Current HIV Research (v.11, #4)

HIV DNA and Immune Alteration During Successful HAART by Diallo Mamadou, Zheng Yu-Huang, Chen Xia, He Bo, Zhou Hua-Ying, He Yan, Chen Zi, Li Qing, Zheng Li-Wen, He Mei, Yao Yun-Hai, Sarr Demba, Delphine Aldebert, Mboup Souleymane (255-262).
Background: Highly Active Antiretroviral therapy (HAART) can effectively reduce the viral load toundetectable levels in most HIV-infected patients. However, some patients may still experience impaired immunologicresponse associated with increased risk of disease progression and death.Objective: The objective of this study was to assess the impact of the HIV DNA load on the immune alteration duringsuccessful HAART.Methods: 40 chronic HIV-infected adults initiating HAART were followed for 24 months. The CD4 count, HIV viralload, HIV DNA load, and levels of γ-cytokines IL-2, IL-7 and IL-21 were monitored at baseline (month 0), month 6, 12,18 and 24 following HAART initiation.Results: The plasma viral load decreased significantly and remained below the detection limits after six months treatment.Likely the HIV DNA load decreased significantly in both cells during 12 months, was undetectable in CD14 monocytesafter 18 months, but remained higher in CD3+ T cells during all the follow up. In addition, the HIV DNA load correlatedpositively between T cells and monocytes in 10 patients who maintained higher HIV DNA load in both cells during 12months. The CD4 count, IL-2, and IL-21 levels increased significantly during 12 months, whereas IL-7 decreasedsignificantly during 18 months, regardless of the HIV DNA load in T cells. Patients with CD4 count below 200/μlmaintained higher HIV DNA load and showed lower increase in CD4 count compared to patients with CD4 count above200/μl. In patients showing undetectable HIV DNA load in both cells, neither IL-2 nor IL-21 correlated with the CD4count even after 24 months despite their partial restoration.Conclusion: These results suggest that the HIV DNA load could continuously hamper the CD4 restoration and γ-cytokinesfunctional activities during HAART. This action seemed to be more severe in patients with pre-HAART CD4 count below200/μl. The CD14 monocytes may contribute to this action as source of T cell infection both before and during HAART.

Mitochondrial Function and Apoptosis of Peripheral Mononuclear Cells (PBMCs) in the HIV Infected Patients by Monika Bociaga-Jasik, Joanna Góralska, Anna Polus, Agnieszka Sliwa, Anna Gruca, Urszula Razny, Anna Zdzienicka, Aleksander Garlicki, Tomasz Mach, Aldona Dembinska-Kiec (263-270).
HIV infection results in the development of immunodeficiency mainly due to the apoptosis of infected andbystander CD4 cells. The aim of the study was to follow the mitochondrial dependent pathway of apoptosis, one of thesuggested mechanisms of above process.The inner mitochondrial membrane potential (MMP), Adenosine-5'-triphosphate (ATP) generation, apoptosis and necrosismarkers of peripheral mononuclear cells (PBMCs) were compared in HIV infected patients and HIV negative controlgroup. The correlation of blood viral load, TNFα concentration, CD4 cells count and duration of ARV therapy wasconsidered. Additionally, group of HIV infected ARV-naive patients was involved for the follow-up study and the effectsof one year of ARV therapy on measured parameters were studied.PBMCs of HIV infected individuals (especially without ARV therapy) demonstrated lower MMP and ATP generation andhigher percentage of apoptotic/necrotic PBMCs. Correlation between blood TNFα level and mitochondrial dysfunctionwas observed. The first months of ARV therapy resulted in most significant restoration of mitochondrial function andliving PBMCs count.HIV infection and ARV therapy have significant impact on mitochondrial function and apoptosis of PBMCs. They aredriven by abnormal mitochondrial function apoptosis of immune cells which seems to be the key element leading toimmunosuppression, thus an early intervention in this process by therapy can be beneficial for symptomatology of HIVinfected patients.

