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Does HIV-1 co-receptor tropism correlate with fibrosis progression in HIV/HCV co-infected patients?
... In addition, the HIV env gp120 was demonstrated to induce hepatocyte apoptosis in vitro through CXCR4 in the absence of infection [13], thus triggering the pro-fibrotic activity of HSC. Whether X4 or R5 viral strains exert a different pro-fibrogenic effect on HSCs in vivo is still unknown [14][15]. ...
... In a previous study [15], in a group of 105 HIV/HCV co-infected patients, treated with ART and tested for CRT on proviral DNA, we retrospectively evaluated the evolution of fibrosis as determined by means of APRI and FIB-4 surrogate scores starting from the first anti-HCV positive testing to the date of CRT assessment. No difference was found in the liver fibrosis evolution between the two groups of R5 and X4 patients. ...
Background
In vitro, gp120 of both X4 and R5 HIV-1 strains activates human hepatic stellate cells, but if it can promote liver fibrosis in vivo is unknown. We aimed to evaluate if patients carrying X4 or R5 strains have a different liver fibrosis (LF) progression over time.
Methods
A total of 1,137 HIV-infected patients in ICONA cohort (21% females, 7% HCV co-infected) with an available determination of HIV-1 co-receptor tropism (CRT), a Fibrosis-4 Index for Liver Fibrosis (FIB-4) <3.25 and at least one-year follow-up were included. CRT was assessed by gp120 sequencing on plasma RNA and geno2pheno algorithm (10% false positive rate) or by Trofile. LF was assessed by means of FIB-4. LF progression was defined as an absolute score increase or a transition to higher fibrosis stratum and/or occurrence of liver-related clinical events.
Results
A total of 249 (22%) patients carried X4 strains, which were associated with older age, lower CD4 count, lower nadir CD4, and intravenous drug use. Overall, X4 and R5 patients had similar baseline FIB-4 scores and similar mean FIB-4 slope after a median follow-up of 35 months. There was no difference between X4 and R5 for time to LF progression (p = 0.925). Estimated risk of LF at 24 months (95% CI) after baseline in X4 and R5 was 10.6% (8.3–12.9) and 9.9% (5.9–14.0), respectively. Age, HCV co-infection, diabetes, HIV-duration, HIV-RNA>100.000 cp/mL, antiretroviral therapy exposure were associated with LF progression at multivariate analysis.
Conclusions
A slight LF progression over time was observed in HIV-infected patients. No difference was demonstrated for X4 and R5 HIV-1 strains in accelerating LF evolution.
... HCV infection does not seem to influence co-receptor tropism, nor is specific co-receptor use associated with HCV infection 13 . Plasma HCV RNA values are comparable between patients infected with R5 and X4 viruses 14,15 . ...
We described short-term HIV tropism changes occurring in peripheral blood mononuclear cells and the correlations with HIV DNA value in HIV-HCV co-infected patients cured for HCV disease and with undetectable HIV viremia or residual viremia (RV). Plasma HIV RNA, cellular HIV DNA and tropism were evaluated pre-HCV treatment (baseline, BL) and at 12(T1) and 24(T2) weeks after HCV treatment start. V3 sequences were interpreted using Geno2pheno and classified as R5 only if all three sequences had an FPR ≥ 10% and as X4 when at least one replicate sequence had an FPR < 10%. Forty-nine patients (21 with X4 and 28 with R5 virus) were enrolled. Five X4 patients and 9 R5 subjects experienced at least one tropism change,11 with RV:1/5 patients with X4 infection at BL switched at T1 versus 8/9 in the R5 group (p = 0.022977) and the difference was confirmed in subjects with RV (p = 0.02);6/9 R5 patients switching at T1 confirmed the tropism change at T2. No significant differences in HIV DNA values between patients with RV starting with a R5 or X4 tropism and experienced tropism switch or not were found. Short-term tropism switch involved almost a third of patients, in all but three cases with HIV RV. Being R5 at BL is associated to a higher instability, expressed as number of tropism changes and confirmed switch at T2.
... This indicates that HCV-induced liver fibrosis mechanisms differed from that mentioned with HBV or HCV. Moreover, some authors believe in the role of oxidative stress, mitochondrial dysfunction and iron accumulation in the pathogenesis of liver fibrosis [176][177][178]. HIV is not fibrogenic per se, but it can accelerate existing liver fibrosis induced by HBV or HCV [179]. ...
... Although no direct correlation has been observed between HCV viral load and fibrosis progression Hep C patients, in patients with HCV/HIV-coinfection, HIV RNA levels may determine the rate of fibrosis progression [58]. However, patients infected with HIV alone do not usually develop significant liver fibrosis, suggesting that HIV infection per se is not profibrogenic, but rather HIV promotes fibrosis following hepatic injury due to hepatotropic viruses [59][60][61]. One proposed mechanism is that HIV gp120 regulates HSCs behavior, including expression of proinflammatory cytokines and directional cell movement [61]. ...
... The prevalence of X4 variants varies significantly between different groups of patients, depending on transmission route, stage of disease, and patient-related characteristics. [48][49][50] To obtain a cohort reflecting the structure of the HIV patient population, we included in the study a group of patients consecutively tested in two different Italian hospitals. In this cohort, according to G2P analysis (FPR = 10%), the majority of samples harbored R5 viruses, while X4 tropic viruses were identified in 26% of samples. ...
