Akul Singhania

An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

Systemic delivery of E6/7 siRNA using novel lipidic particles and its application with cisplatin in cervical cancer mouse models

Small interfering RNA (siRNA) shows great promise in cancer therapy, but its effectiveness in vivo still remains a crucial issue for its transition into the clinics. Although the successful use of polyethylene glycol (PEG)ylated lipidic delivery systems have already been reported, most of the formulation procedures used are labour intensive and also result in unstable end products. We have previously developed a simple yet efficient hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles, in which the products exhibited superior stability. Here, we show that these HFDM-formulated particles are stable in the presence of serum and can deliver siRNA efficiently to tumours after intravenous administration. Using these particles, around 50% knockdown of the target gene expression was observed in tumours. With the use of siRNA targeting the E6/7 oncogenes expressed in cervical cancer, we showed a 50% reduction in tumour size. This level of tumour growth suppression was comparable to that achieved from cisplatin at the clinically used dose. Overall, our results demonstrate the feasibility of using HFDM-formulated particles to systematically administer E6/7-targeted siRNA for cervical cancer treatment. The simplicity of preparation procedure along with superior product stability obtained from our method offers an innovative approach for the in vivo delivery of siRNA.

Whole Serum 3D LC-nESI-FTMS Quantitative Proteomics Reveals Sexual Dimorphism in the Milieu Intérieur of Overweight and Obese Adults

Linking gender-specific differences to the molecular etiology of obesity has been largely based on genomic and transcriptomic evidence lacking endophenotypic insight and is not applicable to the extracellular fluid compartments, or the milieu intérieur, of the human body. To address this need, this study profiled the whole serum proteomes of age-matched nondiabetic overweight and obese females (n = 28) and males (n = 31) using a multiplex design with pooled biological and technical replicates. To bypass basic limitations of immunodepletion-based strategies, subproteome enrichment by size-exclusion chromatography (SuPrE-SEC) followed by iTRAQ 2D-LC-nESI-FTMS analysis was used. The study resulted in the reproducible analysis of 2472 proteins (peptide FDR < 5%, q < 0.05). A total of 248 proteins exhibited significant modulation between men and women (p < 0.05) that mapped to pathways associated with β-estradiol, lipid and prostanoid metabolism, vitamin D function, immunity/inflammation, and the complement and coagulation cascades. This novel endophenotypic signature of gender-specific differences in whole serum confirmed and expanded the results of previous physiologic and pharmacologic studies exploring sexual dimorphism at the genomic and transcriptomic level in tissues and cells. Conclusively, the multifactorial and pleiotropic nature of human obesity exhibits sexual dimorphism in the circulating proteome of importance to clinical study design.

The utility of gene expression in blood cells for diagnosing neuropsychiatric disorders.

Objective diagnostic tools are required for neuropsychiatric disorders. Gene expression in blood cells may provide such a tool and has already been used to construct classifiers capable of diagnosing many human diseases. This chapter discusses the use of microarray gene expression data to construct diagnostic classifiers for neuropsychiatric disorders. The potential pitfalls of microarray gene expression analysis and the experimental design and methods suitable for classifier construction are described in detail. A review of studies that have analyzed gene expression in blood cells from patients with neuropsychiatric disorders is presented with an emphasis on the feasibility of generating a diagnostic classifier for schizophrenia. Finally, the future directions of the field are discussed with respect to using blood gene expression to tailor antipsychotic medications to individual patients, applying microRNA expression for diagnostic purposes, as well as the implications of next-generation sequencing technologies for gene expression analysis.

Suberoylanilide hydroxamic acid induces limited changes in the transcriptome of primary CD4(+) T cells.

