Objective: Liver disease is more progressive in HIV/hepatitis C virus (HCV) co-infection than in HCV infection alone. This accelerated pathogenesis is probably influenced by differences in the composition of infiltrating inflammatory cells and the local release of inflammatory and profibrogenic cytokines.
Methods: Using quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR) we studied intrahepatic messenger RNA levels of cytokines and cellular markers defining distinct subsets of inflammatory cells in liver biopsies from 33 HCV-mono-infected and 40 HIV/HCV-co-infected patients.
Results: Despite their well preserved peripheral blood CD4 cell counts (median 598 cells/μl), HIV/HCV-co-infected patients displayed significantly lower CD4 mRNA levels than HCV-mono-infected patients, whereas increased mRNA levels of CD3ϵ, TCRα, CD8α and CD8β suggested intrahepatic enrichment of CD8 T cells in HIV co-infection. Intrahepatic mRNA levels of the inflammatory cytokines interferon gamma (IFN-γ), regulated upon activation, normal T-cell expressed and secreted (RANTES, CCL5), macrophage inflammatory protein 1 alpha (CCL3) and interferon-inducible protein 10 (CXCL10) were significantly higher in HIV-positive than in HIV-negative patients, whereas mRNA levels of the profibrogenic cytokines macrophage chemoattractant protein 1 (CCL2), secondary lymphochemokine (CCL21) and stroma-derived factor 1 (CXCL12) did not differ between the two groups. All changes were less pronounced in the subgroup of HIV-positive patients receiving antiretroviral treatment (HAART) than in untreated HIV-positive patients.
Conclusion: The accelerated liver disease observed in HIV/HCV-co-infected patients might reflect enhanced intrahepatic inflammatory responses rather than increased local transcription of directly profibrogenic cytokines.
From the aPartners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA
bDepartment of Internal Medicine I, University Hospital of Bonn, Bonn, Germany
cInstitute for Medical Microbiology, Immunology, and Parasitology, University Hospital of Bonn, Bonn, Germany
dClinical HIV Unit and Irsicaixa Foundation, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Barcelona, Spain.
Received 15 December, 2006
Revised 2 October, 2007
Accepted 9 October, 2007
Correspondence to Thomas Kuntzen, MD, Partners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, 149 13th street, 6th floor, Room 6624, Charlestown, MA 02129, USA. Tel: +1 617 643 2838; fax: +1 617 726 5411; e-mail: firstname.lastname@example.org
Liver damage in hepatitis C virus (HCV) infection results from cellular immune responses against HCV-infected hepatocytes rather than direct cytopathic effects of the virus itself [1,2]. It is therefore surprising that despite immunodeficiency HIV/HCV-co-infected patients display enhanced liver disease with accelerated fibrosis progression compared with immunocompetent HCV-mono-infected patients . Possible explanations may lie in the local expression of regulatory cytokines or the cellular composition of inflammatory infiltrates.
In both HCV and HIV mono-infection the chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein 1 alpha (MIP-1α), interferon-inducible protein 10 (IP-10) and IFNγ regulate local inflammation [4–16]. The cytokines macrophage chemoattractant protein 1 (MCP-1), secondary lymphochemokine (SLC) and stroma-derived factor 1 (SDF-1) were linked to hepatic fibrosis in hepatitis C [17–19]. In HIV/HCV co-infection cytokine production is altered by HIV and its immunodeficiency, and also by HAART [20,21].
Using quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR) to amplify gene-specific messenger RNA in liver biopsies from HCV-mono-infected patients, Leroy et al.  found increased expression of IFNγ, RANTES and TNFα, as well as major changes in intrahepatic T-lymphocyte subsets. As this approach closely reflects the in-vivo intrahepatic inflammatory situation, we applied this qRT–PCR to compare mRNA expression patterns of cellular markers and cytokine genes in liver biopsies of HIV/HCV-co-infected and HCV-mono-infected patients.