Abstract Genotype-based algorithms are valuable tools for the identification of patients eligible for CCR5 inhibitors administration in clinical practice. Among the available methods, geno2pheno[coreceptor] (G2P) is the most used online tool for tropism prediction. This study was conceived to assess if the combination of G2P prediction with V3 peptide net charge (NC) value could improve the accuracy of tropism prediction. A total of 172 V3 bulk sequences from 143 patients were analyzed by G2P and NC values. A phenotypic assay was performed by cloning the complete env gene and tropism determination was assessed on U87_CCR5(+)/CXCR4(+) cells. Sequences were stratified according to the agreement between NC values and G2P results. Of sequences predicted as X4 by G2P, 61% showed NC values higher than 5; similarly, 76% of sequences predicted as R5 by G2P had NC values below 4. Sequences with NC values between 4 and 5 were associated with different G2P predictions: 65% of samples were predicted as R5-tropic and 35% of sequences as X4-tropic. Sequences identified as X4 by NC value had at least one positive residue at positions known to be involved in tropism prediction and positive residues in position 32. These data supported the hypothesis that NC values between 4 and 5 could be associated with the presence of dual/mixed-tropic (DM) variants. The phenotypic assay performed on a subset of sequences confirmed the tropism prediction for concordant sequences and showed that NC values between 4 and 5 are associated with DM tropism. These results suggest that the combination of G2P and NC could increase the accuracy of tropism prediction. A more reliable identification of X4 variants would be useful for better selecting candidates for Maraviroc (MVC) administration, but also as a predictive marker in coreceptor switching, strongly associated with the phase of infection.
... Although a correlation between HCV viral load and the progression of fibrosis has not been demonstrated in HCV hepatitis, HIV RNA levels predict the fibrogenic progression of chronic hepatitis in HCV/HIV-co-infected individuals [225,226] . In contrast, patients infected with HIV alone do not show significant liver fibrosis, indicating that HIV infection is not profibrogenic per se but rather accelerates the fibrogenic process in the presence of hepatic damage induced by hepatotropic viruses [225][226][227] . A recent, elegant study by Bruno et al [228] demonstrated that HIV gp120 modulates HSC behavior, including directional cell movement and expression of proinflammatory cytokines. ...
There have been considerable recent advances towards a better understanding of the complex cellular and molecular network underlying liver fibrogenesis. Recent data indicate that the termination of fibrogenic processes and the restoration of deficient fibrolytic pathways may allow the reversal of advanced fibrosis and even cirrhosis. Therefore, efforts have been made to better clarify the cellular and molecular mechanisms that are involved in liver fibrosis. Activation of hepatic stellate cells (HSCs) remains a central event in fibrosis, complemented by other sources of matrix-producing cells, including portal fibroblasts, fibrocytes and bone marrow-derived myofibroblasts. These cells converge in a complex interaction with neighboring cells to provoke scarring in response to persistent injury. Defining the interaction of different cell types, revealing the effects of cytokines on these cells and characterizing the regulatory mechanisms that control gene expression in activated HSCs will enable the discovery of new therapeutic targets. Moreover, the characterization of different pathways associated with different etiologies aid in the development of disease-specific therapies. This article outlines recent advances regarding the cellular and molecular mechanisms involved in liver fibrosis that may be translated into future therapies. The pathogenesis of liver fibrosis associated with alcoholic liver disease, non-alcoholic fatty liver disease and viral hepatitis are also discussed to emphasize the various mechanisms involved in liver fibrosis.
Background
Co-receptor tropism (CRT) in patients with a long history of HIV-1 infection and antiretroviral treatment has been rarely investigated to date. The aim of this study was to determine the prevalence of X4 and R5 strains in patients with a >15-year follow-up and to investigate the demographical, viral, immunological, clinical and therapeutic determinants of CRT in this population. The possible influence of CRT on the inflammation state related to chronic HIV infection was also examined.
Methods
A total of 118 HIV-1 infected patients with an initial HIV-1-positive test before 1997, and still on follow-up, were enrolled and consecutively submitted to blood sampling. Of these, 111 were on antiretroviral therapy and 89/111 (80.2%) had a plasma viral load (pVL) <25 copies/ml at testing. HIV-1 DNA was extracted and amplified from PBMCs for env gp120 sequencing. CRT was assigned by using geno2pheno and isolates were classified as X4 (FPR ≤20%) or R5 (FPR >20%). Level of serological inflammation biomarkers including IL-6, hsPCR, and D-dimers were measured.
Results
An X4 virus was evidenced in HIV-1 proviral DNA of 50 patients (42%) while the remaining 68 patients were classified as R5. The median follow-up was 19 years (range 15–25). No association was observed between CRT and sex, age, nationality, subtype, HIV risk factor, HBV/HCV co-infection, baseline CD4+ cell count and pVL, overall duration of antiretroviral therapy, past exposure to mono-or dual therapies, and duration of NNRTI or PI-based therapy. The presence of an X4 strain was associated with CD4 nadir (p = 0.005), CD4 absolute count over time (p < 0.001), and cumulative positive (copy/years) viremia (p <0.001) during the whole patient history. No differences were found between R5 and X4 patients regarding inflammation marker levels including Il-6, hsPCR and D-dimers.
Conclusions
An archived X4 virus was demonstrated in 42% of patients with a >15-year-history of HIV infection. This presence was clearly associated with a greater exposure to positive viremia and a poorer CD4 trend over time compared to R5, independent of type and duration of antiretroviral treatment. CRT does not seem to influence the inflammation rate of patients aging with HIV.
Hepatic stellate cells, the major producers of extracellular matrix in the liver, and hepatocytes bear CXCR4 and CCR5, the two main co-receptors for entry of the human immunodeficiency virus (HIV). In vitro studies suggest that HIV-envelope proteins can modulate the replication of hepatitis C virus (HCV) and fibrogenesis. We investigated the influence of HIV tropism on liver fibrosis and the concentration of HCV RNA in HIV-HCV co-infected patients.
We used a phenotypic assay to assess HIV tropism in 172 HCV-HIV co-infected patients: one group (75 patients) had mild fibrosis (score ≤F2) and the other (97 patients) had severe fibrosis (score >F2). We also assessed the relationship between HIV tropism and HCV RNA concentration in all these patients. We also followed 34 of these patients for 3 years to determine the evolution of HIV tropism and liver fibrosis, estimated by liver stiffness.
