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Open Access

Peer-reviewed

Research Article

Cannabinoid receptor 2-63 RR variant is independently associated with severe necroinflammation in HIV/HCV coinfected patients

  • Caterina Sagnelli,

    Roles Conceptualization, Data curation, Methodology, Writing – original draft

    Affiliation Department of Mental Health and Public Medicine, Section of Infectious Diseases, University of Campania Luigi Vanvitelli, Naples, Italy

    ⨯
  • Caterina Uberti-Foppa,

    Roles Conceptualization, Data curation, Writing – review & editing

    Affiliation Department of Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy

    ⨯
  • Hamid Hasson,

    Roles Data curation

    Affiliation Department of Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy

    ⨯
  • Giulia Bellini,

    Roles Data curation, Formal analysis

    Affiliation Department of Pediatrics, University of Campania Luigi Vanvitelli, Naples, Italy

    ⨯
  • Carmine Minichini,

    Roles Data curation

    Affiliation Department of Mental Health and Public Medicine, Section of Infectious Diseases, University of Campania Luigi Vanvitelli, Naples, Italy

    ⨯
  • Stefania Salpietro,

    Roles Data curation

    Affiliation Department of Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy

    ⨯
  • Emanuela Messina,

    Roles Data curation

    Affiliation Department of Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy

    ⨯
  • Diletta Barbanotti,

    Roles Data curation

    Affiliation Department of Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy

    ⨯
  • Marco Merli,

    Roles Data curation

    Affiliation Department of Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy

    ⨯
  • Francesca Punzo,

    Roles Data curation, Formal analysis

    Affiliation Department of Pediatrics, University of Campania Luigi Vanvitelli, Naples, Italy

    ⨯
  • Nicola Coppola,

    Roles Data curation, Methodology

    Affiliation Department of Mental Health and Public Medicine, Section of Infectious Diseases, University of Campania Luigi Vanvitelli, Naples, Italy

    ⨯
  • Adriano Lazzarin,

    Roles Conceptualization, Writing – review & editing

    Affiliation Department of Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy

    ⨯
  • Evangelista Sagnelli ,

    Roles Conceptualization, Writing – original draft, Writing – review & editing

    evangelistasagnelli@libero.it, evangelista.sagnelli@unicampania.it

    Current address: Department of Mental Health and Public Medicine, Section of Infectious Diseases, University of Campania Luigi Vanvitelli, Naples, Italy

    Affiliation Department of Mental Health and Public Medicine, Section of Infectious Diseases, University of Campania Luigi Vanvitelli, Naples, Italy

    ⨯
  • Francesca Rossi

    Roles Data curation, Formal analysis, Funding acquisition

    Affiliation Department of Pediatrics, University of Campania Luigi Vanvitelli, Naples, Italy

    ⨯

Abstract

Objective

This is the first study to analyze the impact of the rs35761398 variant of the CNR2 gene leading to the substitution of GLN (Q) of codon 63 of the cannabinoid receptor 2 (CB2) with ARG (R) on the clinical presentation of chronic hepatitis in HIV/HCV coinfected patients.

Methods

Enrolled in this study were 166 consecutive HIV/HCV coinfected patients, naïve for HCV treatment. A pathologist unaware of the patients’ condition graded liver fibrosis, necroinflammation (Ishak) and steatosis. All patients were screened for the CB2 rs35761398 polymorphism.

Results

Of the 166 HIV/HCV coinfected patients, 72.9% were males, 42.5% were infected with HCV-genotype-3 and 60.2% had been intravenous drug users. The median age was 40.6 years and the immunological condition good (median CD4+ cells/mm3 = 507, IQR: 398.0–669.5). Thirty-five (21.1%) patients were naive for ART and 131(78.9%) were on ART. The CB2-RR variant was detected in 45.8% of patients, QR in 38.6% and QQ in 15.7%. Patients with CB2-RR showed a necroinflammation score (HAI) ≥9 more frequently than those with CB2-QQ or CB2-QR (32.9% vs. 11.5% and 14.1%, respectively, p≤0.001). In the multivariate analysis, the CB2-RR variant (p = 0.03) and liver fibrosis were both identified as independent predictors of the entity of liver necroinflammation (p = 0.0001).

Conclusion

This study shows interesting interplay between the CB2-RR variant and liver necroinflammation in chronic hepatitis patients with HIV/HCV coinfection, an observation of clinical value that coincides with the interest in the use of the CB2 agonists and antagonists in clinical practice emerging from the literature.