Materials and methods
Liver biopsies from 33 HCV-mono-infected and 40 HIV/HCV-co-infected patients (Table 1a) naive for anti-HCV treatment were obtained at a single timepoint before anti-HCV treatment. Liver diseases other than hepatitis C were excluded. Among the HIV/HCV-co-infected patients 26 were on HAART (Table 1b). Of note is the fact that immunity was well preserved in co-infected patients: only nine had CD4 T-cell counts below 400 cells/μl. This more severely immunocompromised subgroup did not differ significantly in alanine aminotransferase (ALT) levels, viral loads, histological inflammation and fibrosis scores from the co-infected patients with CD4 T-cell counts greater than 400 cells/μl. The study protocol conformed to the Declaration of Helsinki with a priori approval by the local Institutional Review Board. Written informed consent was obtained from each study participant.
Complimentary DNA synthesis and quantitative real-time reverse transcriptase–polymerase chain reaction
Biopsy specimens were washed in 0.9% sodium chloride to eliminate contamination by blood lymphocytes and then divided into two parts. One part was scored for inflammatory activity and fibrosis by a single histopathologist at each site (Ishak score ). The other part was snap-frozen in liquid nitrogen and stored at −80°C until liver tissue was homogenized and total RNA was extracted including DNAse digestion (RNeasy mini-kit; Qiagen, Hilden, Germany). cDNA was synthesized using the Omniscript RT-Kit (Qiagen) and Oligo-dT-Primer (Promega, Madison, Wisconsin, USA). Quantitative PCR was performed on cDNA transcripts using LightCycler-FastStart-DNAMaster ‘Plus’ SYBRGreen-I (Roche, Mannheim, Germany) according to the manufacturer's instructions on a LightCycler (Roche). Sequences and PCR conditions for CD3ϵ, TCRα, CD4, CD8α, CD8β, CD56 and CD69 were chosen as described  and are summarized for all other markers in supplementary Table 1. Target-gene mRNA concentrations were determined relative to a standard curve using the ΔCt method and were normalized with respect to β-Actin mRNA levels. As a result of the small sample size with limited RNA yields, MCP-1 mRNA levels could not be determined in seven HCV-mono-infected patients, and analysis of CD3ϵ, CD4, CD8α, CD8β, CD69, CD56 and TCRα was limited to 23 and 28 patients with HCV and HIV/HCV infection, respectively.
Quantitative results between different patient groups were compared by the Mann–Whitney-U-test. PCR reactions that failed to amplify a specific product were excluded from the analysis. Pearson's correlations between mRNA levels were calculated using SPSS 12.0 (SPSS Inc., Chicago, Illinois, USA) after logarithmic transformation and subsequent normalization to a mean of 0 to account for non-Gaussian distribution. For ordinal parameters Spearman's rank correlations were used. Bonferroni-type corrections for multiple comparisons were applied based on permutated datasets as described by Moore et al. . All comparisons were two-tailed. P values below 0.05 were considered to indicate statistical significance.
To characterize intrahepatic differences in the composition of inflammatory infiltrates and in cytokine expression patterns we compared mRNA levels of the cellular markers CD3ϵ, TCRα, CD8α, CD8β, CD4, CD68, CD56 and CD69, and of the cytokines IFNγ, MIP-1α, RANTES, IP-10, MCP-1, SDF-1 and SLC in liver biopsies of HCV-mono-infected and HIV/HCV-co-infected patients.
Correlations between cellular surface markers correspond to distinct clusters of inflammatory cells
We first validated and cross-checked the internal consistency of our data by calculating clusters of correlations for cellular markers as described by Leroy et al. . In both patient groups we confirmed highly significant (P < 0.01) correlations between mRNA levels of CD3ϵ and TCRα (HCV r = 0.881 and HIV/HCV r = 0.846), CD8α (r = 0.744 and r = 0.765), and CD8β (r = 0.852 and r = 0.748). We also found a significant relationship between CD3ϵ and CD4 (r = 0.603 and r = 0.651), whereas CD4 was not correlated with CD8α and CD8β. These findings confirmed that clusters of CD3+TCRα+CD8+ and CD3+TCRα+CD4+ T cells could be differentiated reliably. We did not, however, find any correlations between CD3ϵ and CD56.