Initially, most patients (91.8%) received a potent antiretroviral therapy. CXCR4-using viruses were found in 29% of patients. The only factor associated with a CXCR4-using virus infection in multivariate analysis was the nadir of CD4 cells: <200/mm(3) (OR: 3.94, 95%CI: 1.39-11.14). The median HCV RNA concentrations in patients infected with R5 viruses, those with dual-mixed viruses and those with X4 viruses, were all similar. The prevalence of CXCR4-using viruses in patients with mild fibrosis (≤F2) (31%) and those with severe fibrosis (F3-F4) (28%, p = 0.6) was similar. Longitudinal analyses showed that the presence of CXCR4-using viruses did not increase the likelihood of fibrosis progression, evaluated by measuring liver stiffness.
The presence of CXCR4-using viruses in patients receiving a potent antiretroviral therapy does not influence HCV RNA concentration or liver fibrosis.
Background:
The aim of this study was to describe changes in repeated liver stiffness (LS) measurements and to assess the determinants of increase in LS in HIV-HCV-coinfected patients.
Methods:
HIV-HCV-coinfected adults enrolled in the ANRS CO 13 HEPAVIH cohort, for whom two results of LS, evaluated over ≥24 months, were available. Patients with unreliable LS results were not included. LS was measured at baseline and every year thereafter. Determinants of LS increase were assessed using linear (primary outcome: last LS minus first LS value) and logistic (secondary outcome: ≥30% increase in the initial LS value) regression analyses.
Results:
A total of 313 patients (mean age 45 years, 67.4% male) were included. Overall, 93.9% were receiving antiretroviral treatment (ART). The mean baseline CD4(+) T-cell count was 471 cells/mm(3) and 72.2% of patients had undetectable plasma HIV RNA. The mean interval between the first and last LS measurements was 33.5 months. No significant difference was found between baseline and follow-up mean LS values (P=0.39). However, a decrease of ≥30% in LS was observed in 48 (15.3%) patients and an increase of ≥30% in 64 (20.5%) patients. In multivariate linear and logistic analyses, excessive alcohol intake (β coefficient 6.8; P=0.0006) and high HCV viral load (OR 1.7, 95% CI 1.1, 2.5; P=0.01) were independently associated with an increase in LS, whereas time on ART>114.5 months (OR 0.5, 95% CI 0.3, 0.9; P=0.03) and achievement of sustained virological response (OR 0.1, 95% CI 0.01, 0.9; P=0.04) were independently associated with no increase in LS.
Conclusions:
Our findings show that long-term ART and achieving sustained virological response in HIV-HCV-coinfected patients are both significantly associated with lack of increase in LS over a 33-month period.
Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the in vitro impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the in vivo expression of gp120 in HIV/HCV coinfected livers.
Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/- either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies.
X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers.
X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.
HIV coreceptor tropism (CTR) testing is a prerequisite for prescribing a coreceptor antagonist. CTR is increasingly deduced by analyzing the V3 loop sequence of gp120. We investigated the impact of mutations outside V3 on CTR as determined by the enhanced-sensitivity Trofile assay (ESTA). Paired ESTA and gp120 sequencing (population sequencing; from codon 32 of the conserved C1 to the variable V5 domains) were obtained from 60 antiretroviral treatment (ART)-naïve patients (15 with AIDS) infected with subtype B HIV-1. For gp120 sequence analysis, nucleotide mixtures were considered when the second highest electropherogram peak was >25%; sequences were translated into all possible permutations and classified as X4, dual/mixed (DM), and R5 based on coincident ESTA results. ESTA identified R5 and DM viruses in 72 and 28% of patients, respectively; no pure X4 was labeled. Forty percent of AIDS patients had R5 strains. Thirty-two positions, mostly outside V3, were significantly (P < 0.05) different between R5 and DM sequences. According to multivariate analysis, amino acid changes at 9 and 7 positions within the C1 to C4 and V1 to V5 regions, respectively, maintained a statistical significance, as did the net charge of V3 and C4. When analyzing only R5 sequences, 6 positions in the variable regions were found which, along with the V4 net charge, were significantly different for sequences from early- and end-stage disease patients. This study identifies specific amino acid changes outside V3 which contribute to CTR. Extending the analysis to include pure X4 and increasing the sample size would be desirable to define gp120 variables/changes which should be included in predictive algorithms.
The identification of phenotypically distinct HIV-1 variants with different prevalence during the progression of the disease has been one of the earliest discoveries in HIV-1 biology, but its relevance to AIDS pathogenesis remains only partially understood. The physiological basis for the phenotypic variability of HIV-1 was elucidated with the discovery of distinct coreceptors employed by the virus to infect susceptible cells. The role of the viral phenotype in the variable clinical course and treatment outcome of HIV-1 infection has been extensively investigated over the past two decades. In this review, we summarize the major findings on the clinical significance of the HIV-1 coreceptor usage.
The use of maraviroc as part of a simplification of antiretroviral therapy (ART) is hampered by the difficulty of assessing viral tropism in patients with undetectable viraemia. In this context, information on tropism might be obtained from testing either older stored viraemic sera collected before initiation of ART or current proviral DNA in peripheral blood cells.
HIV-1-infected individuals who had initiated ART and had undetectable viraemia for >2 years were identified. V3 genotyping was performed in parallel from plasma HIV-RNA and proviral DNA before starting ART and from proviral DNA while on suppressive ART. Viral tropism was interpreted using geno2pheno (false positive rate = 10%) and an optimized version of position specific scoring matrices (PSSM) with a greater sensitivity to detect X4 variants (PSSM(X4/R5-8)).
A total of 78 HIV-1 infected individuals were examined. Mean time under suppressive ART was 3.5 years (interquartile range: 2.3-4.4). The rate of X4 variants in plasma and proviral DNA samples at baseline was 32.8% and 34.0%, respectively. It was 33.9% after >2 years of suppressive ART in DNA samples. Paired RNA/DNA tropism results at baseline could be obtained for 38 patients, with an overall 82% concordance. After >2 years of suppressed plasma viraemia, HIV tropism was re-assessed in proviral DNA; tropism switches were uncommon, especially comparing baseline and most recent DNA longitudinal specimens (12%).