Citation: Sagnelli C, Uberti-Foppa C, Hasson H, Bellini G, Minichini C, Salpietro S, et al. (2017) Cannabinoid receptor 2-63 RR variant is independently associated with severe necroinflammation in HIV/HCV coinfected patients. PLoS ONE 12(7): e0181890. https://doi.org/10.1371/journal.pone.0181890

Editor: Yury E. Khudyakov, Centers for Disease Control and Prevention, UNITED STATES

Received: May 8, 2017; Accepted: July 7, 2017; Published: July 31, 2017

Copyright: © 2017 Sagnelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: The authors received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; ART, antiretroviral therapy; AST, aspartate aminotransferase; BMI, body mass index; CB2, cannabinoid receptor 2; CHC, chronic hepatitis C; CI, confidence interval; GGT, gamma glutamyl transferase; HAART, highly active antiretroviral therapy; HAI, histological activity index; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IQR, interquartile range; M, Mean; OR, odds ratio; SD, standard deviation

Introduction

One-third of HCV-monoinfected patients with chronic hepatitis C (CHC) progress to cirrhosis in approximately 20 years [1], the risk factors for higher rates and more rapid transition being an older age, alcohol abuse, male sex, and human immunodeficiency virus (HIV) coinfection [2–21].

Several other factors have been found to be associated with CHC progression and severity, such as the duration of HCV infection, coinfection with hepatitis B virus (HBV), insulin resistance, diabetes, high body mass index (BMI), immunosuppression of different etiology, alcohol abuse, drug addiction, interleukin-28B polymorphism and the cannabinoid receptor 2 (CB2)-63 variants [22–28]. The CB2-63 RR variant was identified as an independent predictor of some aggressive autoimmune pathologies [29,30] and of some immune mediated diseases associated with HCV chronic carriers [31].

Two types of CB are known, type 1 (CB1) and type 2 (CB2), which exert mainly anti-inflammatory and immunomodulatory action [32]. CB2 is expressed predominantly in the cells of the immune system [33,34], and particularly in CD4+ cells [35], Kupffer cells and hepatic stellate cells, which all play an essential role in the acute and chronic responses to toxic and infectious agents [36]. The CB2 expression in hepatocytes is induced by various inflammatory processes, such as that induced by HCV replication on the intracellular lipid membrane [33,34].

CB2 may function as a chemotactic modulator that inhibits CXCR4-induced chemotaxis in trafficking T cells [37] and the chemokine receptor 5 (CCR5), a co-receptor for HIV-1 cell entry. By interfering with the action of other chemoattractants [38], as well as the chemotaxis of immune cells, and endothelium and leukocyte infiltration, cannabinoids may reduce the inflammatory processes and injury to the endothelial barriers, such as the blood brain barrier, and HIV-1 infection of susceptible cells [39].

It has also been suggested that in HIV infection, endocannabinoids may interact with different pro-inflammatory events influencing HIV-1 pathogenesis [39], downregulating CCR5 and inhibiting viral expression [40].

A polymorphism at codon 63 of the CB2 gene allows the substitution of glutamine, GLN (Q), with arginine, ARG (R), with a consequent differentiation in the protein polarization. These CB2 variants affect differently the ability of CB2 to perform its inhibitory function [33,41].

To our best knowledge, the present paper is the first to analyze the impact of the rs35761398 variant of the CNR2 gene on the clinical history of biopsy proven chronic hepatitis in HIV/HCV coinfected patients.

We investigated the impact of the rs35761398 variant of the CNR2 gene on the clinical presentation of biopsy proven chronic hepatitis in 166 HIV/HCV coinfected patients.

Material and methods

Ethics statement

All procedures applied in the study were in accordance with the international guidelines, with the standards of human experimentation of the local Ethics Committees and with the Helsinki Declaration of 1975, revised in 1983. At the time of the first observation, each patient signed their informed consent to undergo liver biopsy, for the collection and storage of serum and whole blood and for the collection and use in clinical research of the data obtained, as established by the Ethics Committee of the Azienza Ospedaliera Universitaria—University of Campania Luigi Vanvitelli, Naples, previously named Second University of Naples (protocol number 112/15 March 2013).

Patients

One-hundred and sixty-six consecutive anti-HIV-positive patients with HCV-RNA-positive CHC were enrolled from 1993 to 2008 at the time they underwent their first liver biopsy at one of the two participating Units of Infectious Diseases, one in Milan and the other in Naples, after an observation period of at least 18 months. These two centers have cooperated for several years in numerous clinical investigations applying the same clinical and laboratory approach [14,16,42–44].

All patients enrolled were HBsAg-negative, declared no active alcohol abuse (≥ 40 g/day for males and ≥ 30 g/day for females for at least 5 years) or statin intake and had no evidence of autoimmune or genetic disorders that may induce liver disease. These patients were naive for anti-HCV treatment and none showed ultrasound or serological signs of HCC. None of the 166 patients enrolled had impaired fasting, glycemia or diabetes and had never received anti-tuberculosis or antifungal medications.

At the time of enrolment, 131 (78.2%) patients were receiving antiretroviral therapy (ART) and 35 had been left untreated in accordance with the current DHHS guidelines [45].