Significant differences in intrahepatic inflammatory infiltrates and cytokine expression
In comparing median mRNA levels of leukocyte surface markers between the two patient groups, CD3ϵ, TCRα, CD8α and CD8β were increased more than twofold in HIV/HCV-co-infected patients, whereas CD4 mRNA levels were markedly reduced. In contrast, there was no significant difference in CD68, CD56 and CD69 (Fig. 1). To illustrate changes in the relative proportions of cellular subsets among infiltrating CD3-positive cells we calculated mRNA ratios of CD4 and CD8α/β relative to CD3ϵ levels. Both median CD8α/CD3ϵ (HCV 0.74 versus HIV/HCV 1.0; P = 0.023) and CD8β/CD3ϵ ratios (0.14 versus 0.18; P = 0.042) were approximately 1.3-fold elevated in HIV/HCV-co-infected patients, whereas CD4/CD3ϵ ratios were reduced to approximately 25% (4.8 versus 1.3; P < 0.001).
In examining intrahepatic mRNA levels of inflammatory cytokines, IFNγ, RANTES, MIP-1α and IP-10 were increased 1.8–3.1-fold in HIV/HCV-co-infected patients compared with HCV-mono-infected patients. In contrast, mRNA levels of the profibrogenic chemokines MCP-1, SDF-1 and SLC were not significantly different between the two patient groups (Fig. 1).
Previous studies reporting reduced or maintained intrahepatic cytokine mRNA levels  or HCV-specific T-cell functions in HIV/HCV co-infection [25–29] were mostly carried out on cohorts displaying equal ALT levels between groups. Moreover, HCV genotype 3 infection may be associated with enhanced liver disease in HIV/HCV co-infection . We therefore repeated the above analyses in a subgroup of ALT matched patients (21 HCV mono and 21 HIV/HCV-co-infected patients, mean ALT 2.65 × upper limit of normal each), and in non-GT3-infected patients. Here we found no evidence that our observations were significantly confounded by different ALT levels or HCV genotypes between patient groups (data not shown).
Attenuated inflammatory reactions in HIV-co-infected patients on HAART
When HIV/HCV-co-infected patients on HAART were analysed separately, significantly lower TCRα, CD8α, CD8β and CD68 mRNA levels were detected than in untreated co-infected patients. There was also a trend towards the reduced expression of the activation marker CD69, whereas CD4 and CD56 did not reveal major changes between treated and untreated patients (Fig. 1). Average intrahepatic mRNA levels of IFNγ, MIP-1α and RANTES were markedly lower in the subgroup of HIV/HCV-co-infected patients on HAART than in untreated patients, whereas there were no significant differences in MCP-1, SDF-1 and SLC (Fig. 1).
Correlations between cytokines, cellular subsets, transaminases and fibrosis scores
To disclose functional associations between cellular infiltrates, cytokines and clinical markers of liver damage and fibrosis, we analysed correlations between intrahepatic mRNA levels and aspartate aminotransferase (AST), ALT and histological inflammation and fibrosis scores. The inflammatory cytokines IFNγ, RANTES, MIP-1α and IP-10 were closely correlated with TCRα and CD8α in both patient groups (P < 0.01, r > 0.55 for all). IP-10 was correlated with AST (P = 0.018, r = 0.498) and ALT (P = 0.001, r = 0.594) serum levels in HCV-mono-infected but not in HIV/HCV-co-infected patients, and CD8α showed a weak correlation with AST (P = 0.061) in both patient groups. Beyond that there were no additional correlations between cytokine mRNA and aminotransferase levels. Finally, SLC mRNA was correlated with the histological fibrosis score in HCV-mono-infected patients (r = 0.526; P = 0.003), and considerably less so in HIV/HCV-co-infected patients (r = 0.314; P = 0.055).