HIV tropism switches over time under suppressive ART are rare. There is a relatively good correlation between RNA and DNA tropism estimations using genotypic tests. Thus, HIV-1 tropism might confidently be examined either in older stored viraemic plasma specimens or in current proviral DNA samples.
Most treated HIV-1 patients have undetectable viral loads and the strategies for managing long-term side effects may involve a new class of antiretroviral-like CCR5 antagonists. Tropism determination based on proviral DNA sequence is necessary for patients with a fully suppressed plasma viral load, as assays analysing DNA phenotypes have yet to be developed. We aimed to analyse HIV-1 tropism using proviral DNA sequencing and the associated factors, in a group of patients on antiretroviral (ARV) treatment with an undetectable viral load in plasma.
Blood samples of 140 HIV-1-infected ARV-treated patients with a plasma viral load of <40 copies/mL were studied. All patients had never been treated with CCR5 antagonists. Co-receptor usage was determined using proviral DNA from the V3 env region sequence by Geno2Pheno (false positive rate 10%) and PSSM algorithms.
Among 140 patients treated using ARV therapy with a fully suppressed plasma viral load, at least 70% of patients had proviral R5-tropic virus. Among all the studied factors (time since HIV infection diagnosis, treatment duration, time since viral load undetectability, HIV subtype, current treatment, age, number of treatment types, sex, genotypic susceptibility score, and nadir and current CD4 cell counts), nadir CD4 T cell count alone was associated with tropism during a multivariate analysis. R5X4/X4 tropism was present in all nadir CD4 categories.
Proviral DNA tropism determination should be required, even for patients with a CD4 nadir cell count >350 cells/mm(3), before CCR5 antagonists are used in patients with a fully suppressed plasma viral load.
In patients with hepatitis C virus (HCV)/HIV co-infection, a faster progression of liver fibrosis to cirrhosis has been reported. In this study, an investigation was carried out to determine whether gp120, an HIV envelope protein, modulates the biology of human hepatic stellate cells (HSCs), key cell types in the pathogenesis of fibrosis.
Myofibroblastic HSCs were isolated from normal human liver tissue. Gene expression was measured by real-time PCR. Cell migration was assessed in Boyden chambers. Intracellular signalling pathways were evaluated using phosphorylation-specific antibodies or by transfection of a reporter plasmid.
Transcripts for the chemokine receptors CCR5 and CXCR4, which bind gp120, were detectable in human HSCs. Upon exposure to M-tropic recombinant gp120, which binds CCR5, a significant increase in HSC chemotaxis was observed (1.6+/-0.3-fold, p=0.03). The effects of gp120 were prevented by protein inactivation. gp120 also resulted in a significant increase in secretion (1.5+/-0.3-fold, p=0.03) and gene expression (1.47+/-0.13-fold, p=0.02) of the proinflammatory chemokine monocyte chemoattractant protein-1, and in increased gene expression of tissue inhibitor of metalloprotease-1 and interleukin-6 (2.03+/-0.57-fold, p=0.02). gp120-induced migration required Akt activation. gp120 also induced activation of nuclear factor-kappaB (NF-kappaB) and p38(MAPK). Preincubation of HSCs with TAK779, a CCR5 receptor antagonist, prevented gp120-mediated chemotaxis and monocyte chemoattractant protein-1 secretion. Expression of CCR5 was detectable in areas of inflammation and fibrogenesis in liver biopsies of patients with HCV/HIV co-infection.
This study shows that HIV gp120 modulates different aspects of HSC biology, including directional cell movement and expression of proinflammatory cytokines. These results identify a direct pathway possibly linking HIV infection with liver fibrogenesis via envelope proteins.
To assess the factors associated with liver fibrosis in human immunodeficiency virus and hepatitis C virus (HIV/HCV) co-infected patients eligible for anti-HCV therapy, we performed an observational, single-centred, cross-sectional study of 180 HIV/HCV co-infected patients who underwent liver biopsy between May 1998 and November 2001. A total of 126 patients with a known date of HCV infection were evaluated. Liver fibrosis was defined as a Knodell stage of fibrosis 1-4. The mean age was 36.7 (3.8) years, 81% were male and had a mean age of 20.5 (3.8) years at HCV infection. Mean CD4 cell count and plasma HIV-1 RNA load at the time of biopsy were 552 cell/mm3 (239) and 2.5 log10 (0.9), respectively; 118 patients had been on antiretroviral therapy (ART) for a median of 45 months (Q1-Q3: 21-75) and 84 on protease inhibitor for a median of 12.0 months (Q1-Q3: 0-29.5); 55 had an AIDS event or a CD4 cell count nadir < 200 cells/mm3 prior to biopsy. Median histological activity index was 6 and 27% had a Knodell stage of fibrosis 0. On the multivariate analysis time on ART (OR for 6 months extra: 0.954, 95% CI: 0.859-0.994), CD4 cell count at the time of liver biopsy (OR for 100 cells/mL increase: 0.740, 95% CI: 0.670-0.905), age at HCV infection acquisition (OR for 5 years extra: 2.594, 95% CI: 1.326-5.133) and alcohol intake (> 50 g/day) (OR: 2.73, 95% CI: 1.108-6.731) were associated with liver fibrosis. Hence ART should be a priority in HIV/HCV co-infected patients eligible for anti-HCV treatment as it is a protective factor for liver fibrosis.
