Antonio Chirianni

Premature lesions of the carotid vessels in HIV-1-infected patients treated with protease inhibitors.

ObjectivesTo evaluate the presence of premature atherosclerotic lesions of epiaortic vessels in HIV-1-infected protease inhibitor-(PI) treated patients compared with PI-naive patients and healthy individuals. DesignOne-hundred and two HIV-1-positive patients, including 55 treated with PI for at least 12 months and 47 either naive or treated with PI-sparing regimens, were subjected to epiaortic vessel ultrasonography. These data were compared with those obtained from 104 healthy individuals. MethodsIntima characteristics, pulsation and resistance indexes, and minimal, peak and mean speed were evaluated using a colour power doppler. Atherosclerotic plaques were described. Independent risk factors and values for glycaemia, cholesterolaemia and triglyceridaemia were considered. Statistical analysis included the chi-square test, Mantel–Haenszel test, odds ratio and logistic regression analysis. ResultsOf the PI-treated patients, 29 out of 55 (52.7%) presented acquired lesions of the vascular wall at ultrasonography, whereas similar lesions were found in seven out of 47 (14.9%) PI-naive patients. Of the 104 healthy individuals, seven cases (6.7%) of intimal medial thickness were noted. A slightly significant correlation was found between carotid lesions and age, male sex and hypercholesterolaemia, whereas cigarette smoking, hypertriglyceridaemia and Centers for Disease Control and Prevention stage significantly increased the risk of vascular lesions (P = 0.022, P = 0.017 and P = 0.079 respectively). However, the highest significance regarded use of PI (P = 0.011). These results were confirmed by logistic regression analysis. ConclusionsThese data demonstrate a higher than expected prevalence of premature carotid lesions in the PI-treated compared with PI-naive patients. If confirmed, a periodic ultrasonographic study of the vascular wall should be included in the follow-up of HIV infected patients.

Analysis of HIV-1 load in blood, semen and saliva : evidence for different viral compartments in a cross-sectional and longitudinal study

Objectives: To quantify the HIV‐1 load (measured as copies of viral RNA/ml using competitive reverse transcription‐polymerase chain reaction) in blood, semen and saliva and to look for relationships between the viral burden, the clinical and immunological status and antiretroviral therapy. Methods: Peripheral blood, semen and whole saliva samples were collected from 26 anti‐HIV‐1‐seropositive patients selected for a cross‐sectional study. Nine of the 26 patients provided samples of the three biological fluids for a longitudinal study. Results: HIV‐1 RNA was detected in 26 out of 26 samples of plasma, in 25 out of 26 samples of semen and in 24 out of 25 samples of saliva. The median number of HIV‐1 copies in plasma was 14 817/ml (range: 167‐254 880), in semen was 515/ml (range: 0‐196 050) and in saliva was 162/ml (range: 0‐72 080). The viral load in semen and in saliva was significantly lower than in plasma (P < 0.0001 ). The HIV‐1 RNA levels in plasma and in saliva were correlated (P < 0.05), but levels in semen were not corre‐lated with either plasma or saliva levels. The HIV‐1 copy number in plasma was significantly higher in symptomatic patients than in asymptomatic subjects (P<0.05). Plasma and saliva HIV‐1 RNA levels were higher in subjects with a CD4+ cell count < 200×106/1 than in subjects with a CD4+ cell count > 200×106/1 (P < 0.05). The HIV‐1 RNA load in either plasma, semen or saliva is not related to antiretroviral therapy. Conclusions: The absence of a correlation between plasma and semen loads suggests that semen and blood are distinct viral compartments. Viral load in semen is not related to the clinical stage of HIV infection or to the CD4+ lymphocyte count. Consequently, HIV‐1 ‐infected subjects are potentially infectious at all stages of immunodeficiency and adequate precautions must always be taken to prevent the sexual transmission of HIV.