Samples of serum and whole blood were obtained at the time the liver biopsy was performed, stored at −80°C and never thawed until used for this investigation.

Liver biopsy

Percutaneous liver biopsy (LB) [46] was performed for all patients under US guidance using a disposable modified Menghini needle (16 gauge—external diameter 1.65 mm). A liver specimen of more than 1.5 cm in length was always obtained and at least 11 portal tracts were examined in each sample. Specimens were fixed in formalin, embedded in paraffin and stained with hematoxylin-eosin and Masson’s trichrome stain. Liver biopsies were examined by a skilled pathologist who, blinded for the clinical and laboratory data, used the Ishak scoring system to grade necroinflammation and fibrosis [47]. In the absence of a standardized scoring system to assess liver steatosis, we used a homemade scoring system obtained with a partial modification of Kleiner’s scoring system for NAFLD [48], assigning score 1 to a fatty deposition in 1–10% of hepatocytes, score 2 in 11–31%, score 3 in 31–60% and score 4 in >60%.

Serological determinations

Serum HBsAg was sought by a commercial immunoenzymatic assay (Abbott Laboratories, North Chicago, IL, USA) and the anti-HCV antibody by a 3rd generation commercial immunoenzymatic assay (Ortho Diagnostic Systems, Neckargemund, Germany). Antibodies to HIV 1 and 2 were sought using a commercial ELISA (Abbott Lab., North Chicago, Ill, USA), and positive results were always confirmed by Western blot (Genelabs Diagnostics, Science Park Drive, Singapore), in accordance with the Italian guidelines.

HCV RNA was quantified by a real-time polymerase chain reaction (PCR) in a Light cycler 1.5 (Roche Diagnostics, Branchburg, NJ, USA); by this method, the detection limit in plasma samples is estimated at around 40 IU/mL. HCV genotyping was performed by a Line-Probe assay (INNO-LIPA HCV-II; Innogenetics, Zwigndrecht, Belgium). The HIV viral load was assessed using the Amplicor HIV Monitor 1 test (Roche Molecular Systems Inc., Branchburg, New Jersey).

Lymphocyte subsets (CD4+, CD8+) were evaluated by flow cytofluorimetry using monoclonal antibodies and a fluorescence-activated cell sorter scan (Becton Dickinson, Mountain View, USA). Liver function tests and triglyceride and cholesterol assessment were carried out applying routine methods. The body mass index (BMI: kg/m2) was determined by standard procedure.

The CNR2 rs35761398 polymorphism

Genom ic DNA was extracted from peripheral whole blood with a DNA extraction kit (Roche Diagnostics, Branchburg, NJ, USA) after written informed consent. Molecular screening for the CNR2 rs35761398 polymorphism (CAA/CGG) underlying the CB2Q63R substitution was performed using a TaqMan Assay (Real Master Mix Probe, 5 PRIME, Germany). Primers and probes were the following: sense primer 59-GTGCTCTATCTGATCCTGTC- 39 and anti-sense primer 59-TAGTCACGCTGCCAATC-39; AA-probe 59-CCCACCAACTCCGC- 39 and GG-probe 59-CCCACCGGCTCCG-39 (PRIMM, Milan, Italy). Both PCR and post-PCR allelic discrimination were carried out on an ABI PRISM 9600 apparatus (Applied Biosystems, Foster City, CA). Random samples were confirmed by direct PCR sequencing consisting of 94°C for 4 min followed by 31 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s with forward 59-GAGTGGTCCCCAGAAGACAG-39 and reverse 59-CACAGAGGCTGTGAAGGTCA-39 primers. PCR products were analyzed using an ABI PRISM 3100 automated sequencer (Applied Biosystems, Foster City, CA) and the Big Dye Terminator reaction kit (Applera, Foster City, CA), according to the manufacturer’s instructions. All primers were chosen using Primer3 software (http://primer3.sourceforge.net/). This procedure had been used in previous investigations [49,50].

Statistical analysis

The patients’ allele frequencies were tested for the Hardy-Weinberg equilibrium with Fisher’s exact test.

Continuous variables not normally distributed were summarized as median and interquartile range (IQR), and categorical variables as absolute and relative frequencies. Mood’s Median test was used to compare continuous variables, and the chi-square test for categorical variables. A p value < 0.05 was considered significant.

Multivariate logistic regression analysis was performed to explore the overall effect of the parameters significantly associated with the risk of a histological activity index (HAI) ≥9 at the univariate analysis.

The statistical analysis was performed using Statgraphics Centurion XV.II (Adalta, Arezzo, Italy; Statpoint Technologies Inc., Virginia, USA).