Activated hepatic stellate cells (HSCs) are the main producers of extracellular matrix in the fibrotic liver and are involved in the regulation of hepatic inflammation. The aim of this study was to characterize the role of regulated on activation, normal T-cell expressed, and presumably secreted (RANTES) in activated HSCs. RANTES mRNA and protein secretion were strongly induced after stimulating HSCs with TNF-alpha, IL-1beta, or CD40L. RANTES production was NF-kappaB dependent, because inhibitor-kappaB (IkappaB) superrepressor and dominant-negative IkappaB kinase-2 almost completely blocked RANTES expression. NF-kappaB activation was sufficient to drive RANTES expression as demonstrated by the strong induction of RANTES in HSCs expressing NF-kappaB-inducing kinase. The JNK/activator protein-1 pathway also contributed to RANTES expression as demonstrated by the blocking effects of the JNK inhibitor SP600125. HSCs responded to stimulation with recombinant human (rh)RANTES with an increase in intracellular calcium concentration and a rapid increase in free radical formation. Furthermore, rhRANTES induced ERK phosphorylation, ERK-dependent [3H]thymidine incorporation, and HSC proliferation. Additionally, rhRANTES induced focal adhesion kinase phosphorylation and a substantial increase in HSC migration. HSCs functionally expressed chemokine receptor-5 (CCR5), as shown by flow-cytometric analysis and RT-PCR, and the inhibitory effects of a blocking CCR5 antibody on rhRANTES-induced ERK activation, proliferation, and migration. Diphenylene iodonium and N-acetylcysteine inhibited rhRANTES-induced ERK activation and HSC proliferation, indicating that NADPH oxidase-dependent production of reactive oxygen species was required. In conclusion, RANTES and CCR5 represent potential mediators of 1) HSC migration and proliferation and 2) a cross-talk between HSCs and leukocytes during fibrogenesis.
CD8(+) T-cell responses are an essential antiviral host defense in persistent viral infections, and their sustained effectiveness is thought to be critically dependent on CD4(+) T-helper cells. To determine the relationship between HIV-1-induced CD4(+) T-cell depletion and hepatitis C virus (HCV)-specific CD8(+) T-cell responses during viral persistence, we studied 103 persons positive for HCV, 74 coinfected with HIV-1. CD8(+) T-cell responses to the entire HCV polyprotein were determined by using an interferon-gamma enzyme-linked immunospot (ELISpot) assay. Although HIV-1 infection by itself was not associated with a diminished HCV-specific response, HIV-1-associated CD4(+) depletion was associated with significantly lower HCV-specific CD8(+) T cells (R = 0.48, P < .0001). In contrast, declining CD4(+) counts over the same range were not associated with diminished Epstein-Barr virus (EBV)- (R = 0.19, P = .31) or HIV-1-specific (R = -0.13, P = .60) CD8(+) T-cell responses in persons infected with all viruses. These data indicate that frequencies of circulating HCV-specific CD8(+) T-cell responses are sensitive to absolute CD4(+) T-cell counts and provide a possible explanation for the accelerated HCV disease course in persons coinfected with HIV-1 and HCV.
Here we report a long-term persistence of HIV-1 structural proteins and glycoproteins in germinal centers (GCs) of lymph nodes (LNs) in the absence of detectable virus replication in patients under highly active antiretroviral therapy (HAART). The persistence of viral structural proteins and glycoproteins in GCs was accompanied by specific antibody responses to HIV-1. Seven patients during the chronic phase of HIV-1 infection were analyzed for the presence of the capsid protein (HIV-1p24), matrix protein (HIV-1p17), and envelope glycoproteins (HIV-1gp120/gp41), as well as for viral RNA (vRNA) in biopsy specimens from LNs obtained before initiation of therapy and during HAART that lasted from 5 to 13 months. In parallel, these patients were also monitored for viremia and specific anti-HIV-1 antibody responses to structural proteins and glycoproteins both before and during treatment. Before-therapy viral levels, as determined by RT-PCR, ranged from 3 x 10(3) to 6.3 x 10(5) copies of vRNA per ml, whereas during treatment, vRNA was under detectable levels (<25 copies per ml). The pattern of vRNA detection in peripheral blood was concordant with in situ hybridization results of LN specimens. Before treatment, vRNA associated with follicular dendritic cells (FDCs) was readily detected in GCs of LNs of the patients, whereas during therapy, vRNA was consistently absent in the GCs of LN biopsies of treated patients. In contrast to vRNA hybridization results, viral structural proteins and glycoproteins, evaluated by immunohistochemical staining, were present and persisted in the GC light zone of LNs in abundant amounts not only before initiation of therapy but also during HAART, when no vRNA was detected in GCs. Consistent with immunohistochemical findings, specific antibody responses to HIV-1p17, -p24, and -gp120/gp41, as evaluated by ELISA and virus neutralization, persisted in patients under therapy for up to 13 months of follow-up. The implications of these findings are discussed in relation to HIV-1 persistence in infected individuals and the potential role of chronic antigenic stimulation by the deposited structural proteins in GCs for AIDS-associated B cell malignancies.
Human immunodeficiency virus (HIV) infection is a major cause of acceleration of hepatitis C virus-related liver disease,
cirrhosis, and death. However, studies of liver disease pathogenesis in HIV/HCV coinfection have thus far been limited. Emerging
data support multiple derangements attending HIV coinfection, including increases in profibrogenic cytokine expression and
secretion, generation of enhanced oxidative stress, and increases in hepaotcyte apoptosis. These derangements may be further
augmented in the presence of increased microbial translocation in the setting of HIV disease. New insight into the mechanisms
of HIV/HCV pathogenesis causing accelerated liver fibrosis could lead to new therapeutic strategies designed to retard ths
process.
The aim of the study was to evaluate the use of proviral DNA as a source of viral genetic material for genotypic coreceptor tropism testing (GTT).
GTT consisted of bulk V3 sequencing followed by geno2pheno interpretation with the interpretative cut-off [false positive rate (FPR)] set at 5 and 10%. GTT was performed for 165 patients with a viral load of >500 HIV-1 RNA copies/mL on simultaneously collected plasma RNA and proviral DNA, and for 126 patients with a viral load of <500 copies/mL on current proviral DNA and pretreatment plasma RNA. Phenotypic tropism testing (PTT) results were available for 142 samples.