Low frequency of severe hepatotoxicity and association with HCV coinfection in HIV-positive patients treated with HAART.

Background: Highly active antiretroviral therapy (HAART) is strongly effective in reducing morbidity and mortality in HIV‐1‐positive individuals. Its main drawback is the potential toxicity. Data on the frequency and determinants of severe hepatotoxicity in a clinical setting are still sparse. Methods: This is a prospective study of HIV‐1‐positive individuals with known HBsAg and HCV‐Ab serology. The study end point was progression to alanine aminotransferase (ALT) levels ≥200 IU/L after HAART initiation. Cumulative probability of progression to this end point was estimated by the Kaplan‐Meier method. Crude and adjusted hazard ratios (HR) were estimated by proportional hazards regression model. Results: One thousand two hundred fifty‐five patients were included. HBsAg was found in 91 (7.2%), HCV‐Ab in 578 (46.5%) patients; almost all injection drug users (451 of 482; 93.6%) were HCV‐Ab positive. Sixty‐one individuals progressed to the end point with a probability of 7.9% (95% confidence interval [CI], 5.6‐10.0) of progression at 24 months from starting. Independent factors predicting progression to the end point were baseline ALT levels (HR, 5.29; 95% CI, 3.24‐8.65; every 10 IU/L higher), HCV‐Ab positivity (HR, 4.01; 95% CI, 1.48‐10.85) or both HBsAg and HCV Ab positivity (HR, 3.85, 95% CI, 1.01‐14.61), and previous non‐HAART therapy (HR, 1.84, 95% CI, 1.04‐3.42). Patients receiving stavudine‐containing regimens had a lower risk than those receiving zidovudine‐containing regimens (HR, 0.30, 95% CI, 0.12‐0.71). Conclusions: There was a low risk of ALT ≥200 IU/L in our cohort. Hepatitis C coinfection and elevated ALT levels at HAART initiation are important predictors of progression to ALT ≥200 IU/L; stavudine‐containing regimens were associated with a lower risk compared with zidovudine‐containing regimens.

Colour-Doppler ultrasonography of carotid vessels in patients treated with antiretroviral therapy: A comparative study

Objectives: To evaluate the correlation between antiretroviral therapy (ART) and lesions of the carotid vessels using an ultrasound colour-Doppler technique. Design: A total of 293 HIV-1 infected patients underwent epiaortic vessel ultrasonography: 105 on treatment with protease inhibitors (PI) (group I), 125 PI-naive patients treated with a non-nucleoside reverse transcriptase inhibitor-including regimen (group II), and 63 patients treated with two nucleoside reverse transcriptase inhibitors or naive to ART (group III). Methods: Intima characteristics, pulsation and resistance indexes, and minimal, peak and mean speed were evaluated using a colour power doppler. Atherosclerotic plaques were described. Independent risk factors and values for glycaemia, cholesterolaemia and triglyceridaemia were considered. Statistical analysis included the Wilcoxon tests, the χ2 test, the Cochran Armitage trend test and the Mantel–Haenszel test and, when necessary, logistic regression analysis. Results: Of the 150 group I patients, 55 (52.4%) presented acquired lesions of the vascular wall at ultrasonography, whereas similar lesions were found in 19 out of 125 (15.2%) patients in group II and in nine of 63 (14.3%) in group III. ART, age, smoking and CD4 T-cell count were the main predictive risk factors for vascular lesions. However, the highest significance was with the use of PI. Conclusions: These data confirm the higher prevalence of premature carotid lesions in the PI-treated patients. A periodic ultrasonographic study of the vascular wall should be included in the follow-up of HIV infected patients.

Synthetic full-length and truncated RANTES inhibit HIV-1 infection of primary macrophages.

