Longitudinal evaluation of hepatitis C viral persistence in HIV-infected patients with spontaneous hepatitis C clearance.

Hepatitis C virus (HCV) viral persistence in patients with spontaneous viral clearance is controversial. Several studies have shown HCV-RNA in peripheral blood mononuclear cells (PBMCs) and/or liver tissue among patients who have cleared the virus spontaneously, suggesting that viral persistence is a common situation that could involve the entire population studied. Thus, our aim was to evaluate HCV-RNA persistence in PBMCs and hepatocytes in subjects infected with the human immunodeficiency virus (HIV). A total of 1508 patients were prospectively followed and tested for anti-HCV antibodies and HCV-RNA to identify the patients who achieved spontaneous viral clearance. In all of the patients, the persistence of HCV-RNA in PBMCs was evaluated longitudinally during 2 years of follow-up. Fifty-nine patients fulfilled the inclusion/exclusion criteria and were included in the study. HCV-RNA was not detected in the PBMCs at baseline [59 PBMCs samples tested; 0 %; 95 % confidence interval (CI): 0-3.3 %] or during the follow-up (147 PBMCs samples tested; 0 %; 95 % CI: 0-2.02 %). Our study shows that HCV viral persistence is not a frequent occurrence in HIV-infected patients who have spontaneously resolved an HCV infection. Thus, the lack of serum HCV-RNA should continue to be addressed as the standard of healing.

Association of complement receptor 2 polymorphisms with innate resistance to HIV-1 infection.

HIV-1 induces activation of complement through the classical and lectin pathways. However, the virus incorporates several membrane-bound or soluble regulators of complement activation (RCA) that inactivate complement. HIV-1 can also use the complement receptors (CRs) for complement-mediated antibody-dependent enhancement of infection (C-ADE). We hypothesize that hypofunctional polymorphisms in RCA or CRs may protect from HIV-1 infection. For this purpose, 139 SNPs located in 19 RCA and CRs genes were genotyped in a population of 201 Spanish HIV-1-exposed seronegative individuals (HESN) and 250 HIV-1-infected patients. Two SNPs were associated with infection susceptibility, rs1567190 in CR2 (odds ratio (OR) = 2.27, P = 1 × 10(-4)) and rs2842704 in C4BPA (OR = 2.11, P = 2 × 10(-4)). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Although not significant (P = 0.25, OR = 1.57), similar genotypic proportions were obtained for the CR2 marker rs1567190. The results of the two association analyses were combined through a random effect meta-analysis, with a significant P-value of 2.6 x 10(-5) (OR = 2.07). Furthermore, we found that the protective CR2 genotype is correlated with lower levels CR2 mRNA as well as differences in the ratio of the long and short CR2 isoforms.

Association of low-density lipoprotein receptor genotypes with hepatitis C viral load.

Several data suggest that low-density lipoprotein receptor (LDLR) is a co-receptor for hepatitis C virus (HCV). Soluble LDLR can inhibit HCV infectivity; greater plasma low-density lipoprotein levels are associated with treatment success; LDLR genotypes have a synergistic impact on the likelihood of achieving SVR with Peg-IFN plus RBV, as well as on viral kinetics after starting treatment. The objective of this study was to assess the impact of genetic polymorphisms in genes related to cholesterol synthesis and transport pathways on pre-treatment plasma HCV viral load (VL). A total of 442 patients infected with HCV and treatment naive were prospectively recruited. One hundred forty-four SNPs located in 40 genes from the cholesterol synthesis/transport and IL28B were genotyped and analyzed for genetic association with pre-treatment plasma HCV VL. SNPs rs1433099 and rs2569540 of LDLR showed association with plasma HCV VL (P=4 × 10(-4) and P=2 × 10(-3)) in patients infected with genotypes 1 and 4. A haplotype including the last three exons of LDLR showed association with the cutoff level of 600 000 IU ml(-1) VL for genotypes 1 and 4 (OR=0.27; P=8 × 10(-6)), as well as a quantitative VL (mean±s.d.: 6.19±0.9 vs CC+CG 5.58±1.1 logIU ml(-1), P=8 × 10(-5)). LDLR genotypes are a major genetic factor influencing HCV VL in patients infected with genotypes 1 and 4.

Effects of the genetic pattern defined by low-density lipoprotein receptor and IL28B genotypes on the outcome of hepatitis C virus infection.