In the simultaneous RNA/DNA comparison, concordance in prediction was 95.2% (at FPR 10%) and 96.4% (at FPR 5%). Six RNA-R5/DNA-X4 and two RNA-X4/DNA-R5 discordances were observed at an FPR of 10%, and six RNA-R5/DNA-X4 discordances were observed at an FPR of 5%. In the longitudinal RNA/DNA comparison, concordance was 88.1% (at FPR 10%) and 90.5% (at FPR 5%). Eight RNA-X4/DNA-R5 and seven RNA-R5/DNA-X4 discordances were seen at an FPR of 10%, and 10 RNA-R5/DNA-X4 and two RNA-X4/DNA-R5 discordances at an FPR of 5%. The overall concordance of RNA GTT with PTT was 82% (at FPR 10%) and 83% (at FPR 5%). The overall concordance of DNA GTT with PTT was 85% (at both 10 and 5% FPRs).
GTT produced highly concordant tropism predictions for proviral DNA and plasma RNA. GTT on proviral DNA offers a promising approach for tropism prediction in clinical practice, particularly for the assessment of treated patients with low or suppressed viraemia.
Viral tropism is the ability of viruses to enter and infect specific host cells and is based on the ability of viruses to bind to receptors on those cells. Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. In most European countries, HIV tropism is identified with tropism phenotype testing. New data support genotype analysis of the HIV third hypervariable loop (V3) for the identification of tropism. The European Consensus Group on clinical management of tropism testing was established to make recommendations to clinicians and clinical virologists. The panel recommends HIV-tropism testing for the following groups: drug-naive patients in whom toxic effects are anticipated or for whom few treatment options are available; patients who have poor tolerability to or toxic effects from current treatment or who have CNS pathology; and patients for whom therapy has failed and a change in treatment is considered. In general, an enhanced sensitivity Trofile assay and V3 population genotyping are the recommended methods. Genotypic methods are anticipated to be used more frequently in the clinical setting because of their greater accessibility, lower cost, and faster turnaround time than other methods. For the interpretation of V3 loop genotyping, clinically validated systems should be used when possible. Laboratories doing HIV tropism tests should have adequate quality assurance measures. Similarly, close collaboration between HIV clinicians and virologists is needed to ensure adequate diagnostic and treatment decisions.
HCV related liver disease is one of the most important complications in persons with HIV, with accelerated fibrosis progression in coinfected persons compared to those with HCV alone. We hypothesized that HCV-HIV coinfection increases HCV related hepatocyte apoptosis and that HCV and HIV influence TRAIL signaling in hepatocytes.
We analyzed the effect of HIV in JFH1-infected Huh7.5.1 cells. Apoptosis was measured by Caspase-Glo 3/7 assay and Western blotting for cleaved PARP. TRAIL, TRAIL receptor 1 (DR4), and 2 (DR5) mRNA and protein levels were assessed by real-time PCR and Western blot, respectively. We also investigated activation of caspase pathways using caspase inhibitors and assessed expression of Bid and cytochrome C.
We found increased caspase 3/7 activity and cleaved PARP in JFH1 HCV-infected Huh7.5.1 cells in the presence of heat-inactivated HIV, compared to Huh7.5.1 cells infected with JFH1 or exposed to heat-inactivated HIV alone. Both DR4 and DR5 mRNA and protein expression were increased in JFH1-infected cells in the presence of inactivated HIV compared to Huh7.5.1 cells infected with JFH1 or exposed to heat-inactivated HIV alone. Pancaspase, caspase-8, and caspase-9 inhibition blocked apoptosis induced by HCV, inactivated HIV, and HCV plus inactivated HIV. A caspase-9 inhibitor blocked apoptosis induced by HCV, HIV, and HCV-HIV comparably to pancaspase and caspase-8 inhibitors. HCV induced the activation of Bid cleavage and cytochrome C release. The addition of HIV substantially augmented this induction.
Our findings indicate that hepatocyte apoptosis is increased in the presence of HCV and HIV compared to HCV or HIV alone, and that this increase is mediated by DR4 and DR5 up-regulation. These results provide an additional mechanism for the accelerated liver disease progression observed in HCV-HIV co-infection.
Highly active antiretroviral therapy (HAART)-related hepatotoxicity complicates the management of patients infected with human immunodeficiency virus (HIV), increases medical costs, alters the prescription patterns, and affects the guideline recommendations. Among the clinical consequences derived from HAART-related liver toxicity, hypersensitivity reactions and lactic acidosis are recognized as acute events with potential to evolve into fatal cases, whereas there seems to be other syndromes not as well characterized but of equal concern as possible long-term liver complications. Belonging to the latter category of syndrome, HAART-related nonalcoholic steatohepatitis, liver fibrosis, portal hypertension, and nodular regenerative hyperplasia are discussed in this review. Updated information on liver toxicity of current antiretroviral drugs, including the most recently licensed, is provided. Management and prevention of liver toxicity among HIV-infected patients treated with HAART are reviewed as well.
To investigate any emerging trends in causes of death amongst HIV-positive individuals in the current cART era, and to investigate the factors associated with each specific cause of death.
An observational multicentre cohort study.
All HIV-positive individuals included in one of the cohorts in the Data Collection on Adverse Events of Anti-HIV drugs (D:A:D) Study were included. The association between HIV-specific and non HIV-specific risk factors and death were studied using multivariable Poisson regression.
We observed 2482 deaths in 180,176 person-years (PY) on 33,308 individuals [rate/1000 PY = 13.8 (95% CI 13.2-14.3)]. Primary causes of death were: AIDS (n = 743; rate/1000 PY = 4.12), liver-related (341; 1.89), CVD-related (289; 1.60), non-AIDS malignancy (286; 1.59). The overall rate of death fell from 16.9 in 1999/2000 to 9.6/ 1000 PY in 2007/2008. Smoking was associated with CVD and non-AIDS cancers, HBV and HCV co-infection with liver-related deaths, and hypertension with liver-related and CVD deaths. Diabetes was a risk factor for all specific causes of death except non-AIDS cancers, and higher current HIV RNA for AIDS-related deaths. Lower CD4 cell counts were associated with a higher risk of death from all specific causes of death.
Multiple potentially modifiable traditional and HIV-specific risk factors for death of HIV-infected persons were identified. The maximum reduction in mortality in HIV-infected populations will require that each of these factors be appropriately addressed. No trends in terms of emerging causes of unexpected deaths were observed, although monitoring will continue.