Results

Of the 166 HIV/HCV coinfected patients enrolled in the present study, 72.9% were males, 42.5% were infected with HCV-genotype-3 and 38.1% with genotype 1, 60.2% had a history of previous intravenous drug use (IVDU) but none stated active drug addiction. The median age was 40.5 years and the immunological condition good on the basis of the median CD4+ cells/mm3 count: 507.0 (IQR: 398.0–669.5) at enrolment with a nadir of 258.0 (IQR: 163.5–404.5). Thirty-five (21.1%) patients were naive for ART and 131 (78.9%) were on ART. Untreated patients showed higher CD4+ cell counts than those on ART: 581.0 cells/ mm3 vs. 492.0 cells/ mm3 (p = 0.01) at enrolment and 431.0 cells/ mm3 vs. 230.5 cells/ mm3 (p = 0.01) nadir values.

The CB2-63 RR variant was detected in 45.8% of the patients, QR in 38.5% and QQ in 15.7%. The minor allele frequency was QR and the genotypes were distributed according to the Hardy–Weinberg equilibrium. The mean grade of liver necroinflammation, expressed as HAI scores ranging from 0 to 18, was 5.4 ± 3.0 (SD), the mean stage of liver fibrosis was 2.3 ± 1.6 (range 0–4) and the mean degree of liver steatosis 1.7 ± 1.3 (range 0–4).

Demographic, biochemical and histological characteristics of the patients according to the CB2-63 genotype variants

The 64 subjects with the CB2-63 QR variant and the 76 with the CB2-63 RR variant more frequently than the 26 patients with the CB2-63 QQ variant had a history of previous IVDU (67.2% and 58.0% vs. 50.0%, respectively) (Table 1). Patients with CB2-RR showed a moderate or severe HAI score (9–18) more frequently than those with CB2-63 QQ or CB2-63 QR (32.9% vs. 11.5% and 14.1%, respectively, p = 0.01; Odds Ratio 3.18627; 95% CL 1.47008–6.90597). No other significant difference was observed in the demographic, laboratory or histological data, nor in ART treatment. (Table 1)

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Table 1. Demographic, biochemical and histological characteristics of the 166 HIV/HCV coinfected patients according to the CB2 63 genotype variants.

https://doi.org/10.1371/journal.pone.0181890.t001

Patients’ characteristics according to the grade of liver necroinflammation

Table 2 shows a comparative analysis of the initial characteristics of the 166 patients according to the degree of liver necroinflammation, absent or mild (HAI score 0–8) versus moderate or severe (HAI score 9–18). Compared with the 129 patients with lower HAI scores, the 37 patients with moderate or severe necroinflammation more frequently had the CB2-RR variant (65.6% vs. 47.3%, p = 0.01) and showed higher AST (112.0 vs. 54.0, p = 0.000001), ALT (137.0 vs. 72.0, p = 0.0004), and ALP (205.0 vs. 179.0, p = 0.008) serum levels and higher liver fibrosis scores (3.6 ± 1.5 vs 1.9 ± 1.4, p< 0.0001, and steatosis scores (2.0 ± 1.3 vs 1.6 ± 1.3, p = 0.03) (Table 2). In particular, patients with a moderate or high HAI score, compared to those with a lower score, more frequently showed severe fibrosis score (stage 4–6 in 40.5% vs 11.6%, p = 0.0001) and severe steatosis score (grade 3–4 in 48.7% vs. 29.5%) (Table 2). No other differences in the two necroinflammation subgroups reached statistical significance (Table 2), nor in ART treatment.

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Table 2. Demographic, biochemical and histological characteristics of the 166 HIV/HCV coinfected patients according to necroinflammation (HAI score).

https://doi.org/10.1371/journal.pone.0181890.t002

Multivariate analysis

The association between the CB2-63 RR variant and HAI score 9–18 was analyzed in a multivariate analysis (Table 3) including as covariates the age at enrolment, HIV RNA (≤ 100 vs. > 100 copies/ml), CD4+cells/mL count (≤ 500 vs. > 500 cells/mL), liver fibrosis stage (absent or mild vs. moderate or severe; 0–3 vs. 4–6) and ART regimen (yes vs. no). Homozygous Q63 and heterozygous QR patients were assessed together since the CB2 Q63R variant exerts a significant effect on modulation of necroinflammation only in homozygosity for the R63-encoding allele. The CB2-63 RR variant (p = 0.02) and severe fibrosis (p = 0.001) were both found to be independently associated with severe necroinflammation, with a correlated risk of developing severe necroinflammation about three-fold greater in R63 homozygous carriers compared to QQ and QR carriers.

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Table 3. Logistic regression for HAI score ≥9 for the 166 HIV/HCV coinfected patients.

https://doi.org/10.1371/journal.pone.0181890.t003

Discussion

The present study is the first, to the best of our knowledge, to demonstrate that the CB2-63 RR variant is an independent predictor of moderate/severe liver necroinflammation in chronic hepatitis patients with HIV/HCV coinfection. In accordance, the CB2-63 RR variant was recently found to be independently associated with some aggressive autoimmune pathologies such as celiac disease and childhood immune thrombocytopenic purpura [29,30] and with immune mediated diseases in HCV chronic carriers [31]. In addition, the endocannabinoid system has been demonstrated to play a role in several systemic inflammatory disorders such as rheumatoid arthritis [51]. It has also been reported that in obese children with ultrasound-proven liver steatosis, the ALT serum level is significantly higher in children with the QR or RR variant than in those with the CB2-63 QQ variant [52].