Objective:To assess the off-target effects of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) in human primary CD4+ T cells. Design:A pharmacologically relevant concentration (340 nmol/l) of SAHA was shown to significantly increase histone hyperacetylation by 24 h and this length of treatment was selected to determine its impact on gene expression in primary CD4+ T cells. Methods:Illumina Beadchips for microarray gene expression analysis were used to analyze differential gene expression between cells treated or not with SAHA with a paired analysis using multivariate permutation tests. Gene ontology, biological pathway and protein interaction network analyses were used to identify the higher order biological processes affected by SAHA treatment. Results:Modest modulation by SAHA was observed for 1847 genes with 80% confidence level of no more than 10% false positives. A thousand genes were upregulated by SAHA and 847 downregulated. Pathways and gene ontologies overrepresented in the list of differentially expressed genes included Glycolysis/Gluconeogenesis, tRNA Modification, and the Histone Acetyltransferase Complex. Protein interaction network analysis revealed that transcription factor c-Myc, which was downregulated by SAHA treatment at the mRNA level, interacts with a number of SAHA-responsive genes. Conclusions:The effects on transcription by SAHA were sufficiently modest to support trials to activate HIV replication as part of an eradication strategy. SAHA did not appear to modulate proliferative or apoptotic processes to a great extent, which might impact the ability of patients to eradicate the virus reservoir following activation by HDACi treatment.

Differential gene expression in HIV-infected individuals following ART

Previous studies of the effect of ART on gene expression in HIV-infected individuals have identified small numbers of modulated genes. Since these studies were underpowered or cross-sectional in design, a paired analysis of peripheral blood mononuclear cells (PBMCs), isolated before and after ART, from a robust number of HIV-infected patients (N=32) was performed. Gene expression was assayed by microarray and 4157 differentially expressed genes (DEGs) were identified following ART using multivariate permutation tests. Pathways and gene ontology (GO) terms over-represented for DEGs reflected the transition from a period of active virus replication before ART to one of viral suppression (e.g., repression of JAK-STAT signaling) and possible prolonged drug exposure (e.g., oxidative phosphorylation pathway) following ART. CMYC was the DEG whose product made the greatest number of interactions at the protein level in protein interaction networks (PINs), which has implications for the increased incidence of Hodgkins lymphoma (HL) in HIV-infected patients. The differential expression of multiple genes was confirmed by RT-qPCR including well-known drug metabolism genes (e.g., ALOX12 and CYP2S1). Targets not confirmed by RT-qPCR (i.e., GSTM2 and RPL5) were significantly confirmed by droplet digital (ddPCR), which may represent a superior method when confirming DEGs with low fold changes. In conclusion, a paired design revealed that the number of genes modulated following ART was an order of magnitude higher than previously recognized.

Factors regulated by interferon gamma and hypoxia-inducible factor 1A contribute to responses that protect mice from Coccidioides immitis infection

BackgroundCoccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited. Disseminated infections occur in approximately 5% of cases and may prove fatal. Mouse models of the disease have identified strains that are resistant (e.g. DBA/2) or susceptible (e.g. C57BL/6) to these pathogens. However, the genetic and immunological basis for this difference has not been fully characterized.ResultsMicroarray technology was used to identify genes that were differentially expressed in lung tissue between resistant DBA/2 and sensitive C57BL/6 mice after infection with C. immitis. Differentially expressed genes were mapped onto biological pathways, gene ontologies, and protein interaction networks, which revealed that innate immune responses mediated by Type II interferon (i.e., IFNG) and the signal transducer and activator of transcription 1 (STAT1) contribute to the resistant phenotype. In addition, upregulation of hypoxia inducible factor 1A (HIF1A), possibly as part of a larger inflammatory response mediated by tumor necrosis factor alpha (TNFA), may also contribute to resistance. Microarray gene expression was confirmed by real-time quantitative PCR for a subset of 12 genes, which revealed that IFNG HIF1A and TNFA, among others, were significantly differentially expressed between the two strains at day 14 post-infection.ConclusionThese results confirm the finding that DBA/2 mice express more Type II interferon and interferon stimulated genes than genetically susceptible strains and suggest that differential expression of HIF1A may also play a role in protection.