Noninvasive markers of liver fibrosis correlate with the stage of liver fibrosis, but have not been widely applied to predict liver-related mortality.
We assessed the ability of two indices of liver fibrosis, aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and Fib-4, and two markers of extracellular matrix metabolism, hyaluronic acid (HA) and YKL40, to predict liver mortality in a prospective cohort of hepatitis C virus (HCV)-infected individuals with and without HIV coinfection. These were compared with two established prognostic scores, the Child-Pugh-Turcotte (CPT) and model of end-stage liver disease (MELD) scores.
A total of 303 subjects, of whom 207 were HIV positive at study entry, were followed up for a mean period of 3.1 years. There were 33 deaths due to liver disease. The ability of each test and score to predict 3-year liver mortality was expressed as the area under the receiver operator curve. The area under the receiver operator curve 95% confidence intervals were: HA 0.92 (0.86-0.96), CPT 0.91 (0.79-0.96), APRI 0.88 (0.80-0.93), Fib-4 0.87 (0.77-0.92), MELD 0.84 (71-0.91). In multivariate analyses HA, APRI, and fib-4 were independent predictors of mortality when included in models with MELD or CPT.
Noninvasive markers of liver fibrosis are highly predictive of liver outcome in HCV-infected individuals with and without HIV coinfection. These markers seem to have a prognostic value independent of CPT and MELD.
The influence of antiretroviral therapy on co-receptor tropism remains controversial. To verify if co-receptor tropism shift was affected by HAART, the evolution of proviral DNA V3 genotype after 12 months of a new antiretroviral regimen was compared between responder and non-responder patients. Baseline blood samples were collected from 36 patients infected with HIV-1 subtype-B (18 naïve and 18 experienced) for virus isolation and env V3 genotyping from plasma HIV-1 RNA and PBMC DNA. DNA V3 genotyping was repeated after 12 months from initiating HAART. WebPSSM was used for categorizing V3 sequences into X4 or R5; for analysis purposes, dual/mixed viruses were considered as X4. From the 10 (28%) patients changing their proviral DNA V3 genotype during therapy, six shifted from R5-to-X4 and four from X4-to-R5. The lack of reaching virological suppression was not associated with an X4-to-R5 (P = 0.25) or R5-to-X4 (P = 0.14) shift; time-to-viral suppression and CD4 increase were similar in both groups. No association was found between tropism shift and patient baseline characteristics including age, sex, CDC stage, CD4 count, viral load, exposure and length of previous HAART, enfuvirtide use in the new regimen, number of reverse transcriptase and protease resistance-associated mutations. Conversely, CD4 nadir was correlated to emergence of X4 virus in proviral DNA (mean 27.2 +/- 30.6 in R5-to-X4 shifting patients vs. 161.6 +/- 150.6 in non-shifting patients, P = 0.02). The occurrence of a tropism shift in both directions was independent of HAART use, irrespective of its efficacy. The CD4 count nadir was the only baseline characteristic able to predict an R5-to-X4 viral shift.
Patients co-infected with the human immunodeficiency virus (HIV) and the hepatitis C virus (HCV) are fraught with a rapid fibrosis progression rate and with complications of portal hypertension (PHT) We aimed to assess the influence of immune function [Centers of Disease Control and Prevention (CDC) stage] on development of PHT and disease progression in HIV-HCV co-infection. Data of 74 interferon-naïve HIV-HCV co-infected patients undergoing liver biopsy, measurement of portal pressure and of liver stiffness and routine laboratory tests (including CD4+ cell count, HIV and HCV viral load) were analysed. Time of initial exposure (risk behaviour) was used to assess fibrosis progression. Fibrosis progression, time to cirrhosis and portal pressure were correlated with HIV status (CDC stage). HIV-HCV patients had rapid progression of fibrosis [0.201 +/- 0.088 METAVIR fibrosis units/year (FU/y)] and accelerated time to cirrhosis (24 +/- 13 years), high HCV viral loads (4.83 x 10(6) IU/mL) and a mean HVPG at the upper limit of normal (5 mmHg). With moderate or severe immunodeficiency, fibrosis progression was even higher (CDC-2 = 0.177 FU/y; CDC-3 = 0.248 FU/y) compared with patients with higher CD4+ nadirs (CDC-1 = 0.120 FU/y; P = 0.0001). An indirect correlation between CD4+ cell count and rate of fibrosis progression (R = -0.6654; P < 0.001) could be demonstrated. Hepatic venous pressure gradient (HVPG) showed early elevation of portal pressure with median values of 4, 8 and 12 mmHg after 10, 15 and 20 years of HCV infection for CDC-3 patients. Patients treated with highly active anti-retroviral therapy (HAART) had similar rates of progression and portal pressure values than patients without HAART. Progression of HCV disease is accelerated in HIV-HCV co-infection, being more pronounced in patients with low CD4+ cell count. A history of a CD4+ cell nadir <200/microL is a risk factor for rapid development of cirrhosis and PHT. Thus, HCV treatment should be considered early in patients with HIV-HCV co-infection and largely preserved CD4+ cell counts.
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A few studies have assessed the observed fibrosis progression between serial liver biopsies (LB) in human immunodeficiency virus (HIV) / hepatitis C virus (HCV)-coinfected patients. Approximately half of the patients progressed at least one fibrosis stage over a short period of time. The risk factors for this fast progression need clarification. Because of this, we evaluated the observed fibrosis progression rates of HIV/HCV-coinfected patients and the risk factors for accelerated progression. Overall, 135 HIV-infected patients with positive serum HCV RNA, without other possible causes of liver disease, who underwent two LB, separated at least by 1 year, were included in this retrospective cohort study. The median (Q1-Q3) time between both LBs was 3.3 (2.0-5.2) years. Patients showed the following changes in fibrosis stage: regression >or =1 stage: 23 (17%), no change: 52 (39%), progression 1 stage: 38 (28%), and progression > or =2 stages: 22 (16%). Seventeen (13%) patients had cirrhosis in the second biopsy. Factors independently associated with progression > or =1 stage were undetectable plasma HIV RNA during the follow-up (relative risk [RR] [95% confidence interval, 95% CI] 0.61 [0.39-0.93], P = 0.03), moderate-to-severe lobular necroinflammation (1.77 [1.16-2.7], P = 0.009), time between biopsies (1.11 [1.08-1.2], P = 0.01), and end of treatment response to anti-HCV therapy (0.41 [0.19-0.88], P = 0.02).
