Current HIV Research (v.14, #6)

Meet Our Editorial Board Member by Robert C. Bailey (455-455).

HIV Infection and Myocardial Infarction by Leonardo Calza (456-465).
Background: After the advent of the potent combination antiretroviral therapy (cART) the incidence of acquired immune deficiency syndrome (AIDS) has declined dramatically and HIV infection became a chronic disease with a significant increase in the life expectancy of HIV-positive people. Consequently, chronic comorbidities as coronary heart disease raised an increasing concern in this population.
Objective: Aim of this editorial article is to review the most recent data about the cardiovascular disease risk among HIV-positive persons and to suggest an appropriate clinical management.
Results: An increased risk of myocardial infarction has been reported among HIVinfected subjects compared to the general population, but the pathogenic mechanism of this accelerated atherosclerotic process is complex and certainly multifactorial. The occurrence of myocardial infarction may be the consequence of traditional risk factors (that are overrepresented in the HIVinfected population), direct viral replication, and long-term toxicity of the antiretroviral drugs. Moreover, despite the persistent viral suppression induced by cART usually reduces the cardiovascular risk, several studies show in HIV-positive subjects a condition of chronic inflammation and immune activation that could lead to both accelerated endothelial dysfunction and atherosclerotic disease.
Conclusion: The cardiovascular risk reduction and coronary heart disease prevention are today a leading challenge for all the clinicians involved in the HIV patients' care.

Spatiotemporal Analysis of AIDS Incidence Among Adults in Brazil by Elisangela Aparecida da Silva Lizzi, Altacilio Aparecido Nunes, Edson Zangiacomi Martinez (466-475).
AIDS is the fourth leading cause of death worldwide and, currently, the overall prevalence rate of HIV infection in Brazil is 0.5% among men and 0.3% among women.
Objective: To evaluate the spatiotemporal trend of AIDS in Brazil from 2006 to 2012 and its relationship with human development index (HDI) and their components income, education and life expectancy.
Methods: This ecological study evaluate the spatiotemporal trend of standardized incidence ratio of AIDS among adults in Brazil from 2006 to 2012 and its relationship with HDI by using a Bayesian analysis, considering the Brazilian Federal Units as units of analysis. The proposed statistical model allows obtaining a standardized incidence ratio (SIR, adjusted by gender and age).
Results: Among the men, our results show higher incidence rates in the States of the Southern regions as well as in the state of Amazonas (Northern Brazil). In females, we found other patterns for SIR, with higher incidence rates in the states of Rio de Janeiro (Southeast region), Rio Grande do Sul and Santa Catarina (both in Southern region). Among men it was observed as an expressive association between the SIR values and the overall HDI and income and education components, but it was observed to have an inverse association with the life expectancy component. Among women, it is noted that the SIR values are associated with the overall HDI and the education components only at the beginning of the studied period.
Discussion: AIDS remains a major public health problem in Brazil, mainly in the southern and southeastern regions of the country. Considering its association with HDI, it is noted that the disease still remains related to the pattern observed in the early years of the studied period, at least in the more developed regions of Brazil. This certainly happened because of the chronicity of the disease, thus affecting people with good socioeconomic status.

Discovering molecules capable of binding to HIV trans-activation responsive region (TAR) RNA thereby disrupting its interaction with Tat protein is an attractive strategy for developing novel antiviral drugs. Computational docking is considered as a useful tool for predicting binding affinity and conducting virtual screening. Although great progress in predicting protein-ligand interactions has been achieved in the past few decades, modeling RNA-ligand interactions is still largely unexplored due to the highly flexible nature of RNA. In this work, we performed molecular docking study with HIV TAR RNA using previously identified cyclic peptide L22 and its analogues with varying affinities toward HIV-1 TAR RNA. Furthermore, sarcosine scan was conducted to generate derivatives of CGP64222, a peptide-peptoid hybrid with inhibitory activity on Tat/TAR RNA interaction. Each compound was docked using CDOCKER, Surflex-Dock and FlexiDock to compare the effectiveness of each method. It was found that FlexiDock energy values correlated well with the experimental Kd values and could be used to predict the affinity of the ligands toward HIV-1 TAR RNA with a superior accuracy. Our results based on comparative analysis of different docking methods in RNA-ligand modeling will facilitate the structure-based discovery of HIV TAR RNA ligands for antiviral therapy.

