1. C Sagnelli a, M Merli b, C Uberti-Foppa b, H Hasson b, G Bellini c, C Minichini d, S Salpietro b, E Messina b, N Coppola d, A Lazzarin b, E Sagnelli d, F Rossi c a: Dep. Experimental Medicine and Surgery "F. Magrassi e A. Lanzara", SUN, Naples. b: Dep. Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan. c: Dep. Pediatrics, SUN, Naples. d: Dep. Mental Health and Public Medicine, Section of Infectious Diseases, SUN Naples,. The impact of Cannabinoid Receptor 2-63 variants on the liver histology of 166 patients with HIV infection and chronic hepatitis C and chronic hepatitis C

2. CB1 CB2 ) Cannabinoid receptors (CB1 and CB2) CB1 is found in high concentrations in the brain, but is also present in many peripheral tissues such as the liver, adipose tissue and gut. Straiker A, J Neurphysiol, 2003; Howlett AC, Pharmacol Rev, 2002; Howlett AC, Handb Exp Pharmacol, 2005; Kunos G, J Biol Chem 2008 CB2 is found primarily in the immune system and plays a key role in the modulation of innate immunity. It is also present in peripheral tissues including the liver

3. CB1 was associated with:  liver fibrogenesis  alcoholic and metabolic steatosis  circulatory failure associated with cirrhosis. CB2 was found to have:  antifibrogenic and hepato-protective proprieties  beneficial effects on liver inflammation, alcoholic fatty liver and hepatocyte survival and regeneration. Mallat A, Br J Pharmacol 2010 Endocannabinoid system and the liver Daily cannabis use and HCV - ↑ Fibrosis (Hezode C, Hepatology 2005) - ↑ Steatosis (Hezode C, Gastroenterology 2008) CB1 and HCV CB1 seems to be up-regulated in CHC patients (Van der Poorten D et al, PLoS One. 2010) CB2 and HCV Association to more severe inflammation and hepatocellular necrosis. Coppola N, et al Clin Gastroenterol Hepatol 2013 Coppola N, et al, PlosOne 2014 Osei –Hyiaman D, J Clin Invest 2005; Teixeira-Clerc F, Nat Med 2006; Tam J, Hepatology 2011 Julien B, Gastroenterology 2005; Teixeira-Clerc F, Hepatology 2010; Louvet A, Hepatology 2011; Deveaux V, PlosOne 2009

4. In HIV infection, endocannabinoids may interact with pro- inflammatory events and influence HIV-1 pathogenesis The CB2 agonists may provide an therapeutic target for ablating immuno-pathological processes associated with HIV infection (in reducing neuropathic pain, impaired neurogenesis in the pathogenesis of HIV-associated dementia, reduced AIDS neurological synptoms, ect..). CB2 agonist: -inhibit HIV viral expression, HIV pol and HIV-LTR activity and downregulate CCR5, ect.. -inhibit HIV/Gp120-induced neuronal damage -inhibit macrophage migration TAT induced. -have anti-inflammatory properties. -??????? CB2 and HIV infection

5. AIM This is the first study analyzing the impact of the rs35761398 variant of the CNR2 gene leading to the substitution of Gln (Q) of codon 63 of the CB2 with Arg (R) on the clinical history of chronic hepatitis C in HIV positive patients.

6. Methods Characteristics of patients: naive for anti-HCV treatment, 78.9% under ART, no active alcohol abuse, nor statin assumption, HBsAg-negative, no autoimmune nor genetic liver disease, no US/serologic evidence of HCC. 166 consecutive Caucasian HIV/HCV coinfected patients with asymptomatic chronic hepatitis for 18-36 months were enrolled at the time they underwent their first liver biopsy from 1993 to 2008.