Conclusion:
Fibrosis progresses with high frequency in HIV/HCV-coinfected patients over a period of time of 3 years. Absent-to-mild lobular necroinflammation at baseline, achievement of response with anti-HCV treatment, and effective antiretroviral therapy are associated with slower fibrosis progression.
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, cirrhosis, and death; it is estimated that 180 million persons are infected with HCV worldwide. The consequences of HCV are worse in those who are coinfected with human immunodeficiency virus 1 (HIV-1), which is unfortunately a common scenario because of shared risk factors of the viruses. More studies into effects of HCV/HIV-1 coinfection are needed, but efforts have been hampered by limitations in our understanding of the combined pathogenesis of the 2 viruses. Gaining insight into the mechanisms that underlie the immunopathogenesis of these persistent viral infections could lead to new therapeutic strategies for patients with HCV/HIV-1 coinfection.
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Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell-derived factor-1alpha (SDF-1alpha), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF-1alpha/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF-1alpha is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture-induced activation. Treatment of stellate cells with recombinant SDF-1alpha increases expression of alpha-smooth muscle actin and collagen I and stimulates a dose-dependent increase in HSC proliferation. Inhibitor studies suggest that SDF-1alpha/CXCR4-dependent extracellular signal-regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation.
Conclusion:
HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF-1alpha is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase pathways mediate SDF-1alpha-induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches.
Studies have shown that rates of liver disease are higher in persons who are coinfected with human immunodeficiency virus
(HIV) and hepatitis C virus (HCV) than they are in persons with HCV alone, but estimates of risk vary widely and are based
on data for dissimilar patient populations. We performed a meta-analysis to quantify the effect of HIV coinfection on progressive
liver disease in persons with HCV. Eight studies were identified that included outcomes of histological cirrhosis or decompensated
liver disease. These studies yielded a combined adjusted relative risk (RR) of 2.92 (95% confidence interval [CI], 1.70–5.01).
Of note, studies that examined decompensated liver disease had a combined RR of 6.14 (95% CI, 2.86–13.20), whereas studies
that examined histological cirrhosis had a pooled RR of 2.07 (95% CI, 1.40–3.07). There is a significantly elevated RR of
severe liver disease in persons who are coinfected with HIV and HCV. This has important implications for timely diagnosis
and consideration of treatment in coinfected persons.
Human immunodeficiency virus (HIV) infection is commonly associated with liver dysfunction. The X4 HIV glycoprotein 120 envelope
(env) induces apoptosis in T cells and neurons via the HIV coreceptor CXCR4. Therefore, we investigated whether hepatocyte
death could result from the HIV env signaling through CXCR4 on the hepatocyte. We demonstrated that hepatocytes in humans
express CXCR4 on the cell surface. Furthermore, we established that the X4 HIV env and the entire HIV virion signal hepatocyte
apoptosis through CXCR4. The apoptotic process is dependent on Giα protein signaling, yet it is independent of caspase cascade activation. Thus, HIV can directly cause hepatocyte death in
humans by signaling through CXCR4, without infecting the cell
We have previously shown that hepatocytes exposed to hepatitis C virus (HCV) and human immunodeficiency virus (HIV) envelope
proteins undergo apoptosis. In this article, we further elucidate the signaling mechanisms that mediate this effect. We found
that, in human hepatocellular carcinoma (HepG2) cells, HCV E2 protein and HIV glycoprotein (gp) 120 significantly up-regulated
the Fas ligand (FasL) and enhanced the formation of the Fas death—inducing signaling complex downstream of Fas receptor activation.
Moreover, after stimulation with HCV E2 and HIV gp120, enhanced expression of caspases 2 and 7 and increased caspase 3 activity
were observed. In addition, we showed up-regulation of the proapoptotic molecule Bid and its association with caspase 8 after
treatment with these envelope proteins. We also found that HCV E2 and HIV gp120 induced a partial translocation of Bid to
the mitochondria, which resulted in the release of cytochrome C and the apoptosis-inducing factor. Thus, the results of this
study suggest that FasL and Bid play an important role in HCV and HIV envelope protein—induced apoptosis.
Human immunodeficiency virus (HIV) coinfection increases hepatitis C virus (HCV)-related progression of hepatic fibrosis, increases HCV persistence, and decreases response rates to interferon-based anti-HCV therapy. It has remained unclear how HIV, a nonhepatotropic virus, accelerates the progression of liver disease by HCV.
We explored the possibility that circulating HIV and/or its proteins contribute to the pathogenesis of HCV through engagement of extracellular coreceptors on hepatocytes.
In this study, we found that inactivated HIV or gp120 increases HCV replication and enhances HCV-regulated transforming growth factor (TGF)-beta1 expression in both a replicon and an infectious model of HCV. This proviral effect of HIV and gp120 on HCV replication is neutralized by antibodies to CCR5 or CXCR4. However, HIV and gp120 did not alter type I interferon-mediated signaling in these HCV models, indicating that HIV regulates HCV replication through an alternative mechanism. Interestingly, we found that human TGF-beta1 also enhanced HCV replication. The effect of HIV on HCV replication was blocked by a neutralizing antibody to TGF-beta1, indicating that its effects on HCV replication are TGF-beta1 dependent.
These results suggest a novel mechanism by which HIV not only enhances HCV replication but also contributes to progression of hepatic fibrosis.
Pathogenesis of accelerated fibrosis in HIV/HCV co-infection
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