Objective: To evaluate the role of the selective forces exerted by the host on the HIV-1 structures involved in viral entry. Design and methods: The V3 region of the env gene was analysed in cell-free HIV-1 RNA from 17 infected subjects: 11 long-term non-progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV-1 molecular clone. Results: The intrapatient divergence of the V3-loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non-synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3-loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3-loop structure were more prevalent in LTNP (P = 0.0012). The pNL4-3-derived mutant reproducing a V3-loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte-derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. Conclusions: The results indicate that host factors impose selective constraints on the evolution of the HIV-1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.

Minor Mutations in HIV Protease at Baseline and Appearance of Primary Mutation 90M in Patients for Whom Their First Protease-Inhibitor Antiretroviral Regimens Failed

The association between minor mutations in human immunodeficiency virus (HIV) protease at baseline and development of common primary mutation 90M at virological failure (conferring some resistance to all protease inhibitors [PIs]) was evaluated in 93 previously drug-naive patients experiencing failure of their first PI-based antiretroviral regimens. In logistic regression analysis, the probability of accumulating a new 90M mutation at virological failure was associated with the presence at baseline of minor mutation 36I (naturally occurring in approximately 25% of HIV clade B and in >80% of HIV non-clade-B viruses) (adjusted odds ratio, 13.5 [95% confidence interval, 1.89-95.6]; P=.009) and, possibly, of 10I/V. This suggests a potential role for the presence of 36I at baseline in predicting the appearance of 90M at virological failure.

Evaluation of antitumoral properties of the protease inhibitor indinavir in a murine model of hepatocarcinoma

Purpose: Accumulating evidences show a higher incidence of hepatic neoplasm in HIV/hepatitis C virus (HCV)–coinfected individuals compared with HCV-monoinfected patients. Treatment with HIV-1 protease inhibitors inhibited cancer-promoted angiogenesis in HIV-infected patients affected by Kaposi sarcoma. We aimed to evaluate the antineoplastic potential activities of the protease inhibitor indinavir (Crixivan) in in vitro and in vivo hepatocarcinoma models. Experimental Design: We analyzed effects of indinavir on cell growth and invasiveness in Huh7 and SK-HEP-1 hepatocarcinoma cell lines and on in vivo tumor growth of the same cells in nude mice. Morphologic and molecular analyses on explanted tumors were carried out to evaluate vascularization and apoptosis. Results: We observed a reduced ability to invade an in vitro extracellular matrix for both cell lines treated with indinavir compared with controls (P = 0,001). Moreover, indinavir treatment was able to inhibit matrix metalloproteinase-2 proteolytic activation, whereas there was no effect on cell proliferation. The drug was also able to delay in vivo tumor growth. The inhibition of tumor growth was statistically significant from days 6 to 21 (P = 0.004 and P = 0.003, respectively). Moreover, the drug showed antiangiogenic and proapoptotic actions, as revealed by vessel count and apoptotic index by terminal deoxynucleotide transferase–mediated nick end labeling in explanted tumors. Finally, treatment with indinavir did not block the production of vascular endothelial growth factor in the tumors. Conclusion: Indinavir could be helpful to prevent the development of hepatocarcinomas in HIV/HCV–coinfected individuals. In view of the current trend to substitute protease inhibitors with other antiretroviral agents, this information may have clinical implications.