The aim of this study was to assess the impact of the genetic pattern (GP) defined by the single nucleotide polymorphisms (SNPs) rs14158 of low-density lipoprotein receptor (LDLR) and rs12979860 of interleukin-28B (IL28B) genes on the outcome and features of hepatitis C virus (HCV) infection in patients with and without human immunodeficiency virus (HIV) coinfection. 314 HIV/HCV-coinfected and 109 HCV-monoinfected patients treated with pegylated interferon (Peg-IFN) plus ribavirin (RBV), as well as 51 patients with HCV spontaneous clearance (SC), were included. Variations in both SNPs were determined by the TaqMan polymerase chain reaction (PCR) assay. In the 286 patients chronically infected by HCV genotypes 1 or 4, both rs14158 CC and rs12979860 CC were associated with a higher rate of sustained virological response (SVR), and these effects were complementary in both HCV-monoinfected and HIV/HCV-coinfected patients. Thus, 24 % of patients with rs14158/rs12979860 TT-TC/TT-TC, 33 % with TT-TC/CC, 44.2 % with CC/TT-TC, and 75.8 % harboring CC/CC attained SVR (p < 0.001). SC was associated with the IL28B genotype (66.7 % CC in SC vs. 42.6 % among those with chronic infection, p < 0.001) but not with the LDLR genotype. There was no association between GP and the plasma level of alanine aminotransferase (ALT) or the presence of advanced fibrosis. There is a complementary effect between the IL28B and LDLR genotypes on the probability of achieving SVR after Peg-IFN/RBV therapy in patients with HCV 1 or 4. Thus, the predictive value of IL28B genotype is modulated by the LDLR genotype in both HCV-monoinfected and HIV/HCV-coinfected patients. This complementary effect of both genotypes is also observed on the plasma levels of low-density lipoprotein cholesterol (LDL-C).

Baseline risk factors for relapse in HIV/HCV co-infected patients treated with PEG-IFN/RBV.

PURPOSE: Hepatitis C virus (HCV) viral relapse (VR) after end-of-treatment response (ETR) in human immunodeficiency virus (HIV) co-infected patients is observed in as many as one in three co-infected patients. The aim of the study was to identify baseline risk factors for VR in HIV/HCV co-infected patients treated with pegylated interferon plus ribavirin (PEG-INF/RBV). METHODS: A total of 212 Caucasian HIV-infected patients with chronic hepatitis C naïve for PEG-INF/RBV were followed prospectively. Patients were included in this prospective study if they had completed a full course of therapy with an ETR. We assessed the relationship between VR rate and potential predictors of relapse. RESULTS: Of the patients followed, 130 (61.3 %) attained ETR and 103 (79.2 %) achieved sustained virological response (SVR). Consequently, 27 (20.8 %) showed VR. Patients who relapsed were more often male (p = 0.036), carried the non-CC rs14158 genotype in the low-density lipoprotein receptor (LDLr) gene (p = 0.039), had higher baseline HCV RNA levels (p = 0.012), body mass index (BMI) ≥ 25 kg/m(2) (p = 0.034), significant liver fibrosis (p < 0.001), had been diagnosed with acquired immunodeficiency syndrome (AIDS)-defining criteria in the past (p = 0.001) and bore the HCV genotypes 1/4 (p = 0.046) when compared with SVR patients. The IL28B genotype was not associated with relapse. Multivariate binary logistic regression showed that high baseline HCV RNA, significant liver fibrosis, HCV genotypes 1/4, being overweight and being diagnosed with AIDS-defining criteria in the past were independently associated with relapse. CONCLUSIONS: Our study shows that VR can be accurately predicted in HIV/HCV co-infected patients on the basis of risk factors which can be identified before treatment.

Different distributions of hepatitis C virus genotypes among HIV-infected patients with acute and chronic hepatitis C according to interleukin-28B genotype.