7. Initial characteristics: according to CB2 variants CB2 QQCB2 QRCB2 RRp-value N° of patients266476 Males, N° (%)23 (88.5)41 (64.1)57 (75.0)0.053 Age, years, median (IQR)40.6 (36.4-43.4)40.0(37.1-42.6)42.0 (37.9-46.6)0.12 BMI, m2/kg, median (IQR)23.3 (21.7-25.4)22.5(21.1-24.16)23.2 (21.5-25.2)0.3 IVDU, N°(%)13 (22.4)43 (76.8)44 (73.3) 0.27 Nadir CD4, cell/mmc, median (IQR)231.0 (82.50-322.5)261.5 (160.2-427.0)283.0 (182.5-378.7)0.29 HIV-RNA cps/mL, median (IQR) Negative (< 100 cps /mL), N° (%) 10870.0 (2450.5-44338.0) 15 (57.69) 3800.0 (1007.0-18687.0) 38 (59.37) 13301.0 (3991.0-37219.0) 32 (42.10) 0.37 0.36 CD4, cell/mmc, median (IQR)474.0 (369.5-557.3)570.0(420.3-725.3)492.0(394.8-659.0)0.33 AST, IU/mL, median (IQR)58.0 (41.75-71.00)56.0 (39.5-87.8)67.00 (40.00-132.0090.41 ALT, IU/mL, median (IQR)89.5 (58.25-127.0)73.5 (44.0-114.0)111.50 (49.00-158.75)0.15 Bilirubin, mg/dL, median (IQR)0.7(0.53-0.92)0.7 (0.5-0.5)0.70 (0.47-1.04)0.91 GGT, IU/mL, median (IQR)87.0 (42.75-157.5)70.5(38.0-154.3)75.00 (36.0-189.0)0.61 ALP, IU/mL, median (IQR)174.0 (133.00-240.5)198.0(139.0-253.0)187.50 (136.5-249.8)0.39 Glucose, mg/dL, median (IQR)95.0 (80.50-104.5)85.5 (79.0-92.8)88.0 (81.0-97.5)0.13 Creatinine, mg/dL, median (IQR)0.79 (0.71-0.84)0.8 (0.7-0.9)0.78 (0.69-0.86)0.83 Triglycerides, mg/dL, median (IQR)125.5 (98.75-153.00)122.0 (81.3-153.0)133.0 (88.5-184.0)0.59 Tota. cholesterol, mg/dL, median(IQR)170.5 (149.75-195.0)160.0 (129.0-186.0)162.0 (129.5-194.8)0.2 HCV RNA, IUx10 3, median (IQR)445956.0 (115463.3-1575000.0) 452925.0 (104103.0-1771436.5) 730500.0 (336750.0-1411766.5) 0.76 Duration of HIV infection, years, median (IQR) 15.9 (10.9-17.7)14.39(7.7-18.1)11.7 (6.9-18.5)0.19 HAART, N°(%):treated HAART-naive 22 (76.9) 4 (15.3) 52 (81.3) 12 (18.8) 57 (75.0) 19 (25.0) 0.49 Duration of ART, years, median (IQR)6.46 (3.4-12.8)7.69 (5.87-10.4)8.2 (6.3-11.2)0.41 HCV genotype, N° (%): 1 2 3 4 missing, N° 10 (40.0) 1 (4.0) 10 (40.0) 4 (16.0) 1 22 (35.5) 2 (3.3) 27 (43.5) 11 (17.7) 2 29 (39.7) 5 (6.9) 31 (42.5) 8 (10.9) 3 0.89

8. Initial characteristics: according to CB2 variants CB2 QQCB2 QRCB2 RRp-value N° of patients266476 HAI, score (M ± SD) 5.81 ± 2.615.30 ± 2.676.79 ± 3.37 0.056 Fibrosis, score (M ± SD)2.35 ± 1.412.22 ± 1.702.37 ± 1.560.85 Degree of fibrosis, N° (%):0-3 4-6 22 (84.62) 4 (15.38) 53 (82.81) 11 (17.19) 61 (80.26) 15 (19.74) 0.86 Steatosis, score (M ± SD)1.92 ± 1.351.69 ± 1.281.61 ± 1.330.57 Degree of steatosis, N° (%):0-2 3- 4 15 (57.69) 11 (42.31) 45 (60.31) 19 (29.69) 50 (65.79) 26 (34.21) 0.51 P=0,001

