Genome-Wide Host Gene Expression Analysis Before and After the Initiation of Highly Active Antiretroviral Therapy And Natural Control of HIV in Therapy Naïve HIV+ Non-Progressors

Since its discovery 25 years ago, the HIV virus has infected more than 34 million people in the world; the infection caused by this virus has led the world to its most terrible pandemic, and the greatest global health crises of our times. The host-virus interaction and natural history of the disease are influenced by the distinctive interface the virus has with each infected individual. The infection with HIV-1 is characterized by the destruction of CD4+ T-cells during the typical course of the infection, but HIV has the arsenal to infect practically all the major blood leukocytes. Taken from these observations, it is apparent that HIV has the innate ability to subvert and manipulate the host gene machinery at the transcriptomic level. The highly active anti-retroviral therapy (HAART) consists of a combination of powerful drugs, which serve as potent defence mechanism against the ways in which the HIV virus attacks the human bod y. Although these drugs are not able to rid the body of HIV virus, they can significantly delay the onset of AIDS and reduce the incidence of opportunistic infections, morbidity and mortality related to HIV infection. After the introduction of HAART treatment, it is observed that most patients with good adherence respond to HAART, which is defined by a decrease of plasma viral load to undetectable levels and an immune reconstitution with a significant increase of CD4+ T cell levels. Around 30% of the patients fail to achieve this response and continue to express high plasma viral load and low CD4+ T cell numbers. In contrast, some rare HIV+ patients maintain below detectable levels of plasma viremia without the treatment. These are termed long-term nonprogressors(LTNPs), less commonly called elite controllers. These rare individuals are infected with HIV, but have the natural ability to control the infection with the strength of their immune system. Many of th ese patients have been HIV positive for 30 years or more and! off the rapy for the entire duration of their infection, showing high CD4+T cells counts and no progression to the disease. In this context, it is important to mention that the genomic basis of this natural effective immunological control of viremia in LTNPs, as opposed to drug-mediated control of HIV, remains unknown. The development of high throughput microarray platforms and bioinformatic platforms to visualize and analyse the complex dataset has enabled considerable progress in the field of viral genomics, and also the visualization of host-virus interactions at the molecular level. In chapter III, we carried out a comparative genome-wide (encompassing all 25,000 human genes) pharmacogenomic study using whole primary peripheral blood mononuclear cells (PBMC) derived from 14 HIV+ patients at two time points: pre-HAART (TP-1 with detectable viremia) and post (TP2: below detectable level (BDL) of plasma HIV <40 copies of HIV RNA/mL plasma), to ascertain how genomically distinct viremic phase is from the phase in which virus is fully controlled with HAART. Another goal was to define the underlying pharmacogenomic basis of HIV control during HAART. In the second study shown in Chapter IV, we compared the two time points against the 9 LTNPs to unravel the genomic basis of natural control of viremia in therapy naïve LTNPs showing below detectable levels of viremia (<20 copies of HIV RNA/mL plasma) and high and stable CD4+T cell counts. Genomic RNA extracted from the PBMCs was used in genome-wide microarray analysis, using HT-12v3 Illumina chips. Quantile normalization was performed to normalize the data and inter-patient variability. Illumina®BeadStudio Data Analysis Software wa s used to obtain differentially expressed (DE) genes. Only the significant genes with p value <0.01 and FDR of <5% (for the comparison between TP1 and TP2) and FDR <1% (for the comparison between LTNP vers us TP1 and LTNP versus TP2) were considered appropriate for ! analysis . Pathway analysis was performed in MetaCoreTM from GeneGo, Inc to derive functional annotations. Functionally significant genes were validated by quantitative real time PCR. Between TP1 and TP2, 234 genes were differentially expressed. Within these genes, 212 were down-regulated and 22 upregulated. Between the comparison between LTNP vs TP1, 965 genes were differently expressed (706 genes were up-regulated and 259 genes were down-regulated), and when LTNP was compared to the TP2 group, we found 1181 DE genes (with 727 genes up-regulated and 454 genes downregulated). In the first part of this study, comparing the TP1 and TP2 only, we found that of the top 10 pathways, 8 belonged to the immune response system. This was the most significant pathway up-regulated in TP1 when compared to TP2. This comprised of genes that were involved in antiviral action of interferon (IFN) and their signalling function, antiviral response, dendritic cell maturation a nd migration, and cell metabolism. Map folder and enrichment analysis corroborated with our findings, thereby confirming an intrinsic role of the immune, inflammatory and interferon response family-related genes during HIV viremia in the absence of treatment. But a closer examination of this contrast also showed a mirror down-regulation of genes involved in innate and adaptive immunity, inflammation, apoptosis and antiviral functions. This directly implies a functional relevance of these pathways, through their modulation in TP1 and TP2 stages. Although these data are intuitive and expected, such analysis has never been performed before. The second part of this study, on comparisons between LTNP and TP1 and LTNP against TP2, we show the first evidence demonstrating that the natural control of HIV in LTNP is guided by the genes enriched in the immune response, cytoskeleton remodelling, apoptosis and T cell signalling pathways. Another striking ob servation was that, even though the LTNP and TP2 groups maintai ned BDL of plasma viremia (<40 copies), the LTNP group was genomically distinct from the TP2, which controlled viremia with HAART. This highlights the qualitative distinction and critical role of enriched pathways in natural control of viremia in the LTNPs. Seventeen genes encompassing all these pathways were validated by q-PCR, which showed consistent trends between microarray and q-RT PCR. One gene in particular, the thrombospondin (THBS1) (R2= 0.942) was identified as a biomarker in our study, discriminating between viremic patients and LTNPs at the genomic (R2= 0.942, p<2.654e.08) and proteomic levels (p<0.003761). The levels of expression of THBS1 showed excellent correlation with plasma viremia (R2=0.81557;p<.0.0003761), the first description of such an important protein. This is the most unique finding of this work, which has significance in HIV disease prognostics and diagnostics, in addition to predicting the strength of the ho st immune system, as evident from its down-regulation and low expression in the natural controllers. Overall, through these analyses, we have shown that, although there are common set of key genes associated with HIV at all stages, each stage also showed unique molecular signatures. This was demonstrated with the identification of molecular signatures for the control of virus with HAART therapy, as well as for the natural control of HIV in LTNPs. Especially for the LTNPs, the downregulation of the apoptosis was the most significant feature, which may have important implications in therapy, especially in the context of using apoptosis as a target for future therapies