OBJECTIVES: The C allele of the single nucleotide polymorphism rs12979860, located near the interleukin-28B (IL-28B) gene, has a strong impact on hepatitis C virus (HCV) treatment response, as well as on spontaneous viral clearance. In patients with chronic hepatitis C (CHC), genotype CC carriers harbour HCV genotype 3 more commonly than those with non-CC genotypes. The aim of this study was to compare the HCV genotype distributions, according to IL-28B genotype, in HIV-infected patients with CHC and those with acute hepatitis C (AHC). METHODS: The rs12979860 genotype was determined by polymerase chain reaction (PCR) in two subpopulations of HIV-infected patients. The first consisted of 80 German patients with AHC. The second consisted of 476 patients with CHC, belonging to one German and two Spanish cohorts. RESULTS: In the AHC group, 31 (81.6%) rs12979860 CC carriers were infected with HCV genotype 1 or 4 vs. 32 (76.2%) among non-CC carriers (P=0.948). In patients with CHC, among those with the CC genotype, 119 (54.6%) were infected with HCV genotype 1 or 4 and 99 (45.4%) with genotype 2 or 3, whereas in the subset with non-CC genotypes, 200 (77.5%) harboured HCV genotype 1 or 4 and 58 (22.5%) genotype 2 or 3 (P<0.001). CONCLUSIONS: Among HIV-infected patients with CHC, those bearing the IL-28B genotype CC were more commonly infected with genotype 3 than subjects with non-CC genotypes, whereas in HIV-infected subjects with AHC this finding was not obtained. These results strongly suggest that the protective effect of the CC genotype against evolution to CHC is mainly exerted in patients infected with HCV genotype 1 or 4.

c/EBPbeta is a major regulatory element driving transcriptional activation of the CXCL12 promoter.

CXCL12 is considered a constitutively expressed chemokine with homeostatic functions. However, induction of CXCL12 expression and its potential role in several pathologic conditions have been reported, suggesting that CXCL12 gene expression can be induced by different stimuli. To elucidate the molecular mechanisms involved in the regulation of CXCL12 gene expression, we aim to define the molecular factors that operate at the transcriptional level. Basal, constitutive expression of CXCL12 was dependent on basic helix-loop-helix factors. Transcriptional up-regulation of the CXCL12 gene was induced by cellular confluence or inflammatory stimuli such as interleukin-1 and interleukin-6, in a CCAAT/enhancer binding protein beta (c/EBPbeta)-dependent manner. Chromatin immunoprecipitation assays confirmed c/EBPbeta binding to a specific response element located at -1171 of the promoter region of CXCL12. Our data show that c/EBPbeta is a major regulatory element driving transcription of the CXCL12 gene in response to cytokines and cell confluence.

Spotted fever group Rickettsia in brown dog ticks Rhipicephalus sanguineus in southwestern Spain.

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.

Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function.

The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNA-damaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.

Stromal-cell derived factor is expressed by dendritic cells and endothelium in human skin.

Stromal-cell derived factor or SDF-1 is a CXC chemokine constitutively expressed by stromal bone marrow cell cultures that binds to the G-protein-coupled receptor CXCR4. SDF-1/CXCR4 represents a unique, nonpromiscuous ligand/receptor pair that plays an essential role in prenatal myelo- and lymphopoiesis as well as in cardiovascular and neural development. SDF-1 prevents entry of CXCR4-dependent (X4) HIV viruses in T lymphocytes, by binding and internalizing CXCR4. The expression pattern of SDF-1 protein in normal tissues is not known. Here we describe an analysis of SDF-1 mRNA and protein in normal and inflamed skin by in situ hybridization and immunohistochemistry, using a novel anti-SDF-1 monoclonal antibody. We also describe the expression pattern of CXCR4 receptor by immunohistochemistry. Our results show that SDF-1 protein and mRNA are normally expressed by endothelial cells, pericytes, and either resident or explanted CD1a+ dendritic cells. Epithelial cells of sweat glands but not keratinocytes also express SDF-1. In various inflammatory skin diseases, a large number of mononuclear cells and fibroblasts in close contact with CXCR4-positive lymphocytic infiltrates also express SDF-1. CXCR4 was also detected in many different normal cell types, including endothelial and epithelial cells, which points to a role for SDF-1/CXCR4 cell signaling in vascular and epithelial homeostasis. The demonstration of SDF-1 expression in dendritic and endothelial cells provides new insights into the mechanisms of normal and pathological lymphocyte circulation and makes it possible to envisage a role for locally secreted SDF-1 in the selective incapacity of mucosal dendritic cells to support and propagate infection by X4 HIV isolates.

Activation of blood T lymphocytes down-regulates CXCR4 expression and interferes with propagation of X4 HIV strains.