In our previous study performed on 253 anti-HIV negative subjects with HCV chronic infection the CB2-63 QQ variant and an older age were found to be independently associated with an asymptomatic, inactive condition characterized by persistently normal alanine-aminotransferase (PNALT) values at a two-monthly determination for 18 months or more [50], whereas in a subsequent investigation on 169 anti-HIV-negative patients with HCV-related chronic hepatitis, the CB2-63 QQ variant was associated with more severe liver necroinflammation [53]. Based on the observation that CB2-mediated inhibition of T-cell proliferation has been shown to be normal in T cells deriving from QQ subjects and reduced two-fold in T cells from subjects with the RR homozygous variant [54], these associations were interpreted as the result of differences in the strength and duration of the inhibition of the T cells expressing CB2-63 QQ or CB2-63 RR and of the consequent lesser or more vigorous immune response against infected hepatocytes. Interestingly, the mean age of the 53 PNALT subjects in the first study was higher than that of the second study (58.5 vs 49.6 years), suggesting that a substantial percentage of PNALT subjects may have reached this status after a variety of immunological conditions ranging from a strong cellular immune response in the HCV acute infection and the initial phase of chronic hepatitis to a failure of the cellular immune response in the more advanced inactive phase of the illness [53].

The anti-HIV-positive patients in the present study were much younger (mean age 40.5 years) than those with chronic HCV monoinfection (PNALT subjects or chronic hepatitis patients) mentioned above and plausibly exposed to HCV and its associated immunological pressure for a shorter time, a difference that might influence the expression of the CB2-63 variants. Noteworthy, HIV replication is responsible for a variety of immunological reactions favouring more severe HCV-related necroinflammation [53] and may interact with the CB2-63 genetic expression. Indeed, the specific activation of CB2 receptors may inhibit not only the production of autoantibodies, proinflammatory cytokines and matrix metalloproteinases, but also bone erosion, an immune response mediated by T cells and the proliferation of fibroblast-like synoviocytes [51]. In addition, CB2 exerts an inhibitory effect on inflammatory processes [55], including macrophage migration [56], and provides an important therapeutic target for reducing/ablating some immunopathological processes associated with HIV-1 infection [56], a beneficial effect confirmed by the observation that CBR2 agonists reduce AIDS symptoms [57].

The activation of CB2 inhibits CXCR4-tropic HIV infection by altering the CD4+ T-cell actin dynamic and reduces the frequency of infected cells by 30–60% [57]. The level of CB2 activation able to inhibit the HIV virus does not actually alter the CXCR4 surface expression, but significantly reduces CXCR4-mediated G-protein binding and downstream signaling [57], factors accompanied by a reduction in F-actin accumulation [58] and the prevention of the cortical actin rearrangements required for reverse transcription and migration of the viral preintegration complex into the nucleus [58]. Taken together, these data suggest that CB2 cross-regulates CXCR4 and that this inhibitory cross-talk may decrease HIV infection [57]. Therefore, the reciprocal effects exerted by HIV infection and the CB2 receptor may explain the different association of severe liver necroinflammation and the CB2 variants: CB2 RR in HIV/HCV coinfected patients who show an aggressive disease with a rapid progression to liver cirrhosis and hepatocellular carcinoma and CB2 QQ in HCV-monoinfected patients with an asymptomatic indolent course of the liver disease.

Concluding on this point, the data from this study identified the CB2 RR variant as an independent predictor of more severe liver necroinflammation in HIV/HCV coinfected patients with chronic hepatitis.

The number of patients investigated in the present study may be considered barely sufficient for a genetic association, a limitation offset by the gold-standard method used to detect liver lesions (liver biopsy examination by a skilled pathologist) and by the novelty of the data reported. In addition, in-vitro studies are needed to ascertain how the CB2-63 variants modify the mechanism of liver cell injury in HCV infection, both in HIV-infected and non-infected patients. Further clinical investigations on this topic are welcome, particularly because of the increasing interest in the use of CB2 agonists and antagonists in clinical practice.