The Role of CNTNAP2 in the Development & Evolution of the Human Cerebral Cortex

The differing cognitive abilities of humans and other primates are accompanied by changes to forebrain neuronal circuit composition and function. This is underpinned by species-specific features of their development. Excitatory neurons in the human cerebral cortex have longer neurites and more elaborate neurite branching than chimpanzees or macaques, which is thought to contribute to differences in neural network performance. To study how differences in expression of single genes contributes to inter-species differences in neuronal form and function, my research focused on Contactin-Associated Protein-like 2 (CNTNAP2), a gene important for neuronal differentiation and synapse formation, for which there is accumulating evidence for differential use in the human cerebral cortex compared with other primates. Previous work in mice has found that loss of Cntnap2 function reduces neurite branching and dendritic spine density in vitro and in vivo. The potential evolutionary significance of this gene is highlighted by the presence of eight human accelerated regions (HARs), suggesting that there are human-specific aspects to its temporal and spatial expression during cortical development. The research presented in this thesis used human and non-human primate stem cell- derived forebrain neurons to study several aspects of CNTNAP2’s function in human cerebral cortex development. We present the first study of neurite outgrowth and neuronal activity in forebrain neurons generated from a human CNTNAP2 knockout (KO) pluripotent stem cell line. Differentiated human CNTNAP2 KO neurons have reduced neurite branching relative to wild type cells. Strikingly, the KO neurons were significantly more active - bursting more strongly and more frequently. We also applied a combination of bioinformatic and experimental approaches to show one or more of the CNTNAP2 HARs may be a gene enhancer. Our results indicate that loss of function mutations in CNTNAP2 contribute to human neurodevelopmental diseases through altering neuronal activity. This may be due to changes in neurite branching, and ultimately, to neuronal connectivity. These discoveries suggest a mechanism by which CNTNAP2 mutations causes disease in affected children, and may have contributed to the evolution of human-specialized brain function. This model will now be invaluable in deciphering the downstream molecular events caused by CNTNAP2 mutations, and may provide molecular targets for novel treatments in CNTNAP2 patients