The chemokine receptor CXCR4 serves as a coreceptor for HIV-1 entry into CD4+ cells, in particular for strains emerging late in the infection. Cell surface expression of CXCR4 has, therefore, important implications for HIV-1 pathogenesis. Using blood lymphocytes cultured under various conditions, we studied the expression and regulation of CXCR4. Flow cytometry showed that only about 20% of freshly isolated lymphocytes expressed CXCR4 on the cell surface whereas in 80% of resting blood lymphocytes CXCR4 was located intracellularly. Within a few hours in culture, the intracellular CXCR4 was translocated to the surface and was expressed in the large majority of both naive and memory lymphocytes. A decrease in surface expression of CXCR4 was found when lymphocytes cultured overnight for maximal receptor expression were stimulated with phytohemagglutinin, anti-CD3 antibodies, phorbol 12-myristate 13-acetate and stromal cell-derived factor-1. The superantigen staphylococcal enterotoxin A, a more selective stimulus, induced a marked decrease in CXCR4 expression preferentially in cells positive for the CD25 activation marker. Confocal laser scanning microscopy demonstrated the presence of CXCR4 in the cytosol and on the surface of resting lymphocytes and also showed CXCR4 redistribution after activation. The number of cells infected by the X4 HIV strain NL4.3 paralleled the expression of CXCR4 in CD4+ T lymphocytes. Sustained reduction of CXCR4 cell surface expression upon activation with phytohemagglutinin correlated with a low number of CD4+ T lymphocytes expressing HIV p24 gag antigen. Our results indicate that activation of CD4+ T lymphocytes reduces surface expression of CXCR4 in part by receptor internalization and that cell activation-dependent CXCR4 down-regulation limits spread of infection by X4 viruses.

Genomic organization and promoter characterization of human CXCR4 gene.

CXCR4 is the receptor for the CXC chemokine SDF1 that has essential functions on embryo organogenesis, immunological functions and T lymphocyte trafficking. Recently, CXCR4 has drawn unexpected attention as it was recently identified as a co-factor required for entry of lymphotropic HIV isolates in CD4+ T lymphocytes. CXCR4 is the only SDF1 receptor identified so far. This suggests that CXCR4 expression is critical for the biological effects of SDF1. To investigate the mechanisms controlling both the constitutive and induced expression of CXCR4 receptors we have isolated and characterized the promoter region and determined the genomic structure of the human gene. The CXCR4 gene contains two exons separated by an intronic sequence. A 2.6 kb 5'-flanking region located upstream the CXCR4 open reading frame contains a TATA box and the transcription start site characteristic of a functional promoter. This region also contains putative consensus binding sequences for different transcription factors, some of them associated with the hemopoiesis and lymphocyte development.

Viraemia and p24 antigenaemia are independent risk factors for the emergency of a zidovudine-resistant genotype in nucleoside analogue-treated HIV-1 infection.

We aimed to determine, in an observational retrospective study, whether baseline HTV-1 RNA is an independent predictive factor for the emergence of a genotype associated with zidovudine resistance and whether previously identified predictive factors remain independent when viraemia is taken into account. Fifty nucleoside-naive HIV-1-infected individuals initiating zidovudine therapy (in 11 cases associated with didanosine) were submitted to clinical, immunological and virological monitoring at entry and every 12 weeks thereafter. The critical endpoint of the study was the influence of key baseline characteristics (CD4 cell counts, clinical stage, HIV-1 p24 antigen, virus phenotype and viraemia) upon the time to development of mutation at codon 215. The presence of serum p24 antigen, syncytium-inducing (S1) phenotype, a HIV-1 RNA load greater than the median (32495 RNA copies/ml), CD4 cell counts lower than 200/mm3 and clinical CDC category C were all baseline features associated with more rapid development of the mutant RT215 genotype in the univariate analysis. However, a multivariate Cox proportional hazard stepwise regression analysis showed that only baseline p24 antigenaemia, SI phenotype and a HIV-1 RNA load greater than 32495 RNA copies/ml were sequentially selected as independent predictive factors for the development of the mutant genotype. The present study suggests that baseline HIV-1 RNA load is an independent predictive factor for the development of a zidovudine resistance genotype. Likewise, it reinforces the independent predictive value of serum p24 antigenaemia and SI phenotype, even when viraemia is taken into account.

Human immunodeficiency virus infection modifies the natural history of chronic parenterally-acquired hepatitis C with an unusually rapid progression to cirrhosis.