References

  1. 1. Alter MJ, Margolis HS, Krawczynski K, Judson FN, Mares A, Alexander WJ, et al. The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. N Engl J Med. 1992;327(27):1899–1905. pmid:1280771
  2. 2. Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, DOSVIRC groups. Lancet. 1997;349(9055):825–832. pmid:9121257
  3. 3. Wiley TE, McCarthy M, Breidi L, Layden TJ. Impact of alcohol on the histological and clinical progression of hepatitis C infection. Hepatology. 1998;28(3):805–809. pmid:9731576
  4. 4. 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(4):562–9. pmid:11462196
  5. 5. HH , Yi Q, Dore GJ, Krahn MD. Natural history of hepatitis C virus infection in HIV-infected individuals and the impact of HIV in the era of highly active antiretroviral therapy: a meta-analysis. AIDS. 2008;22(15):1979–1991. pmid:18784461
  6. 6. Herman KE, Rouster SD, Chung RT, Rajicic N. Hepatitis C virus prevalence among patients infected with human immunodeficiency virus: a cross-sectional analysis of the US adult AIDS Clinical Trials Group. Clin Infect Dis. 2002;34(6):831–7. pmid:11833007
  7. 7. Frederick T, Burian P, Terrault N, Cohen M, Augenbraun M, Young M, et al. Factors associated with prevalent hepatitis C infection among HIV-infected women with no reported history of injection drug use: the Women’s Interagency HIV Study (WIHS). AIDS Patient Care STDS. 2009;23(11):915–23. pmid:19877800
  8. 8. Chu C, Umanski G, Blank A, Meissner P, Grossberg R, Selwyn PA. Comorbidity-related treatment outcomes among HIV-infected adults in the Bronx, NY. J Urban Health. 2011;88(3):507–16. pmid:21302140
  9. 9. Raymond HF, Hughes A, O’Keefe K, Stall RD, McFarland W. Hepatitis C prevalence among HIV-positive MSM in San Francisco: 2004 and 2008. Sex Transm Dis. 2011;38(3):219–20. pmid:20938373
  10. 10. Benhamou Y, Bochet M, Di Martino V, Charlotte F, Azria F, Coutellier A, et al. Liver fibrosis progression in human immunodeficiency virus and hepatitis C virus coinfected patients. The Multivirc Group. Hepatology. 1999;30(4):1054–8. pmid:10498659
  11. 11. Kim A, Wiesch J, Kuntzen T, Timm J, Kaufmann D, Duncan J, et al. Impaired hepatitis C virus-specific T cell responses and recurrent hepatitis C virus in HIV Coinfection. PLoS Med. 2006;3(12):e492. pmid:17194190
  12. 12. Koziel M. Influence of HIV co-infection on hepatitis C immunopathogenesis. J Hepatol. 2006;44(1 Suppl):S14–8. pmid:16338026
  13. 13. Lo Re V, Kallan MJ, Tate JP, Localio AR, Lim JK, et al. Hepatic Decompensation in Antiretroviral-Treated HIV/Hepatitis C-Coinfected Compared to Hepatitis C-Monoinfected Patients: A Cohort Study. Ann Intern Med. 2014; 160(6): 369–379. pmid:24723077
  14. 14. Sagnelli C, Uberti-Foppa C, Pasquale G, De Pascalis S, Coppola N, Albarello L, et al. Factors influencing liver fibrosis and necroinflammation in HIV/HCV coinfection and HCV monoinfection. Infection. 2013;41(5):959–67. pmid:23839212
  15. 15. Schiavini M, Angeli E, Mainini A, Uberti-Foppa C, Zerbi P, Sagnelli C, et al. Fibrosis progression in paired liver biopsies from HIV/HCV co-infected patients. Hepat Mon. 2011;11(7):525–31. pmid:22706343
  16. 16. Sagnelli C, Uberti-Foppa C, Galli L, Pasquale G, Coppola N, Albarello L, et al. Liver histology in HIV/hepatitis C-coinfected and HCV-monoinfected patients with persistently normal alanine aminotransferases. J Acquir Immune Defic Syndr. 2010;54(1):107–8. Erratum in: J Acquir Immune Defic Syndr. 2010;54(3):338. Galli, Laura [added]. pmid:20418725
  17. 17. Angeli E, Mainini A, Cargnel A, Uberti-Foppa C, Orani A, Carbone R, et al. Predictability of sustained virological response to pegylated interferon alpha-2b Plus ribavirin therapy by week-8 viral response in HIV-positive patients with chronic hepatitis C virus infection. Curr HIV Res. 2009;7(4):447–55. pmid:19601782
  18. 18. Uberti-Foppa C, De Bona A, Galli L, Sitia G, Gallotta G, Sagnelli C, et al. Liver fibrosis in HIV-positive patients with hepatitis C virus: role of persistently normal alanine aminotransferase levels. J Acquir Immune Defic Syndr. 2006;41(1):63–7. pmid:16340475