Background: Current antiretroviral therapy (ART) cannot cure HIV-1 infection due to the presence of latent viral reservoirs. The “shock and kill” strategy is a promising approach to eliminate the viral reservoir. However, there are various limits existing in current latency-reversing agents, searching for new activators are urgently needed.
Objective: The present study aimed at investigating the ability of hymecromone and scoparone for activating HIV-1 from latent reservoirs.
Methods: Jurkat T cell model of HIV-1 latently were used to evaluate the effect of hymecromone and scoparone. The percentage of green florescence protein expression as a marker for reactivation of HIV-1 promoter was measured via FACScan. The expression of CD25 and CD69 in human peripheral blood mononuclear cells was measured by flow cytometry at 72 h post-treatment with hymecromone or scoparone or prostratin using antibodies against CD25 and CD69.
Results: Hymecromone and scoparone can induce HIV-1 LTR reactivation in a dose and timedependent. We further show that hymecromone and scoparone can reactivate latent virus without inducing the activation of global T cells. We also found that scoparone acts by NF-&kgr;B signal pathway.
Conclusion: Hymecromone and scoparone can effectively reactivate latent HIV-1 with low cellular toxicity, indicating hymecromone and scoparone might be potential drugs for HIV-1 reservoir eradication strategies in the future.

Baseline CD4/CD8 T-Cell Ratio Predicts Prompt Immune Restoration Upon cART Initiation by Francesco Bellissimo, Marilia Rita Pinzone, Benedetto Maurizio Celesia, Bruno Cacopardo, Giuseppe Nunnari (491-496).
Introduction: The reversal of CD4/CD8 ratio is considered an independent predictor of death in the general population, where the ratio physiologically decreases with aging. Despite effective cART, CD4/CD8 normalization does not always occur in HIV-positive subjects. In the setting of HIV, low CD4/CD8 T-cell ratio correlates with immune activation and non-AIDS events. The aim of the study was to evaluate the rate and predictors of CD4/CD8 ratio normalization in a cohort of HIV-positive subjects starting combination antiretroviral therapy (cART).
Methods: This is a retrospective-prospective observational cohort study conducted at the Unit of Infectious Diseases of the University of Catania. Our cohort included naive individuals who initiated cART from January 2007 to December 2013.
Results: A total of 123 individuals were enrolled. The median age was 38 years (IQR 29-44). The median baseline CD4+ T-cell count was 288 cells/?l (IQR 105-400). 83 (67.5%) had a CD4+ T-cell count <350/?l; baseline median CD4/CD8 ratio was 0.24 (IQR 0.13-0.4); 65 patients (52.8%) had a HIV viral load >100,000 copies/ml. At 24 months, 33 individuals (26.8%) normalized their CD4/CD8 ratio, with a median time to CD4/CD8 ratio normalization of 17 months (IQR 12-30). In univariate analysis, a baseline CD4+ T-cell count >350/?l (p <0.01), a baseline CD4/CD8 ratio >0.5 (p <0.01), CDC stage A (p<0.01) and an efavirenz-based first-line regimen (p<0.05) were associated with CD4/CD8 ratio normalization. In multivariate logistic analysis, the only predictor of CD4/CD8 normalization was a baseline ratio >0.5 (OR 4.3 (1.7-11.2), p=0.003).
Conclusion: Starting cART with a ratio >0.5 is associated with an increased likelihood to normalize CD4/CD8 ratio. Early diagnosis should be encouraged in order to treat patients promptly and favor a more robust immunological reconstitution.

Effects of Antiretroviral Molecules on Survival and Gene Expression of An Osteoblast-like Cell Line by Anna Miserocchi, Giuseppina Musumeci, Isabella Bon, Silvia Morini, Serena Longo, Marco Borderi, Davide Gibellini, Maria Carla Re (497-505).
Background: The advent of combined antiretroviral therapy effectively undermined the evolution of HIV disease. Nevertheless, clinical observations indicated a clear association between therapy and the impairment of bone mineral density.
Objective: We selected some antiretroviral compounds used in clinical practice, to study their impact on bone health and their possible implication in the onset of bone disease.
Method: Scalar concentrations of several antiretroviral drugs (used in single and in combination) were tested on an osteoblast-like cell line, HOBIT cells, to analyse cell survival and gene expression of selected bone markers.
Results: None of the tested concentrations of Tenofovir, Emtricitabine, Nevirapine, Maraviroc or Raltegravir induced any significant apoptosis activation at our experimental conditions. Only some protease inhibitors and Efavirenz, at high concentration, determined a significant activation of programmed cell death. In parallel experiments, protease inhibitors used in combination with Tenofovir and Emtricitabine, increased apoptosis. Furthermore, we performed a study of mRNA expression of specific genes involved in osteoblast biology and in bone synthesis and observed that some protease inhibitors induced a selective decrease of some osteogenic markers.
Conclusion: All the protease inhibitors included in this study trigger apoptosis at the highest concentration analysed, suggesting great caution in HIV-patients co-infected with HBV or HCV, where elevated plasma concentrations of drugs could be reached as a consequence of liver failure. Lastly, an increased apoptosis rate and an impairment of osteogenic markers were recorded only in the presence of Nelfinavir, suggesting a role of protease inhibitors in the alteration of osteoblast biology.