BACKGROUND/AIMS: To investigate the possible role of HIV infection in the natural history of chronic parenterally-acquired hepatitis C. METHODS: A multicenter cross-sectional study was performed in 547 patients with chronic parenterally-acquired hepatitis C with or without HIV infection (116 HIV-positive and 431 HIV-negative). Approximate duration of HCV infection was estimated in all patients included, and histologic diagnoses made at different time intervals following HCV infection were analyzed in both groups. Factors related to serum HCV-RNA levels were also investigated. RESULTS: Histologic findings were similar in liver biopsies from both HIV-infected and noninfected patients. However, in the first 10 years, 13 out of 87 (14.9%) HIV-positive subjects developed cirrhosis, in comparison with 7 out of 272 (2.6%) in the HIV-negative group (p < 0.01). Similar results were found in the first 5 and 15 years, respectively, and most of the HIV-negative patients with cirrhosis (42 out of 56) developed cirrhosis in a time interval longer than 15 years. Consequently, mean interval from estimated time of HCV infection to cirrhosis was significantly longer in HIV-negative than HIV-positive patients (23.2 vs. 6.9 years; p < 0.001). Chronic active hepatitis (with and without cirrhosis) and long duration of HCV infection were significantly associated with higher HCV load (p < 0.05). Finally, HIV-positive patients with CD4+ cell counts > 500 cells/ml showed a lower HCV load than those with < 500 cells/ml (p < 0.05). CONCLUSIONS: HIV infection modifies the natural history of chronic parenterally-acquired hepatitis C with an unusually rapid progression to cirrhosis. HIV-related immunodeficiency may be a determinant of higher hepatitis C viremia levels and more severe liver damage.

HIV-1 viraemia changes in patients with and without syncytium-inducing phenotype treated with nucleoside analogues: a case-control study.

The objective of the present study was to investigate the effect of the nucleoside analogue treatment on serum viraemia, CD4+ cell count and disease progression in patients with and without syncytium-inducing (SI) HIV-1 variants. To achieve this in a case-control study, 11 nucleoside-naive patients harbouring SI variants who started treatment with zidovudine or zidovudine plus didanosine were matched with 11 control patients who never formed SI variants during a follow-up of 48 weeks. The matching criteria were age, CD4+ cell count and CDC clinical category at the start of the study and exposure to the same antiretroviral treatment. During the follow-up there were no significant differences in the changes of serum HIV-1 RNA viral load and CD4+ cell counts between the two groups. In contrast, AIDS or new AIDS-defining events were observed in five SI cases but in none of the non-SI controls (P = 0.002). The emergence of a zidovudine-resistant mutation at codon 215 was observed in all the patients harbouring SI strains and in six of the subjects with non-SI variants (P = 0.03). The results of the present study show that in patients carrying SI virus, measurements of CD4+ count or RNA viral burden are neither related to the virulence of the virus strains nor able to predict the clinical course of the disease, at least under anti-retroviral drug conditions. Thus, determination of SI phenotype should be considered in the evaluation and monitoring of HIV-1 therapies.

A pilot case-control study of zidovudine compared with zidovudine plus didanosine in patients with advanced HIV-1 disease and no previous experience with antiretrovirals.

Although zidovudine (ZDV) is effective in HIV-1-infected patients, the duration of its efficacy may be short when treatment is started in advanced HIV disease. This pilot prospective case-control study was designed to evaluate the combination of ZDV plus didanosine [ddI] compared with ZDV monotherapy as an initial therapeutic strategy. 'Control' patients (ZDV monotherapy) were matched with 'case' patients (ZDV plus ddI combination therapy) according to the presence or absence of AIDS-defining criteria at entry and CD4 cell count. The case patient group consisted of 35 consecutive HIV-1-infected individuals with < or = 300 CD4 cells/mm3, no previous experience of antiretroviral therapy and who accepted treatment with a combination of ZDV plus ddI. The control patient group consisted of 35 consecutive patients with similar characteristics, but who preferred to start treatment with ZDV alone. Control patients received 250 mg ZDV bid and case patients received ZDV at the same dose plus ddI (200 mg bid). Primary study endpoints were virological (serum HIV-1 RNA) and immunological (CD4 cell count) responses. Viral phenotype (syncytium-inducing (SI) or non-syncytium-inducing (NSI)), development of mutations at codons 215, 41 and 74 and clinical progression (new AIDS-defining event or death) were also assessed. Virological and CD4 cell count responses were significantly greater and more sustained in the group treated with ZDV plus ddI than in the control group, with peak responses of -1.2 +/- 0.7 log10 versus -0.3 +/- 0.4 log10 at 1 month (P = 0.0003) and 61 +/- 52 cells/mm3 versus 19 +/- 25 cells/mm3 at 2 months (P = 0.001), respectively. In both groups the percentage of patients developing a mutation at codon 215 was around 80 per cent at 12 months. A mutation at codon 74 was detected in 30 per cent of case patients at 12 months. Five case patients (14 per cent) versus 12 control patients (34 per cent) showed signs of clinical progression (P = 0.09). In a multivariate model, clinical progression was significantly associated with a baseline