Two HIV-1 non B isolates, 98US_MSC5007 and 98US_MSC5016, which have been identified amongst the USArmy personnel serving abroad, are known to have originated from other nations. Notwithstanding, they are categorizedas American strains. This is because their countries of origin are unknown. American isolates are basically B subtype.98US_MSC5007 belongs to Circulating Recombinant Form (CRF02_AG) while 98US_MSC5016 is of the C clade. Bothsub-groups are recognized to have originated from African and Asian continents. It has become necessary to properlydetermine the countries of origin of microbes and viruses. This is because diversity and cross-subtyping have been foundto mitigate the designing and development of vaccine and therapeutic interventions. The aim of this study therefore is toidentify the countries of origin of the two American isolates found amongst US Army personnel serving abroad.A Digital Signal Processing-based Bioinformatics technique called Informational Spectrum Method (ISM) has been engaged.ISM entails translating the amino acids sequences of the protein into numerical sequences (signals) by means of one biologicalparameter (Amino Acids Scale). The signals are then processed using Discrete Fourier Transform (DFT) in order to uncoverand present the embedded biological information as Informational Spectra (IS). Spectral Position of Maximum BindingInteraction (SPMBI) is used. Several approaches including Phylogeny have preliminarily been employed in the determination ofevolutionary trends of organisms and viruses. SPMBI has preliminarily been used to re-establish the semblance and commonoriginality that exist between human and Chimpanzee, evolutionary roadmaps in the Influenza and HIV viruses.The results disclosed that 98US_MSC5007 shared same semblance and originality with a Nigeria isolate (92NG083)while 98US_MSC5016 with the Zairian isolates (ELI, MAL, and Z2/CDC-34). These results appear to demonstrate thatthe American soldiers harboring these strains may have been infected by isolates from Nigeria and Zaire, respectively.This is because 98US_MSC5007 and the Nigerian isolate share SPMBI at position 44. Additionally, 98US_MSC5016,which has SPMBI at position 148, may have come from Zaire as it has similar SPMBI with the Zairian isolates at 150.SPMBI is a demonstration of Bio-functionality arising from maximum affinity by the proteins from different sources to acommon protein. To help validate the findings, the experiment was further repeated using ISM-based Phylogenetictechnique. The outcome appears not to be in complete accord with the results obtained in this study.It is therefore recommended that the countries in which these US Army personnel are deployed be identified and wherethe findings made and the locations of the Army personnel appropriately correlate, this novel procedure be engaged in theidentification of the nations of origins of all other such HIV isolates across all clades and nations.

14-3-3 Protein in CSF Reflects SIV-Mediated Pre-Synaptic Damage by Kristi L. Helke, Suzanne E. Queen, Joseph L. Mankowski (281-287).
HIV-associated neurocognitive disorders (HAND) remain prevalent despite effective combined anti-retroviraltherapy (cART). Cognitive function has been shown to inversely correlate with decreased synaptic and dendritic density.In this study, macaques inoculated with SIV were examined over a 3-month course of infection to characterize theappearance of the neuronal damage marker 14-3-3 protein in CSF and to determine whether CSF 14-3-3 levels directlyreflected synaptic alterations. SIV-infected macaques with 14-3-3 in CSF had significantly lower levels of the presynapticprotein synaptophysin in cortical grey matter. Synaptophysin levels were inversely correlated with amount ofSIV RNA in the CNS. In contrast, levels of 14-3-3 in CSF did not correspond with either alterations in levels of the postsynapticprotein PSD-95 or viral replication in the brain. These findings suggest that the appearance of 14-3-3 in CSFduring asymptomatic infection reflects pre-synaptic damage in SIV-infected macaques and thus may serve as a marker ofthe early synaptic alterations that underlie HIV-induced neurocognitive impairment.

Transmission of Drug-Resistant HIV-1 Variants Among Individuals with Recent Infection in Southern Poland by Joanna Smoleń- Dzirba, Magdalena Rosińska, Piotr Kruszyński, Jolanta Bratosiewicz- Wąsik, Janusz Janiec, Marek Beniowski, Monika Bociąga- Jasik, Elżbieta Jabłonowska, Bartosz Szetela, Tomasz J. Wąsik (288-294).
Transmitted drug resistance (TDR) is an important public health issue, because it may affect the outcome ofantiretroviral treatment. The prevalence of human immunodeficiency virus type 1 (HIV-1) with TDR mutations definedaccording to the list of the World Health Organization was investigated among 53 therapy-naive persons with confirmedrecent HIV-1 infection diagnosed in Poland, in the years 2008-2010. Proviral DNA was amplified, sequenced, andscreened for the TDR mutations in the pol gene fragments coding for the whole protease and the initial 256 residues of thereverse transcriptase. The frequency of sequences with at least one TDR mutation was 11.3%. In four (7.5%) sequences atleast one resistance mutation related to reverse transcriptase inhibitors was identified, and in further two (3.8%) sequencesone mutation related to protease inhibitors' resistance was present. The moderate rate of TDR highlights the need for acontinuous surveillance and resistance testing among treatment-naive individuals to optimize treatment effects within acountry.