In the present study we determined differences in gene expression levels for a large panel of leukocyte markers and cytokines in liver biopsies from HCV-mono-infected and HIV/HCV-co-infected patients. As a result of limited sample sizes, immunohistochemistry studies to evaluate directly the amount and localization of expressed proteins could not be performed. It is, however, well documented that mRNA levels enable an indirect assessment of the expression of cellular surface molecules [31–33] and cytokine secretion . Here, increased CD8 and reduced CD4 mRNA levels in HIV/HCV-co-infected patients probably reflect HIV-induced CD8 T-cell expansion and CD4 T-cell depletion, whereas CD3ϵ, TCRα, CD68 and CD56 mRNA levels also suggested a general intrahepatic enrichment of T cells, but not macrophages or natural killer cells in HIV/HCV-co-infected patients. Enhanced expression of MIP-1α, RANTES, IFNγ, and IP-10 was probably related to effector functions of CD8 T cells as suggested by correlations between these cytokines and CD8. Importantly, IP-10, which is induced in hepatocytes by IFNγ and Fas-ligand [10,11,13] during HCV infection, was also correlated with aminotransferase levels in HCV-mono-infected patients, indicating a role of CD8 T cells and their effector cytokines in the pathogenesis of HCV-related tissue damage. Here, MIP-1α and RANTES are also of interest as they may be released together with granzymes from cytolytic granules of CD8 T cells [5,12]. These observations are in line with findings by Leroy et al. , who identified CD8 T cells rather than natural killer cells as the predominant inflammatory cell type in HCV mono-infection.
A previous publication hypothesized a defect in intrahepatic cytokine activation , and functional studies found equivalent or reduced HCV-specific T-cell responses in HIV/HCV-co-infected compared with mono-infected patients [25–29]. As HCV-specific cytokine production is influenced by progressive immunodeficiency [26,29], the relatively well-preserved CD4 cell counts in our HIV-positive patients may explain contrasting results. Moreover, our method measures the collective intrahepatic cytokine expression. As chemotactic stimuli from the HCV-infected liver attract lymphocytes regardless of their antigen specificity, local accumulation of HIV-specific, activated lymphocytes probably also contributes to increased cytokine levels in our study. As a result of the lack of liver tissue from a control group of HIV-mono-infected patients we could not, however, quantitate this effect. It remains to be determined whether bystander cytotoxicity directly caused by inflammatory cytokines [35–38] from HIV-specific T cells could be involved in the pathogenesis of enhanced liver disease in HIV/HCV-co-infected patients.
Recent studies reported beneficial effects of HAART on HCV disease progression in HIV/HCV-co-infected patients [21,39–42]. In line with a recent publication , inflammatory cytokine expression and the composition of cellular infiltrates in our HAART-treated patients appeared to match more closely our findings in HCV-mono-infected individuals. Although longitudinal samples before and after treatment initiation are needed to address this point adequately, our cross-sectional data support the idea that enhanced intrahepatic inflammatory processes in co-infected patients might be reversible under HAART.
MCP-1 and SLC may be involved in hepatic fibrogenesis by the attraction and activation of hepatic stellate cells in hepatitis C [17,18]. Profibrogenic properties of SDF-1 were deduced from its expression in neo-blood vessels at inflammatory foci . Positive correlations between SLC and fibrosis scores in our study support a functional role for SLC in HCV-induced fibrogenesis. Considering the lack of differential regulation observed here it appears unlikely, however, that SLC, MCP-1 and SDF-1 directly influence the enhanced fibrosis progression in HIV/HCV co-infection.