Role of NEDD8 in HIV-associated lipodystrophy

The pathogenetic bases of HAART-associated lipodystrophy are still poorly known, even if it is clear that adipose tissue and its metabolism are sensitive to antiretroviral therapy alone and/or in combination with HIV infection. The NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression, morphogenesis and tumorigenesis. We investigated the possible involvement of NEED8 in adipogenesis and, consequently, in HIV-related lipodystrophy. One hundred HIV-1-infected patients were included in the study. Using an in vitro model of adipogenesis we evaluated the effects on adipogenesis of the forced expression of NEDD8 together with efavirenz, stavudine, saquinavir, amprenavir and indinavir, belonging to the three main classes of anti-HIV medications. We showed that NEDD8 expression level is higher in the peripheral blood of HIV patients developing lipodystrophy. Coherently, forced expression of NEDD8 in an in vitro model of adipogenesis was able to perturb expression of some key proteins involved in adipogenesis, such as C/EBPalpha and PPARgamma, possibly acting throughout the NEDD8/p27/beta-catenin pathway. Moreover, three out of five evaluated drugs were able to affect adipocyte differentiation: efavirenz, stavudine and saquinavir. Finally, we have shown that NEDD8 was expressed in the fat tissue of lipodystrophic patients, being significantly higher in the lipodystrophic patients with respect to the controls, thus further confirming the altered NEDD8 expression in the fat tissue of HIV-infected patients affected by lipodystrophy. Taken together, our data support the hypothesis of an implication of NEDD8 through p27 and beta-catenin pathways in the disruption of adipogenesis and consequent lipodystrophy in patients affected by HIV infection under HAART therapy with qualitative and quantitative differences according to diverse antiretroviral treatments. These evidences indicate the NEDD8/beta-catenin/p27 pathway as a possible molecular target for prevention of lipodystrophy development in patients under HAART therapy.

From current status to optimization of HCV treatment: Recommendations from an expert panel

Chronic hepatitis C virus (HCV) infection is a major public health problem at a global level, causing an enormous burden of hepatic and extra-hepatic morbidity and mortality. Treatment of chronic HCV (CHC) has been revolutionized in the last few years by the introduction of highly effective and well tolerated direct acting antiviral agents (DAAs) able to achieve >90% rates of sustained virological response (SVR) in many groups of patients, including those previously excluded from interferon-based regimens. For such reason interferon-free regimens are now the treatments of choice for all patients. Successful anti-HCV treatment can stop liver disease progression and can solve the HCV-related extra hepatic manifestations, eventually reducing both liver-related and overall mortality. Together with the rapidly accumulating data about the evolution of treatment landscape, different guidelines from national and international Liver Scientific Societies have been published until today. However, these recommendations may not be applied worldwide as, due to high treatment costs, most of them identify as priority groups only patients with advanced liver disease. Moreover some types of patients pose clinical management problems for which even the guidelines do not always provide useful answers. With the aim of treatment optimization by filling some of the gaps of the current guidelines and addressing the remaining unmet needs in practice, a group of Italian experts, experienced on treatment of HCV infection, met in Stresa in February 2016. The summary of all the considerations arising from this two-day meeting and the final statements are reported in this position paper.

Amprenavir inhibits the migration in human hepatocarcinoma cell and the growth of xenografts

The introduction of HAART (highly‐active‐antiretroviral‐therapy) has resulted in extended survival of HIV positive patients. Conversely, due to the prolonged expectancy of life and the ageing of the HIV positive population, tumors are now one of the major cause of death, and among them hepatocellular carcinoma (HCC) has become a growing concern in these patients. Considering the potential anti‐tumoral effects of HIV protease inhibitors, we decided to evaluate the anti‐tumoral activity of Amprenavir on liver carcinoma and to evaluate its potential synergistic effects in combination with standard chemoterapic drugs, such as Doxorubicin. Our results indicate that Amprenavir had direct inhibitory effects on invasion of Huh‐7 hepatocarcinoma cell lines, inhibiting MMP proteolytic activation. Amprenavir was able to delay the growth of hepatocarcinoma xenografts in nude mice and had a synergistic effect with Doxorubicin. Furthermore, Amprenavir was able to promote regression of hepatocarcinoma growth in vivo by anti‐angiogenetic and overall anti‐tumor activities, independently by PI3K/AKT related pathways that at today is one of the more suggestive hypothesis to explain the anti‐tumor effects of the different protease inhibitors. In summary these findings suggest novel anti‐neoplastic action of Amprenavir on liver cancer showing the possibility of novel combination therapies. J. Cell. Physiol. 228: 640–645, 2013.