A longitudinal study of the experiences and psychological well-being of Indian surrogates

Study question: What is the psychological well-being of Indian surrogates during and after the surrogacy pregnancy? Summary answer: Surrogates were similar to a matched group of expectant mothers on anxiety and stress. However, they scored higher on depression during and after pregnancy. What is known already: The recent ban on trans-national commercial surrogacy in India has led to urgent policy discussions regarding surrogacy. Whilst previous studies have reported the motivations and experiences of Indian surrogates no studies have systematically examined the psychological well-being of Indian surrogates, especially from a longitudinal perspective. Previous research has shown that Indian surrogates are motivated by financial payment and may face criticism from their family and community due to negative social stigma attached to surrogacy. Indian surrogates often recruited by agencies and mainly live together in a “surrogacy house.” Study design, size, duration: A longitudinal study was conducted comparing surrogates to a matched group of expectant mothers over two time points: (a) during pregnancy (Phase1: 50 surrogates, 70 expectant mothers) and (b) 4–6 months after delivery (Phase 2: 45 surrogates, 49 expectant mothers). The Surrogates were recruited from a fertility clinic in Mumbai and the matched comparison group was recruited from four public hospitals in Mumbai and Delhi. Data collection was completed over 2 years. Participants/materials, setting, methods: Surrogates and expectant mothers were aged between 23 and 36 years. All participants were from a low socio-economic background and had left school before 12–13 years of age. In-depth faceto-face semi-structured interviews and a psychological questionnaire assessing anxiety, stress and depression were administered in Hindi to both groups. Interviews took place in a private setting. Audio recordings of surrogate interviews were later translated and transcribed into English. Main results and the role of chance: Stress and anxiety levels did not significantly differ between the two groups for both phases of the study. For depression, surrogates were found to be significantly more depressed than expectant mothers at phase 1 (p = 0.012) and phase 2 (p = 0.017). Within the surrogacy group, stress and depression did not change during and after pregnancy. However, a non-significant trend was found showing that anxiety decreased after delivery (p = 0.086). No participants reported being coerced into surrogacy, however nearly all kept it a secret from their wider family and community and hence did not face criticism. Surrogates lived at the surrogate house for different durations. During pregnancy, 66% (N = 33/50) reported their experiences of the surrogate house as positive, 24% (N = 12/50) as negative and 10% (N = 5/50) as neutral. After delivery, most surrogates (66%, N = 30/45) reported their experiences of surrogacy to be positive, with the remainder viewing it as neutral (28%) or negative (4%). In addition, most (66%, N = 30/45) reported that they had felt “socially supported and loved” during the surrogacy arrangement by friends in the surrogate hostel, clinic staff or family. Most surrogates did not meet the intending parents (49%, N = 22/45) or the resultant child (75%, N = 34/45). Limitations, reasons for caution: Since the surrogates were recruited from only one clinic, the findings may not be representative of all Indian surrogates. Some were lost to follow-up which may have produced sampling bias. Wider implications of the findings: This is the first study to examine the psychological well-being of surrogates in India. This research is of relevance to current policy discussions in India regarding legislation on surrogacy. Moreover, the findings are of relevance to clinicians, counselors and other professionals involved in surrogacy. Trial registration number: N/A