  19. 19. Pineda JA, Garcia-Garcia JA, Aguilar-Guisado M, Rios-Villegas MJ, Ruiz-Morales J, Rivero A, et al. Clinical progression of hepatitis C virus-related chronic liver disease in human immunodeficiency virus-infected patients undergoing highly active antiretroviral therapy. Hepatology. 2007;46(3):622–30. pmid:17659577
  20. 20. Brau N, Salvatore M, Rios-Bedoya CF, Fernandez-Carbia A, Paronetto F, Rodriguez-Orengo JF, et al. Slower fibrosis progression in HIV/HCV-coinfected patients with successful HIV suppression using antiretroviral therapy. J Hepatol.2006;44(1):47–55. pmid:16182404
  21. 21. Qurishi N, Kreuzberg C, Luchters G, Effenberger W, Kupfer B, Sauerbruch T, et al. Effect of antiretroviral therapy on liver-related mortality in patients with HIV and hepatitis C virus coinfection. Lancet. 2003;362(9397):1708–13. pmid:14643119
  22. 22. Coppola N, Pisaturo M, Sagnelli C, Onorato L, Sagnelli E. Role of genetic polymorphisms in hepatitis C virus chronic infection. World J Clin Cases. 2015;3(9):807–22. pmid:26380828
  23. 23. Sagnelli E, Coppola N, Pisaturo M, Masiello A, Tonziello G, Sagnelli C, et al. HBV Superinfection in HCV Chronic Carriers: A Disease That Is Frequently Severe but Associated with the Eradication of HCV. Hepatology. 2009: 49: 1090–7. pmid:19263473
  24. 24. 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. pmid:11481613
  25. 25. Adinolfi LE, Gambardella M, Andreana A, Tripodi MF, Utili R, Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology. 2001: 33: 1358–64. 18. pmid:11391523
  26. 26. Ishida JH, Jin C, Bacchetti P, Tan V, Peters M, Bacchetti P, et al. Influence of cannabis use on severity of hepatitis C disease. Clin Gastroenterol Hepatol. 2008: 6: 69–75. pmid:18166478
  27. 27. Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O'Huigin C, et al. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature. 2009: 461: 798–801. 20. pmid:19759533
  28. 28. Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature. 2009: 461: 399–40. pmid:19684573
  29. 29. Rossi F, Mancusi S, Bellini G, Roberti D, Punzo F, Vetrella S, et al. CNR2 functional variant (Q63R) influences childhood immune thrombocytopenic purpura. Haematologica. 2011: 96: 1883–5. pmid:21828121
  30. 30. Rossi F, Bellini G, Tolone C, Luongo L, Mancusi S, Papparella A, et al. The cannabinoid receptor type 2 Q63R variant increases the risk of celiac disease: implication for a novel molecular biomarker and future therapeutic intervention. Pharmacol Res. 2012:66: 88–94. pmid:22465144
  31. 31. Coppola N, Zampino R, Bellini G, Stanzione M, Capoluongo N, Marrone A, et al. CB2-63 polymorphism and immune-mediated diseases associated with HCV chronic infection. Dig Liver Dis. 2016;48(11):1364–1369. pmid:27476469
  32. 32. Miller AM, Stella N. CB2 receptor-mediated migration of immune cells: it can go either way. Br J Pharmacol. 2008;153(2):299–308. pmid:17982478
  33. 33. Cabral GA, Griffin-Thomas . Cannabinoids as therapeutic agents for ablating neuroinflammatory disease. Endocr Metab Immune Disord Drug Targets. 2008: 8: 159–72. pmid:18782012
  34. 34. Louvet A, Teixeira-Clerc F, Chobert MN, Deveaux V, Pavoine C, Zimmer A, et al. Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice. Hepatology. 2011: 54: 1217–26. pmid:21735467
  35. 35. Cencioni MT, Chiurchiu V, Catanzaro G, Borsellino G, Bernardi G, Battistini L, et al. Anandamide suppresses proliferation and cytokine release from primary human T-lymphocytes mainly via CB2 receptors. PLoS One. 2010: 14; 5(1): e8688. pmid:20098669
  36. 36. Basu PP, Aloysius MM, Shah NJ, Brown RS. Review article: the endocannabinoid system in liver disease, a potential therapeutic target. Aliment Pharmacol Ther. 2014: 39: 790–801. pmid:24612021
  37. 37. Ghosh S, Preet A, Groopman JE, Ganju RK. Cannabinoid receptor CB2 modulates the CXCL12/CXCR4-mediated chemotaxis of T lymphocytes. Mol Immunol. 2006:43: 2169–2179. pmid:16503355
  38. 38. Montecucco F, Burger F, Mach F, Steffens S. CB2 cannabinoid receptor agonist JWH-015 modulates human monocyte migration through defined intracellular signaling pathways. Am J Physiol Heart Circ Physiol. 2008; 294:H1145–1155. pmid:18178718