Distinct Geographical Clustering of HIV-1 and a Signature Amino Acid at Position 41 of the p24 Unveiled by gag Variability in India by Arpan Acharya, Salil Vaniawala, Harsh Parekh, Anju Nagee, Rabindra N. Misra, Minal Wani, Pratap N. Mukhopadhyaya (295-303).
A portion of the gag gene cDNA for p24 protein from 30 Indian HIV-1 proviral DNA was amplified by PCRand sequenced. Phylogenetic analysis with reference samples of A1, A2, B, C, D, F1, F2, G, H, J, K, N and O subtypesrevealed that 29 test samples aligned with subtype C reference strain while 1 matched with HIV-1 subtype A. Multiplealignment of predicted amino acid sequence of the Indian test samples and reference C subtype of HIV-1 samples fromother countries indicated a molecular signature by way of rigid conservation of the amino acid 'S' at position 41 of thegag p24 protein in all Indian HIV-1 samples analyzed in this study as opposed to 'T'in the same position in C subtypesequences from other parts of the world. A phylogenetic analysis and visualization of the resulting tree in radial positionshowed distinct clubbing of all Indian C subtypes and formation of a cluster when compared to C subtype sequences fromother countries with a single Chinese sample as an exception which was found in the Indian cluster. The use of a portionof p24 gene sequence as tool for subtyping as well as phylogenetic grouping with special reference to its geographicallocation is discussed.

Virulence Factors of Candida albicans Isolates from the Oral Cavities of HIV-1-Positive Patients by Tatiany O.A. Menezes, Luciana C.S. Gillet, Sílvio A.F. Menezes, Rosimar N.M. Feitosa, Marluísa O.G. Ishak, Ricardo Ishak, Sílvia H. Marques-da-Silva, Antonio C.R. Vallinoto (304-308).
The present study assessed the phenotypic aspects of oral-cavity Candida albicans isolates from 300 HIV-1-positive patients, relating the most commonly investigated virulence factors (enzyme typing and germ-tube formation) tothe most common morphotypes. The samples were seeded into specific media for isolation and subsequent identificationusing the automated Vitek 2 system. The following assays were performed for phenotypic characterization:morphotyping, germ-tube formation and enzyme typing. Out of 300 collected samples, 144 tested positive for yeasts ofthe Candida genus, 98 (32.7 %) of which were identified as C. albicans. The latter samples were attributed to sevendifferent morphotypes; the three most common morphotypes were 7208 (49 %), 7308 (14.3 %) and 3208 (13.3 %). All ofthe C. albicans isolate samples formed germ tubes and produced the enzymes proteinase and phospholipase, with anactivity classified as intermediate to high. Due to the identification of virulence factors among the analyzed samples,monitoring of HIV-1-positive patients colonized by different morphotypes must be established because these morphotypesare extremely pathogenic and can trigger severe fungal infections.

Lamivudine-PEGylated Chitosan: A Novel Effective Nanosized Antiretroviral Agent by Mohammad R. Aghasadeghi, Hossein Heidari, Seyed M. Sadat, Shiva Irani, Safieh Amini, Seyed D. Siadat, Mohammad E. Fazlhashemy, Rezvan Zabihollahi, Seyed M. Atyabi, Seyed B. Momen, Mohammad S. Khosraavy, Mehdi Davari, Tahmineh Darvishmohammadi, Mohammad Doroudian, Mehdi S. Ardestani (309-320).
Due to the fact that a definite treatment for AIDS disease has not been discovered so far, antiretroviral drugsare relatively significant in controlling the disease. In this study, Lamivudine- which is an old and effective anti-AIDSdrug- was connected to PEGylated chitosan nanoparticle in order to produce a new and greater version of Lamivudine.First, physicochemical studies such as HNMR, FTIR spectroscopy and CHN analysis were performed to ensure the propersynthesis. Following that, Lamivudine-PEGylated Chitosan (LPC) drug was evaluated in terms of its inhibitory capabilityin producing HIV virions and its cytotoxicity in different doses. HIV virions were created by transfection of psPAX2,PmzNL4-3 and pMD2.G plasmids into HEK293T cell line. Also, assessment of the P24 amounts of cell supernatant wasperformed using ELISA method. Among the different doses of LPC drug (0.1, 1, 10 and 100μM), it was found that 0.1μMwas the most effective and least toxic dose compared to Lamivudine in the same dose. Results of this study indicate thatLPC drug has the ability to inhibit HIV virus replication in a more significant way in comparison to the old drug. Besides,the drug has a low cytotoxicity and is effective with a lower dose.