In summary, our results indicate an accumulation of cytotoxic CD8 T cells with an increase in inflammatory mediators, possibly leading to enhanced tissue damage in HIV/HCV-co-infected patients, whereas we found no differential regulation of profibrogenic cytokines. Reduced inflammatory reactions in HIV-positive patients receiving HAART may explain the protective effect of HIV-specific treatment on liver disease in HIV/HCV co-infection.
The authors would like to thank Martin Wolff for sample donation, and Georg Lauer, Victoria Kasprowicz and Galit Alter for reviewing this manuscript.
Authors' contributions: T.K. collected sample material, designed and performed research, analysed and interpreted data and drafted the manuscript; C.T., B.C., B.K. provided samples and clinical data; B.L. performed the statistical analysis; G.F. and H.D.N. provided samples and primer sequences and assisted with the method; T.S. and J.K.R. provided samples, interpreted data and reviewed the manuscript; U.S. designed research, analysed and interpreted data and drafted the manuscript.
Sponsorship: This work was supported by the Heinz-Ansmann-Foundation.
Conflicts of interest: J.K.R. declared relations (consulting, speaker's bureau) with Roche, Schering, Gilead, GSK, BI and Abbott. U.S. received travel grants from Roche and Essex. C.T. declared relations (speakers bureau, advisory board) with Roche. All other authors have no conflicts of interest.
1. Rodriguez-Inigo E, Bartolome J, de Lucas S, Manzarbeitia F, Pardo M, Arocena C, et al
. Histological damage in chronic hepatitis C is not related to the extent of infection in the liver. Am J Pathol 1999; 154:1877–1881.
2. Takaku S, Nakagawa Y, Shimizu M, Norose Y, Maruyama I, Wakita T, et al
. Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes. Biochem Biophys Res Commun 2003; 301:330–337.
3. Graham CS, Baden LR, Yu E, Mrus JM, Carnie J, Heeren T, et al
. Influence of human immunodeficiency virus infection on the course of hepatitis C virus infection: a meta-analysis. Clin Infect Dis 2001; 33:562–569.
4. Soo HM, Garzino-Demo A, Hong W, Tan YH, Tan YJ, Goh PY, et al
. Expression of a full-length hepatitis C virus cDNA up-regulates the expression of CC chemokines MCP-1 and RANTES. Virology 2002; 303:253–277.
5. Wagner L, Yang OO, Garcia-Zepeda EA, Ge Y, Kalams SA, Walker BD, et al
. Beta-chemokines are released from HIV-1-specific cytolytic T-cell granules complexed to proteoglycans. Nature 1998; 391:908–911.
6. Apolinario A, Majano PL, Alvarez-Perez E, Saez A, Lozano C, Vargas J, et al
. Increased expression of T cell chemokines and their receptors in chronic hepatitis C: relationship with the histological activity of liver disease. Am J Gastroenterol 2002; 97:2861–2870.
7. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells. Science 1995; 270:1811–1815.
8. Trifilo MJ, Bergmann CC, Kuziel WA, Lane TE. CC chemokine ligand 3 (CCL3) regulates CD8(+)-T-cell effector function and migration following viral infection. J Virol 2003; 77:4004–4014.
9. Taub DD, Lloyd AR, Conlon K, Wang JM, Ortaldo JR, Harada A, et al
. Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells. J Exp Med 1993; 177:1809–1814.
10. Narumi S, Tominaga Y, Tamaru M, Shimai S, Okumura H, Nishioji K, et al
. Expression of IFN-inducible protein-10 in chronic hepatitis. J Immunol 1997; 158:5536–5544.
11. Mihm S, Schweyer S, Ramadori G. Expression of the chemokine IP-10 correlates with the accumulation of hepatic IFN-gamma and IL-18 mRNA in chronic hepatitis C but not in hepatitis B. J Med Virol 2003; 70:562–570.
