Signature genes associated with immunological non-responsiveness to anti-retroviral therapy in HIV-1 subtype-c infection.

OBJECTIVE: HIV-infected individuals undergoing therapy may show an immunological-discordant response to therapy, with poor CD4+ T cells recovery, despite viral suppression below the detection limit. The present study was carried out to delineate the underlying molecular mechanisms of immunological non-responsiveness to HIV therapy. DESIGN: We conducted microarray-based whole gene expression profiles of 30 subjects infected with HIV-1 subtype C, in peripheral blood to discern the signature genes associated with immunological non-responsiveness. After a thorough analysis and comparison of gene-expression profiles, microarray data was validated via qRT-PCR approach. RESULTS: Overall, we found 10 genes significantly up-regulated and 60 genes down-regulated (≥2-fold change) in immunological non-responders as compared to responders. Based on these results and pathway analysis of the protein-protein interaction, 20 genes were shortlisted for validation in human infected cases. We found statistically significant differences in expression levels of twelve genes IL-1α, IL-1ß, IL-7R, TNF-α, FoxP3, PDCD5, COX7B, SOCS1, SOCS3, RPL9, RPL23, and LRRN3 respectively among immunological non-responders compared to therapy responders, confirming their an intimate relationship with immunological responsiveness to therapy. CONCLUSIONS: Altogether, microarray and qRT-PCR validation results indicated that the aberrant expression of key genes involved in the regulation of T cell homeostasis, immune activation, inflammatory cytokine production, apoptosis, and immune-regulatory processes are possibly associated with immunological non-responsiveness in HIV-1 C infected individuals on ART.

Mycobacterium tuberculosis modulates the gene interactions to activate the HIV replication and faster disease progression in a co-infected host.

Understanding of the chronic immune activation, breakdown of immune defense and synergistic effect between HIV and Mycobacterium tuberculosis (Mtb) may provide essential information regarding key factors involved in the pathogenesis of HIV disease. In this study, we aimed to highlight a few of the immunological events that may influence and accelerate the progression of HIV disease in the presence of co-infecting Mtb. A cross-sectional study was performed on cohorts, including anti-tubercular therapy (ATT) naïve active pulmonary tuberculosis (PTB) patients, antiretroviral therapy (ART) naïve HIV-1 infected individuals at different stages of disease, ATT and ART naïve HIV-PTB co-infected individuals and healthy controls. A significantly higher T-regulatory cell (Treg) frequency coupled with the high FoxP3 expression in the CD4 T-cells indicated an immunosuppressive environment in the advance stage of HIV-1 infection. This is further substantiated by high HO-1 expression favoring TB co-infection. Functionally, this change in Treg frequency in HIV-1 infected individuals correlated well with suppression of T-cell proliferation. Mtb infection seems to facilitate the expansion of the Treg pool along with increased expression of FoxP3, specifically the variant-1, as evident from the data in HIV-1 co-infected as well as in patients with only PTB. A significantly lower expression of HO-1 in co-infected individuals compared to patients with only HIV-infection having comparable CD4 count correlated well with increased expression of CCR5 and CxCR4 as well as NF-κB and inflammatory cytokines IL-6 and TNF-α, which collectively may contribute to enhanced viral replication and increased cell death, hence faster disease progression in co-infected individuals.

Down-regulation of NF-κB signalling by polyphenolic compounds prevents endotoxin-induced liver injury in a rat model.

Activation of NF-κB has been reported to play a key role in causing endotoxin-induced hepatic damage through enhanced production of reactive oxygen species and pro-inflammatory mediators. In this context, the potential of polyphenolic phytochemicals in preventing endotoxin-induced liver damage remains unclear. Here, we demonstrate that catechin and quercetin have the potential to down-regulate the initial signalling molecule NF-κB which may further inhibit the downstream cascade including TNF-α and NO. These results were confirmed using N-nitro-L-arginine methyl ester (L-NAME), the inhibitor of inducible nitric oxide synthase (iNOS) along with the biochemical and histological alterations occurring in the presence and absence of supplementation with both the polyphenols. However, catechin was found to be more effective than quercetin against endotoxin-induced liver injury. These findings suggest that these polyphenols may form a pharmacological basis for designing a therapeutic agent against endotoxin-mediated oxidative damage.

Prediction of drug-resistance in HIV-1 subtype C based on protease sequences from ART naive and first-line treatment failures in North India using genotypic and docking analysis.