Gender-dependent differences in plasma matrix metalloproteinase-8 elevated in pulmonary tuberculosis

Tuberculosis (TB) remains a global health pandemic and greater understanding of underlying pathogenesis is required to develop novel therapeutic and diagnostic approaches. Matrix metalloproteinases (MMPs) are emerging as key effectors of tissue destruction in TB but have not been comprehensively studied in plasma, nor have gender differences been investigated. We measured the plasma concentrations of MMPs in a carefully characterised, prospectively recruited clinical cohort of 380 individuals. The collagenases, MMP-1 and MMP-8, were elevated in plasma of patients with pulmonary TB relative to healthy controls, and MMP-7 (matrilysin) and MMP-9 (gelatinase B) were also increased. MMP-8 was TB-specific (p<0.001), not being elevated in symptomatic controls (symptoms suspicious of TB but active disease excluded). Plasma MMP-8 concentrations inversely correlated with body mass index. Plasma MMP-8 concentration was 1.51-fold higher in males than females with TB (p<0.05) and this difference was not due to greater disease severity in men. Gender-specific analysis of MMPs demonstrated consistent increase in MMP-1 and -8 in TB, but MMP-8 was a better discriminator for TB in men. Plasma collagenases are elevated in pulmonary TB and differ between men and women. Gender must be considered in investigation of TB immunopathology and development of novel diagnostic markers.

Are you also what your mother eats? Distinct proteomic portrait as a result of maternal high-fat diet in the cerebral cortex of the adult mouse

Epidemiological studies suggest an association between maternal obesity and adverse neurodevelopmental outcomes in offspring. Our aim was to compare the global proteomic portrait in the cerebral cortex between mice born to mothers on a high-fat or control diet who themselves were fed a high-fat or control diet. Male mice born to dams fed a control (C) or high-fat (H) diet 4 weeks before conception and during gestation, and lactation were assigned to either C or H diet at weaning. Mice were killed at 19 weeks and their cerebral cortices were analysed using a two-dimensional liquid chromatography-mass spectrometry methodology. In total, 6 695 proteins were identified (q<0.01), 10% of which were modulated in at least one of the groups relative to controls. In silico analysis revealed that mice clustered based on the diet of the mother and not their own diet and that maternal high-fat diet was significantly associated with response to hypoxia/oxidative stress and apoptosis in the cerebral cortex of the adult offspring. Maternal high-fat diet resulted in distinct endophenotypic changes of the adult offspring cerebral cortex independent of its current diet. The identified proteins could represent novel therapeutic targets for the prevention of neuropathological features resulting from maternal obesity.

Dose-responsive gene expression in suberoylanilide hydroxamic acid-treated resting CD4+ T cells.

Design:Persistent latently infected CD4+ T cells represent a major obstacle to HIV eradication. Histone deacetylase inhibitors (HDACis) are a proposed activation therapy. However, off-target effects on gene expression in host immune cells are poorly understood. We hypothesized that HDACi-modulated genes would be best identified with a dose–response analysis. Methods:Resting primary CD4+ T cells were treated with 0.34, 1, 3, or 10 &mgr;mol/l of the HDACi, suberoylanilide hydroxamic acid (SAHA), for 24 h and subjected to microarray gene expression analysis. Genes with dose-correlated expression were filtered to identify a subset with consistent up or downregulation at each SAHA dose. Histone modifications were characterized in six SAHA dose-responsive genes by chromatin immunoprecipitation (ChIP-RT-qPCR). Results:A large number of genes were shown to be upregulated (N = 657) or downregulated (N = 725) by SAHA in a dose-responsive manner (FDR-corrected P-value ⩽ 0.5, fold change ≥|2|). Several genes (e.g. CINNAL1, DPEP2, H1F0, IRGM, PHF15, and SELL) are potential in-vivo biomarkers of SAHA activity. SAHA dose-responsive genes included transcription factors, HIV restriction factors, histone methyltransferases, and host proteins that interact with HIV. Pathway analysis suggested net downregulation of T-cell activation with increasing SAHA dose. Histone acetylation was not correlated with host gene expression, but plausible alternative mechanisms for SAHA-modulated gene expression were identified. Conclusion:Numerous genes in CD4+ T cells are modulated by SAHA in a dose-responsive manner, including genes that may negatively influence HIV activation from latency. Our study suggests that SAHA influences gene expression through a confluence of several mechanisms, including histone modification, and altered expression and activity of transcription factors.