12. Iijima W, Ohtani H, Nakayama T, Sugawara Y, Sato E, Nagura H, et al
. Infiltrating CD8+ T cells in oral lichen planus predominantly express CCR5 and CXCR3 and carry respective chemokine ligands RANTES/CCL5 and IP-10/CXCL10 in their cytolytic granules: a potential self-recruiting mechanism. Am J Pathol 2003; 163:261–268.
13. Harvey CE, Post JJ, Palladinetti P, Freeman AJ, Ffrench RA, Kumar RK, et al
. Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation. J Leukoc Biol 2003; 74:360–369.
14. Patzwahl R, Meier V, Ramadori G, Mihm S. Enhanced expression of interferon-regulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization. J Virol 2001; 75:1332–1338.
15. Napoli J, Bishop GA, McGuinness PH, Painter DM, McCaughan GW. Progressive liver injury in chronic hepatitis C infection correlates with increased intrahepatic expression of Th1-associated cytokines. Hepatology 1996; 24:759–765.
16. Leroy V, Vigan I, Mosnier JF, Dufeu-Duchesne T, Pernollet M, Zarski JP, et al
. Phenotypic and functional characterization of intrahepatic T lymphocytes during chronic hepatitis C. Hepatology 2003; 38:829–841.
17. Marra F, Romanelli RG, Giannini C, Failli P, Pastacaldi S, Arrighi MC, et al
. Monocyte chemotactic protein-1 as a chemoattractant for human hepatic stellate cells. Hepatology 1999; 29:140–148.
18. Bonacchi A, Petrai I, Defranco RM, Lazzeri E, Annunziato F, Efsen E, et al
. The chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic hepatitis C. Gastroenterology 2003; 125:1060–1076.
19. Wald O, Pappo O, Safadi R, Dagan-Berger M, Beider K, Wald H, et al
. Involvement of the CXCL12/CXCR4 pathway in the advanced liver disease that is associated with hepatitis C virus or hepatitis B virus. Eur J Immunol 2004; 34:1164–1174.
20. Pacifici R, Di Carlo S, Bacosi A, Pichini S, Zuccaro P. Cytokine production in saquinavir treated mice. Int J Immunopharmacol 1997; 19:243–248.
21. Benhamou Y, Di Martino V, Bochet M, Colombet G, Thibault V, Liou A, et al
. Factors affecting liver fibrosis in human immunodeficiency virus-and hepatitis C virus-coinfected patients: impact of protease inhibitor therapy. Hepatology 2001; 34:283–287.
22. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et al
. Histological grading and staging of chronic hepatitis. J Hepatol 1995; 22:696–699.
23. Moore CB, John M, James IR, Christiansen FT, Witt CS, Mallal SA. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science 2002; 296:1439–1443.
24. Blackard JT, Komurian-Pradel F, Perret M, Sodoyer M, Smeaton L, Clair JB, et al
. Intrahepatic cytokine expression is downregulated during HCV/HIV co-infection. J Med Virol 2006; 78:202–207.
25. Alatrakchi N, Graham CS, He Q, Sherman KE, Koziel MJ. CD8+ cell responses to hepatitis C virus (HCV) in the liver of persons with HCV-HIV coinfection versus HCV monoinfection. J Infect Dis 2005; 191:702–709.
26. Kim AY, Lauer GM, Ouchi K, Addo MM, Lucas M, Wiesch JS, et al
. The magnitude and breadth of hepatitis C virus-specific CD8+ T cells depend on absolute CD4+ T-cell count in individuals coinfected with HIV-1. Blood 2005; 105:1170–1178.
27. Morishima C, Shuhart MC, Yoshihara CS, Paschal DM, Silva MA, Thomassen LV, et al
. Preservation of intrahepatic hepatitis C virus (HCV)-specific CD4+ T cell responses despite global loss of CD4+ T cells in HCV/HIV coinfection. J Infect Dis 2007; 196:577–586.
28. Graham CS, Curry M, He Q, Afdhal N, Nunes D, Fleming C, et al
. Comparison of HCV-specific intrahepatic CD4+ T cells in HIV/HCV versus HCV. Hepatology 2004; 40:125–132.