Toxic Shock Syndrome and Persistent Immune Activation in an HIVPositive Patient by Michal Holub, Simona Arientová, David Jilich, Alzbeta Davidová, Ondrej Beran, Hanus Rozsypal (321-325).
The case of an HIV-positive treatment-naive male with toxic shock syndrome (TSS) is presented herein. Thecourse of TSS was favorable; however, the patient had extremely high plasma levels of MCP-1 and CD38 and HLA-DRexpression on CD8+ T cells during the acute illness. Furthermore, the numbers of CD8+ T cells were reduced and CD4+T cells remained stable during acute illness in comparison to baseline values. MCP-1 and HLA-DR gradually decreased,but they were still elevated after a month, whereas the number of circulating CD8+ T cells increased more than fivefold.CD38 expression remained stable during this period. A further decrease in CD38, HLA-DR and MCP-1 was noted fivemonths after the initiation of antiretroviral therapy.

Enteric Pathogens, Immune Status and Therapeutic Response in Diarrhea in HIV/AIDS Adult Subjects from North India by Arun Kumar Jha, Beena Uppal, Sanjim Chadha, Prabhav Aggarwal, Roumi Ghosh, Richa Dewan (326-332).
Intestinal infection causing diarrheal disease is a dominant contributor to high morbidity and mortality indeveloping countries. This intervention study aimed to assess the response of specific anti-microbial and anti-retroviraltherapy (ART) on enteropathogens identified in HIV/AIDS adult subjects from northern India. Seventy five ART naive(group 1) and seventy five ART adherent (group 2) HIV/AIDS adult subjects with diarrhea were enrolled. Stool samplesfrom all subjects were examined for enteropathogens by wet mount, staining methods, culture and ELISA. Subjects withenteropathogens were started on specific therapy as per National AIDS Control Organisation, Government of India'sguidelines. Follow-up stool samples were examined after 2-4 weeks of completion of therapy for persistence/clearing ofenteropathogens. CD4+ T lymphocyte count was done for all subjects. At enrollment, group 1 had 26.13% bacterial,57.66% parasitic & 16.22% fungal pathogens while group 2 had 11.9%, 69.05% & 19.05% pathogens, respectively.Parasitic diarrhea was more common than bacterial diarrhea. The coccidian parasites (Cryptosporidium spp. & Isosporabelli) were the common parasites identified. Clearance of enteric pathogens was significant after specific anti-microbialtherapy (p = 0.0001). Persistence of enteropathogens was seen primarily for coccidian parasites. Clearance ofenteropathogens after specific therapy and the diagnostic yield of stool specimens were influenced by the CD4+ counts.Immune competence coupled with specific anti-microbial therapy displays the best response against enteric pathogens.

Long-Term Gender-Based Outcomes for Atazanavir/Ritonavir (ATV/r)- Containing Regimens in Treatment-Experienced Patients with HIV by Veronica Svedhem-Johansson, Pascal Pugliese, Norbert H. Brockmeyer, Anders Thalme, Claudia Michalik, Stefan Esser, Marie-Helene Barlet, Tina Nakonz, Maria J. Jimenez-Exposito (333-341).
Clinical data on antiretroviral effectiveness in women are limited, especially long-term data, because womenare usually underrepresented in clinical trials. This sub-analysis of a large European non-comparative, retrospective,observational cohort study evaluated gender differences in long-term outcomes in antiretroviral-experienced adult patientswith HIV-1 infection switched to an ATV/r-based regimen between October 2004 and March 2007. Data were extractedfrom 3 European HIV databases every 6 months (maximum follow-up 5 years). Time to virological failure (VF), definedas two consecutive HIV-1 RNA≥50 c/mL or one HIV-1 RNA≥50 c/mL followed by treatment discontinuation (TD), andtime to TD were analyzed using the Kaplan-Meier method. Associations of gender with VF and TD were analyzed usingmultivariate Cox proportional models. Safety and tolerability were evaluated. In total, 1294 patients (336 women, 958men) were analyzed. No gender differences in time to VF were observed; at 3 years, the probability of not having VF was0.59 (95%CI: 0.52, 0.65) and 0.63 (95%CI: 0.59, 0.67) for women and men, respectively. In multivariate analyses,women had a higher risk of TD than men (hazard ratio [HR], 1.54; 95%CI: 1.28, 1.85) but no increased risk of VF (HR,1.06; 95%CI: 0.85, 1.33). Safety and tolerability were comparable between genders. In a clinical setting, long-termefficacy and safety outcomes of ATV/r-based regimens were similar by gender. Women had a higher risk of TD but noincreased risk of VF. ATV/r is an effective and well-tolerated therapeutic option for treatment-experienced men andwomen with HIV-1 infection.