Naringin Ameliorates HIV-1 Nucleoside Reverse Transcriptase Inhibitors- Induced Mitochondrial Toxicity by Adebiyi Oluwafeyisetan, Adebiyi Olubunmi, Owira Peter (506-516).
Background: Mitochondrial reactive oxygen species (ROS) generation and defective oxidative phosphorylation (OXPHOS) have been proposed as possible mechanisms underlying the development of nucleoside reverse transcriptase inhibitors (NRTIs)-induced mitochondrial toxicities. Available options in managing these complications have, so far, produced controversial results, thus necessitating further research into newer agents with promise. Antioxidant and free-radical scavenging effects of naringin, a plant-derived flavonoid, have previously been demonstrated.
Objective: This study was designed to investigate the effects of naringin on NRTIs-induced mitochondrial toxicity.
Methods: Wistar rats were randomly divided into Zidovudine (AZT)-only (100 mg/kg body weight BW); AZT+Naringin (100+50 mg/kg BW); AZT+Vitamin E (100+100 mg/kg BW); Stavudine (d4T)- only (50 mg/kg BW); d4T+Naringin (50+50 mg/kg BW); d4T+Vitamin E (50+100 mg/kg BW) and Vehicle (3.0 mL/kg BW)-treated groups, respectively. After 56 days of oral daily dosing, rats were euthanized by halothane overdose, blood collected by cardiac puncture and livers promptly excised for further biochemical and ultrastructural analyses.
Results: AZT- or d4T-only caused significant mitochondrial dysfunction and mitochondrial ultrastructural damage compared to controls, while either naringin or vitamin E reversed indices of mitochondrial dysfunction evidenced by significantly reduced mitochondrial malondialdehyde (MDA) and blood lactate concentrations, increased liver manganese superoxide dismutase (MnSOD) activity and upregulate expression of mitochondrial-encoded subunit of electron transport chain (ETC) complex IV protein compared to AZT- or d4T-only treated rats. Furthermore, naringin or vitamin E, respectively, ameliorated mitochondrial damage observed in AZT- or d4T-only treated rats.
Conclusion: Naringin ameliorated oxidative stress and NRTI-induced mitochondrial damage and might, therefore, be beneficial in managing toxicities and complications arising from NRTI use.

Prevalence of HIV-1 Subtypes and Antiretroviral Drug Resistance Mutations in Nepal by Nirajan Bhusal, Ruengpung Sutthent, Navin Horthongkham, Niracha Athipanyasilp, Wannee Kantakamalakul (517-524).
Background: There have been very few reports of HIV-1 subtypes and drug resistance mutations (DRMs) from Nepal which is geographically located between two high-prevalence HIV-1 infection countries, China and India.
Objective: The aim of this study was to determine prevalence of acquired and transmitted DRMs and HIV-1 subtypes in Nepal.
Methods: Thirty-five HIV-1 seropositive samples from central region of Nepal were collected in 2011. The subjects were divided into two groups, antiretroviral (ARV) drug naïve group (n=15) and antiretroviral treatment (ART) group (n=20), 90% (18/20) of them received zidovudine, lamivudine and nevirapine (AZT/3TC/NVP) regimen. HIV pol (protease and reverse transcriptase regions) nucleotide sequences were analyzed by Viroseq HIV-1 Genotyping System. Nearly full-length genomic (NFLG) sequences of 10 samples were performed.
Results: NFLG genotyping revealed that 80% of samples were infected with subtype C and 20% with recombinants (C/D/H and C/A). Phylogenetic analysis of 35 pol sequences from Nepal were subtype C. The prevalence of acquired DRMs to NNRTIs and NRTIs was 15% (3/20). DRMs to NVP, K103N and V179D, and to NRTIs were observed at 11.1% (2/18) and 5% (1/20), respectively. The prevalence of DRMs to rilpivirine for E138A/G was 5.7%. The minor protease inhibitors (PI) associated mutations (A71T/V and T74S) were observed in 5/35 (14.3%) subjects.
Conclusion: This is the first report of NFLG HIV-1 genomic sequences and DRMs from Nepal. National surveillance of HIV DRMs to ARVs and molecular epidemiology study should be done annually for better prevention and treatment of HIV infection in Nepal.