29. Dutoit V, Ciuffreda D, Comte D, Gonvers JJ, Pantaleo G. Differences in HCV-specific T cell responses between chronic HCV infection and HIV/HCV co-infection. Eur J Immunol 2005; 35:3493–3504.
30. Sanchez-Conde M, Berenguer J, Miralles P, Alvarez F, Carlos Lopez J, Cosin J, et al
. Liver biopsy findings for HIV-infected patients with chronic hepatitis C and persistently normal levels of alanine aminotransferase. Clin Infect Dis 2006; 43:640–644.
31. Ellmeier W, Sawada S, Littman DR. The regulation of CD4 and CD8 coreceptor gene expression during T cell development. Annu Rev Immunol 1999; 17:523–554.
32. Gao MH, Walz M, Kavathas PB. Posttranscriptional regulation associated with control of human CD8A expression of CD4+ T cells. Immunogenetics 1996; 45:130–135.
33. Bernard K, Auphan N, Granjeaud S, Victorero G, Schmitt-Verhulst AM, Jordan BR, et al
. Multiplex messenger assay: simultaneous, quantitative measurement of expression of many genes in the context of T cell activation. Nucl Acids Res 1996; 24:1435–1442.
34. Dumoulin FL, Leifeld L, Honecker U, Sauerbruch T, Spengler U. Intrahepatic expression of interleukin-1beta and tumor necrosis factor-alpha in chronic hepatitis C. J Infect Dis 1999; 180:1704–1708.
35. Crispe IN, Dao T, Klugewitz K, Mehal WZ, Metz DP. The liver as a site of T-cell apoptosis: graveyard, or killing field? Immunol Rev 2000; 174:47–62.
36. Bowen DG, Warren A, Davis T, Hoffmann MW, McCaughan GW, Fazekas de St Groth B, et al
. Cytokine-dependent bystander hepatitis due to intrahepatic murine CD8 T-cell activation by bone marrow-derived cells. Gastroenterology 2002; 123:1252–1264.
37. Gremion C, Grabscheid B, Wolk B, Moradpour D, Reichen J, Pichler W, et al
. Cytotoxic T lymphocytes derived from patients with chronic hepatitis C virus infection kill bystander cells via Fas–FasL interaction. J Virol 2004; 78:2152–2157.
38. Bertoletti A, Maini MK. Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection? Curr Opin Immunol 2000; 12:403–408.
39. Marine-Barjoan E, Saint-Paul MC, Pradier C, Chaillou S, Anty R, Michiels JF, et al
. Impact of antiretroviral treatment on progression of hepatic fibrosis in HIV/hepatitis C virus co-infected patients. AIDS 2004; 18:2163–2170.
40. Tural C, Fuster D, Tor J, Ojanguren I, Sirera G, Ballesteros A, et al
. Time on antiretroviral therapy is a protective factor for liver fibrosis in HIV and hepatitis C virus (HCV) co-infected patients. J Viral Hepat 2003; 10:118–125.
41. Kramer JR, Giordano TP, Souchek J, Richardson P, Hwang LY, El-Serag HB. The effect of HIV coinfection on the risk of cirrhosis and hepatocellular carcinoma in U.S. veterans with hepatitis C. Am J Gastroenterol 2005; 100:56–63.
42. Mehta SH, Thomas DL, Torbenson M, Brinkley S, Mirel L, Chaisson RE, et al
. The effect of antiretroviral therapy on liver disease among adults with HIV and hepatitis C coinfection. Hepatology 2005; 41:123–131.
43. Sitia G, De Bona A, Bagaglio S, Galli L, Paties CT, Uberti-Foppa C, et al
. Naive HIV/HCV-coinfected patients have higher intrahepatic pro-inflammatory cytokines than coinfected patients treated with antiretroviral therapy. Antivir Ther 2006; 11:385–389.