  39. 39. Rom S, Persidsky Y. Cannabinoid receptor 2: Potential role in immunomodulation and neuroinflammation. J Neuroimmune Pharmacol. 2013; 8(3): 608–620. pmid:23471521
  40. 40. Rock RB, Gekker G, Hu S, Sheng WS, Cabral GA, Martin BR, et al. J Neuroimmune WIN55, 212-2-mediated inhibition of HIV-1 expression in microglial cells: involvement of cannabinoid receptors. Pharmacol. 2007;2(2):178–83.
  41. 41. Carrasquer A, Nebane NM, Williams WM, Song ZH. Functional consequences of non synonymous single nucleotide polymorphisms in the CB2 cannabinoid receptor. Pharmacogenet Genomics. 2010:20: 157–166. pmid:20124950
  42. 42. Sagnelli C, Uberti-Foppa C, Galli L, Pasquale G, Coppola N, Albarello L, et al. Anti-hepatitis C virus treatment may prevent the progression of liver fibrosis in non-responder human immunodeficiency virus/hepatitis C virus coinfected patients. Braz J Infect Dis. 2014; 18:164–9. pmid:24650995
  43. 43. Sagnelli C, Merli M, Uberti-Foppa C, Hasson H, Grandone A, Cirillo G, et al. The TM6SF2 E167K variant predicts severe liver fibrosis for HIV/HCV coinfected patients, and severe steatosis only for a non-3 HCV genotype. World Journal of Gastroenterology. 2016:22(38):8509–8518. pmid:27784963
  44. 44. Sagnelli C, Merli M, Uberti-Foppa C, Hasson H, Cirillo G, Grandone A, et al. Impact of PNPLA3 variants on liver histology of 168 patients with HIV infection and chronic hepatitis C. Clin Microbiol Infect. 2016;22(4):372–8. pmid:26806136
  45. 45. http://aidsinfo.nih.gov/guidelines/archive/adult-and-adolescent-guidelines/
    • 46. Sagnelli C, Martini S, Pisaturo M, Pasquale G, Macera M, Zampino R et al. Liver fibrosis in human immunodeficiency virus/hepatitis C virus coinfection: Diagnostic methods and clinical impact. World J Hepatol. 2015;7(24):2510–21. pmid:26523204
    • 47. 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. pmid:7560864
    • 48. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005; 41: 1313–21. pmid:15915461
    • 49. Coppola N, Zampino R, Bellini G, Macera M, Marrone A, Stanzione M, et al. Cannabinoid receptor 2–63 QQ variant is associated with severe necroinflammation in chronic hepatitis C. Clin Gastroenetrol Hep. 2014:12: 334–340.
    • 50. Coppola N, Zampino R, Sagnelli C, Bellini G, Marrone A, Stanzione M, et al. Cannabinoid receptor 2–63 QQ variant is associated with persistently normal aminotransferase serum levels in chronic hepatitis C. PLoS One. 2014;9(6):e99450. pmid:24940753
    • 51. Gui H, Tong Q, Qu W, Mao CM, Dai SM. The endocannabinoid system and its therapeutic implications in rheumatoid arthritis. Int Immunopharmacol. 2015:26(1):86–91. pmid:25791728
    • 52. Rossi F, Bellini G, Nobili B, Maione S, Perrone L, Del Giudice EM. Association of the cannabinoid receptor 2 (CB2) Gln63Arg polymorphism with indices of liver damage in obese children: an alternative way to highlight the CB2 hepatoprotective properties. Hepatology. 2011:54: 1102–1103. pmid:21608006
    • 53. Coppola N, Zampino R, Bellini G, Macera M, Marrone A, Pisaturo M, et al. Association between a polymorphism in cannabinoid receptor 2 and severe necroinflammation in patients with chronic hepatitis C. Clin Gastroenterol Hepatol. 2014;12(2):334–40. pmid:23707465
    • 54. Sipe JC, Arbour N, Gerber A, Beutler E. Reduced endocannabinoid immune modulation by a common cannabinoid 2 (CB2) receptor gene polymorphism: possible risk for autoimmune disorders. J Leukoc Biol. 2005:78: 231–8. pmid:15845647
    • 55. Cabral GA, Staab A. Effects on the immune system. Handb. Exp. Pharmacol. 2005;168:385–423.
    • 56. Raborn ES, Jamerson M, Marciano-Cabral F, Cabral GA. Cannabinoid inhibits HIV-1 Tat-stimulated adhesion of human monocyte-like cells to extracellular matrix proteins. Life Sci. 2014;104(1–2):15–23. pmid:24742657
    • 57. Costantino CM, Gupta A, Yewdall AW, Dale BM, Devi LA, Chen BK. Cannabinoid Receptor 2-Mediated Attenuation of CXCR4-Tropic HIV Infection in Primary CD4+ T Cells. PLoS One. 2012;7(3):e33961. pmid:22448282
    • 58. Yu D, Wang W, Yoder A, Spear M, Wu Y. The HIV envelope but not VSV glycoprotein is capable of mediating HIV latent infection of resting CD4 T cells. PLoS Pathog. 2009;5: e1000633. pmid:19851458