9. Initial characteristics: according to necroinflammation score Degree of HAIP-VALUE 0-89-18 N° of patients12937 Males, N° (%)91 (70.5)30 (81.1)0.20 Age, years, median (IQR)40.9 (37.24-44.2)41.0 (38.0-44.3)0.85 BMI, m2/kg, median (IQR)22.8 (21.2-24.9)23.6 (21.5-25.4)0.72 Nadir of CD4, cell/mmc, median (IQR)258.0 (165.25-422.0)272.0 (149.0-374.0)0.82 IVDA, N°(%) 77 (71.96)23 (76.7)0.8 HIV-RNA, cps/mL, median (IQR) Negative (< 100 cps /mL), N° (%) 8321.0 (1541.5-36329.8) 72 (55.8) 12000.0(3183.5-31052.0) 13 (35.1) 0.19 0.37 CD4, cell/mmc, median (IQR)509.5 (404.5-691.0)466.00 (355.0-608.5)0.37 AST, IU/mL, median (IQR)54.0 (38.3-77.0)112.00 (76.0-166.5)0.0000016 ALT, IU/mL, median (IQR)72.0 (43.5-120.0)137.00(87.5-223.5)0.00039 Bilirubin, mg/dL, median (IQR)0.7 (0.5-1.1)0.81 (0.5-1.05)0.7 GGT, IU/mL, median (IQR)68.5(33.0-155.3)99.00 (60.0-189.0)0.06 ALP, IU/mL, median (IQR)179.0 (132.0-238.3)205.00 (177.0-274.0)0.008 Glucose, mg/dL, median (IQR)87.0 (79.0-95.0)91.00 (83.3-99.8)0.015 Creatinine, mg/dL, median (IQR)0.78 (0.7-0.9)0.79 (0.7-0.8)0.64 Triglycerides, mg/dL, median (IQR)127.0 (86.5-169.8)123.00 (169.8-182.0)1 Total cholesterol, mg/dL, median (IQR)163.0 (137.0-194.0)154.00 (121.5-180.5)0.53 HCV RNA, IUx10 3, median (IQR)511250.0 (115550.0-1432735.0) 732000 (342000.0-1487013.0) 0.30 Duration of HIV infection, years, median (IQR)14.39 (7.67-18.18)13.3 (7.5-18.3)0.37 HAART, N°(%):treated HAART-naive 103 (82.2) 26 (20.2) 28 (75.7) 9 (24.3) 0.58 Duration of ART, years, median (IQR)8.2 (6.15-11.3)6.79 (4.5-10.3)0.2 HCV genotype, N° (%):1 2 3 4 missing, N° 47 (38.2) 7 (5.7) 48 (39.0) 21 (17.1) 6 14 (37.8) 1 (2.7) 20 (54.0) 2 (5.4) 0 0.19

10. Initial characteristics: according to necroinflammation score P= 0.010

11. Initial characteristics: according to necroinflammation score Degree of HAIP-VALUE 0-89-18 N° of patients12937 Liver histology: Fibrosis, score (M ± SD) 1.9 ± 1.4 3.6 ± 1.5 <0.000001 Degree of fibrosis, N° (%): 0-3 4-6 114 (88.4) 15 (11.6) 22 (67.6) 15 (40.5) 0.0001 Steatosis, score (M ± SD)1.6 ± 1.32.0 ± 1.30.03 Degree of steatosis, N° (%): 0-2 3- 4 91 (70.1) 38 (29.5) 19 (51.4) 18 (48.7) 0.03

12. Multivariate logistic regression analysis to confirm the association between the CB2-RR variants and a HAI > 9 was performed, including as covariants the CB2 variant, the CD4+> 500 cell/mL, fibrosis, HAART regimen and age. Both the severe fibrosis (p=0.0001) and the CB2-RR variant (p=0.03) were found to be independently associated with severe necroinflamation.

13. Conclusion The CB2-RR variant was identified as an independent predictor of severe necroinflamation in HIV/HCV coinfected patients with chronic hepatitis. An association of clinical value that warrants further investigation to better define their role in clinical practice.

14. Results Peg-IFN ± RBVSVR CB2-QQ (26 cases) 168 (50%) CB2-QR (64 cases) 3915 (38.5%) CB2-RR (76 cases) 5331 (58.3%) P= 0.16