanine transaminase level: complete and sustained response (n = 15), complete response followed by relapse (n = 10), and no response (n = 10). Patients with complete and sustained response had lower pre-IFN serum HCV RNA level (median: RT-PCR+, bDNA-) compared to the complete response to relapse group (median: 5.25 × 10⁶ genome equivalent/ml [eq/ml], P < 0.001) and the no response group (median: 10.63 × 10⁶ eq/ml, P < 0.001). Seven (46.7%) of the complete and sustained response patients had HCV genotype 2a and three patients had a mixture of genotypes 1b and 2a. In contrast, all 10 patients in the complete response to relapse group had genotype 1b whereas 8 of 10 patients in the non-response group had genotype 1b and 2 had genotype 2b. The patients with HCV genotype 2a had lower serum HCV RNA level than those with 1b (P = 0.002). When the HCV viremia were controlled by stratifying them into < and > 10⁶ eq/ml, patients with genotype 1 had a similar complete and sustained response rate compared to genotype 2. These data indicated that pre-IFN serum HCV RNA is also a good predictor of subsequent complete and sustained response in Japanese patients with chronic HCV infection. © 1994 Wiley-Liss, Inc.     

Sequence and structural analysis of the 5′ noncoding region of hepatitis C virus in patients with chronic infection

Hepatitis C virus (HCV), exhibits considerable genetic diversity, but presents a relatively well conserved 5′ noncoding region (5′ NCR) among all genotypes. In this study, the structural features and translational efficiency of the HCV 5′ NCR sequences were analyzed using the programs RNAfold, RNAshapes and RNApdist and with a bicistronic dual luciferase expression system, respectively. RNA structure prediction software indicated that base substitutions will alter potentially the 5′ NCR structure. The heterogeneous sequence observed on 5′ NCR led to important changes in their translation efficiency in different cell culture lines. Interactions of the viral RNA with cellular transacting factors may vary according to the cell type and viral genome polymorphisms that may result in the translational efficiency observed. J. Med. Virol. 81:1212–1219, 2009. © 2009 Wiley‐Liss, Inc.     

Loss of serum HCV RNA at week 4 of interferon-α therapy is associated with more favorable long-term response in patients with chronic hepatitis C

To determine the virological factors associated with a favorable long-term response to interferon-α (IFN) therapy in chronic hepatitis C virus (HCV) infection, 61 Japanese patients with chronic HCV infection were treated with IFN for 24 weeks (780 million units in total) and followed for 8 to 16 months after cessation of therapy. Ten patients dropped out because of severe side effects. Of the 51 patients who completed IFN therapy, 23 showed complete and sustained response (CR→SR), 13 complete response with early relapse (CR→Rel), and 15 no response to IFN (NR). For the pretreatment serum HCV RNA level, 20/23 who had CR→SR had <l0⁶ eq/ml compared to 3/13 CR→Rel and 1/15 NR (P< 0.01). Serologically defined HCV type 2 infection was also associated with a better opportunity to develop CR→SR compared to CR→Rel of NR (P<0.01). Loss of serum HCV RNA at week 4 of IFN therapy was also associated with a more favorable long-term response [17/19 CR→SR were HCV RNA negative compared to 3/11 CR→Rel (P<0.01)and2/13NR(P<0.01)]. n contrast, normalization of serum alanine ami-notransferase (ALT) levels at week 4 was found in 9/19 CR→SR compared to 8/11 CR→Re1 (P= NS), and 0/13 in NR (P<0.01). Six months after cessation of IFN therapy, 3/25 CR→SR patients were HCV RNA positive despite normalization of serum ALT levels. These data indicated that in addition to pretreatment serum HCV RNA levels and HCV type, the kinetics of response to IFN (at week 4) were also predictive of subsequent long-term response to IFN in patients with chronic HCV infection. © 1995 Wiley-Liss, Inc. © 1995 Wiley-Liss, Inc.     

Virological analysis and phenotypic characterization of peripheral blood lymphocytes of hepatitis C virus-infected patients with and without mixed cryoglobulinaemia

In clinical and pathological terms hepatitis C virus (HCV)-infected patients can be subdivided into two main groups with and without mixed cryoglobulinaemia (MC). Involvement of blood mononuclear cells by HCV has potentially important implications. To this end, HCV-RNA levels in peripheral blood lymphocytes (PBL) preparations of 20 chronically HCV-infected patients with MC were measured and compared with those found in a group of 20 patients without MC matched for age, serum HCV-RNA, infectious genotype, source and presumable duration of infection. Phenotypic abnormalities of PBL subsets in each group of patients were determined by cell surface marker expression and compared. Results showed a significant enrichment of HCV-RNA in PBL of MC patients compared with a non-MC group (P = 0.01). Different distribution of HCV-RNA was accompanied by evidence of an increased frequency of circulating B cells. These data indicate that MC patients are characterized distinctly by a higher quota of cell-associated viral load.     

Liver distribution of γδ‐T‐cells in patients with chronic hepatitis of different etiology

The γδ T cells represent a minor unique T‐cell subpopulation long been considered as innate‐like immune cells. They are found in increased numbers in tissues from various inflammatory conditions. Their role in chronic hepatitis, however, is still discussed controversially. Fresh frozen tissues from 50 patients (18 cases hepatitis B infection, 25 hepatitis C, three cases with co‐infection of hepatitis B and C and four patients with autoimmune hepatitis) were investigated. Immunohistochemistry with primary antibodies detecting αβ and γδ TCR was used to evaluate their incidence and distribution in the different histological structures of the liver. The inflammatory infiltrate in all cases of chronic hepatitis was dominated by αβ T cells and was mainly localized in the portal tracts with formation of an interface hepatitis (95.3%αβ T cells; 4.7%γδ T cells). There were neither significant differences between inflammatory infiltrate nor the amount or percentage of γδ T cells between hepatitis B, C or autoimmune hepatitis. No accumulation of γδ T cells could be observed in cases of chronic hepatitis of different etiologies. The immune‐mediated phenomena in chronic hepatitis are dominated by αβ T cells. Thus, the adapted immune system is responsible for the inflammatory processes in chronic hepatitis.