Genotyping reveal emergence of drug resistance (DR)-related mutations in HIV-1 protease (PR) gene in the first-line treatment failure patients as per Stanford DR database. In order to have a subtype C specific prediction model, a three dimensional structure of local wild type C variant is created and the identified mutations were introduced to assess the mutational effects on protease inhibitors (PI) in a homology model. We estimated viral load, CD4 count and conducted DR genotyping in HIV isolates from 129 therapy naive and 20 first-line treatment failure individuals. Several genotypic variations, as compared to subtype B sequence in the Stanford gene database were detected in HIV-1 subtype C isolates from treatment naive individuals. Among these, nine mutations (12S, 15V, 19I, 36I, 41K, 63P, 69K, 89M, 93L) occurred in more than 60% of the isolates and were considered as local wild type for molecular modelling studies. No major mutations were seen in the PR sequences in isolates from treatment-naive individuals, although isolates from two patients had T74S mutation, known to be associated with reduced susceptibility to nelfinavir (NFV) and a combination of M36I, H69K and L89M mutations found in isolates from 77 patients (59.7%), considered to be conferring resistance to tipranavir (TPV) according to ANRS algorithm. Among the first-line treatment failures, an isolate from one patient showed L33F, I47T, M46G, and G48E mutations conferring intermediate resistance to saquinavir (SQV) and lopinavir (LPV). Though the docking energy scores are in agreement with this interpretation for SQV, it, however, indicated these mutations to be causing intermediate to high level resistance to atazanavir (ATV) and tipranavir (TPV) but making it susceptible to LPV. The patient finally responded to a second-line regimen containing 3TC, AZT and LPV with significant viral suppression. All the DR genotyping studies analyse the results using available databases which are all based on subtype B specific sequences. The proposed homology model in this study is unique, as it may predict subtype C specific susceptibility criteria for the available PIs.

Experimentally induced various inflammatory models and seizure: understanding the role of cytokine in rat.

BACKGROUND: The mechanism of epileptogenesis is not well established. There is higher incidence of seizures among patients with chronic inflammatory disease. Cytokines are rapidly induced in the brain after a variety of stimuli including inflammation. Aim of this study was to produce various inflammatory models and seizure to understand the role of TNFalpha in above mentioned models. MATERIALS AND METHODS: A total of 54 male rats were included in the study. Animals were divided into 3 groups of colitis, arthritis, and cotton wool granuloma. Each group had 3 subgroups of control, model and treatment. At the end of 3 days in colitis, 17 days in arthritis and 7 days in cotton wool granuloma groups a subconvulsive dose of PTZ (40 mg/kg i.p) was injected to note seizure onset and seizure score. Brain samples were subjected to DNA fragmentation testing. Presence of inflammation was confirmed by morphology and histology. Plasma and brain TNFalpha levels were measured. RESULTS: The models of colitis, arthritis and CWG were effectively produced as evidenced by morphology and histology scores (p<0.001). Seizure onset was reduced and grade was increased (p<0.001). Thalidomide reduced the morphological, histological (p<0.002), DNA fragmentation and seizure grade (p<0.001) while increased seizure onset (p<0.001) in the arthritis group. TNFalpha levels in both plasma and brain were reduced following thalidomide treatment (p<0.002) in arthritis group. There were no significant findings in colitis or cotton wool granuloma groups. CONCLUSION: Inflammation was associated with decreased threshold to PTZ induced seizure. Thalidomide is effective in reducing the extent of arthritis as well as reducing the seizure scoring and increasing seizure onset in the adjuvant arthritis group. Thalidomide was also effective in reducing TNFalpha levels thus contributing to its antiepileptic activity.

Drug resistance-associated genotypic alterations in the pol gene of HIV type 1 isolates in ART-naive individuals in North India.

ABSTRACT We genotyped the RT and PI regions of the pol gene of HIV-1 from treatment-naive infected individuals in North India and evaluated their possible physiological relevance and association with drug resistance. Plasma samples from 52 newly diagnosed HIV-1-infected drug-naive individuals were subjected to CD4(+) cell count and plasma viral load. For genotyping, the protease and RT regions of the pol gene were amplified from cDNA reverse transcribed from plasma viral RNA by single or nested polymerase chain reaction (PCR). Sequences of amplified products were analyzed for mutations using the Stanford DR and REGA database. Two out of 49 amplicons showed mutations at known "major" subtype B drug resistance positions (one each in protease and RT). In the protease region it showed a major drug resistance mutation at M46I as well as "minor" positions F53L and T74P. In the RT gene, one patient showed a mutation at major NNRTI position G190V. Forty-nine percent had mutations in the hinge (M36I, R41K, H69K) and alpha-helix (L89M) regions of the C-virus protease, which has been linked to increased catalytic activity. Our study indicates that a number of major mutations associated with resistance to PIs, NNRTIs, and NRTIs do exist, though at a low frequency, among HIV-1 isolates from treatment-naive individuals in North India. Many minor or accessory mutations related to resistance to PIs and NRTIs are also present as the variants. These results point to the greater biochemical fitness of subtype C protease and faster decrease in drug sensitivity.