Analysis of multiply spliced transcripts in lymphoid tissue reservoirs of rhesus macaques infected with RT-SHIV during HAART.

Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4⁺ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads <50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load

Audit of Antenatal Testing of Sexually Transmissible Infections and Blood Borne Viruses at Western Australian Hospitals

In August 2007, the Western Australian Department of Health (DOH) released updated recommendations for testing of sexually transmissible infections (STI) and blood-borne viruses (BBV) in antenates. Prior to this, the Royal Australian & New Zealand College of Obstetricians & Gynaecologists (RANZCOG) antenatal testing recommendations had been accepted practice in most antenatal settings. The RANZCOG recommends that testing for HIV, syphilis, hepatitis B and C be offered at the first antenatal visit. The DOH recommends that in addition, chlamydia testing be offered. We conducted a baseline audit of antenatal STI/BBV testing in women who delivered at selected public hospitals before the DOH recommendations. We examined the medical records of 200 women who had delivered before 1st July 2007 from each of the sevenWAhospitals included in the audit. STI and BBV testing information and demographic data were collected. Of the 1,409 women included, 1,205 (86%) were non-Aboriginal and 200 (14%) were Aboriginal. High proportions of women had been tested for HIV (76%), syphilis (86%), hepatitis C (87%) and hepatitis B (88%). Overall, 72% of women had undergone STI/BBV testing in accordance with RANZCOG recommendations. However, chlamydia testing was evident in only 18% of records. STI/BBV prevalence ranged from 3.9% (CI 1.5– 6.3%) for chlamydia, to 1.7% (CI 1–2.4%) for hepatitis C, 0.7% (CI 0.3–1.2) for hepatitis B and 0.6% (CI 0.2–1) for syphilis. Prior to the DOH recommendations, nearly three-quarters of antenates had undergone STI/BBV testing in accordance with RANZCOG recommendations, but less than one fifth had been tested for chlamydia. The DOH recommendations will be further promoted with the assistance of hospitals and other stakeholders. A future audit will be conducted to determine the proportion of women tested according to the DOH recommendations. The hand book from this conference is available for download Published in 2008 by the Australasian Society for HIV Medicine Inc © Australasian Society for HIV Medicine Inc 2008 ISBN: 978-1-920773-59-

Combination antiretroviral therapy -associated lipodystrophy : insights into pathogenesis and treatment

Introduction: Combination antiretroviral therapy (cART) has decreased morbidity and mortality of individuals infected with human immunodeficiency virus type 1 (HIV-1). Its use, however, is associated with adverse effects which increase the patients risk of conditions such as diabetes and coronary heart disease. Perhaps the most stigmatizing side effect is lipodystrophy, i.e., the loss of subcutaneous adipose tissue (SAT) in the face, limbs and trunk while fat accumulates intra-abdominally and dorsocervically. The pathogenesis of cART-associated lipodystrophy is obscure. Nucleoside reverse transcriptase inhibitors (NRTI) have been implicated to cause lipoatrophy via mitochondrial toxicity. There is no known effective treatment for cART-associated lipodystrophy during unchanged antiretroviral regimen in humans, but in vitro data have shown uridine to abrogate NRTI-induced toxicity in adipocytes. Aims: To investigate whether i) cART or lipodystrophy associated with its use affect arterial stiffness; ii) lipoatrophic SAT is inflamed compared to non-lipoatrophic SAT; iii) abdominal SAT from patients with compared to those without cART-associated lipoatrophy differs with respect to mitochondrial DNA (mtDNA) content, adipose tissue inflammation and gene expression, and if NRTIs stavudine and zidovudine are associated with different degree of changes; iv) lipoatrophic abdominal SAT differs from preserved dorsocervical SAT with respect to mtDNA content, adipose tissue inflammation and gene expression in patients with cART-associated lipodystrophy and v) whether uridine can revert lipoatrophy and the associated metabolic disturbances in patients on stavudine or zidovudine based cART. Subjects and methods: 64 cART-treated patients with (n=45) and without lipodystrophy/-atrophy (n=19) were compared cross-sectionally. A marker of arterial stiffness, heart rate corrected augmentation index (AgIHR), was measured by pulse wave analysis. Body composition was measured by magnetic resonance imaging and dual-energy X-ray absorptiometry, and liver fat content by proton magnetic resonance spectroscopy. Gene expression and mtDNA content in SAT were assessed by real-time polymerase chain reaction and microarray. Adipose tissue composition and inflammation were assessed by histology and immunohistochemistry. Dorsocervical and abdominal SAT were studied. The efficacy and safety of uridine for the treatment of cART-associated lipoatrophy were evaluated in a randomized, double-blind, placebo-controlled 3-month trial in 20 lipoatrophic cART-treated patients. Results: Duration of antiretroviral treatment and cumulative exposure to NRTIs and protease inhibitors, but not the presence of cART-associated lipodystrophy, predicted AgIHR independent of age and blood pressure. Gene expression of inflammatory markers was increased in SAT of lipodystrophic as compared to non-lipodystrophic patients. Expression of genes involved in adipogenesis, triglyceride synthesis and glucose disposal was lower and of those involved in mitochondrial biogenesis, apoptosis and oxidative stress higher in SAT of patients with than without cART-associated lipoatrophy. Most changes were more pronounced in stavudine-treated than in zidovudine-treated individuals. Lipoatrophic SAT had lower mtDNA than SAT of non-lipoatrophic patients. Expression of inflammatory genes was lower in dorsocervical than in abdominal SAT. Neither depot had characteristics of brown adipose tissue. Despite being spared from lipoatrophy, dorsocervical SAT of lipodystrophic patients had lower mtDNA than the phenotypically similar corresponding depot of non-lipodystrophic patients. The greatest difference in gene expression between dorsocervical and abdominal SAT, irrespective of lipodystrophy status, was in expression of homeobox genes that regulate transcription and regionalization of organs during embryonal development. Uridine increased limb fat and its proportion of total fat, but had no effect on liver fat content and markers of insulin resistance. Conclusions: Long-term cART is associated with increased arterial stiffness and, thus, with higher cardiovascular risk. Lipoatrophic abdominal SAT is characterized by inflammation, apoptosis and mtDNA depletion. As mtDNA is depleted even in non-lipoatrophic dorsocervical SAT, lipoatrophy is unlikely to be caused directly by mtDNA depletion. Preserved dorsocervical SAT of patients with cART-associated lipodystrophy is less inflamed than their lipoatrophic abdominal SAT, and does not resemble brown adipose tissue. The greatest difference in gene expression between dorsocervical and abdominal SAT is in expression of transcriptional regulators, homeobox genes, which might explain the differential susceptibility of these adipose tissue depots to cART-induced toxicity. Uridine is able to increase peripheral SAT in lipoatrophic patients during unchanged cART.Johdanto: Ihmisen immuunikatoviruksen (HIV) hoitoon käytetyt lääkeyhdistelmät ovat vähentäneet HIV-positiivisten henkilöiden sairastuvuutta ja kuolleisuutta. Yhdistelmähoitoon liittyy kuitenkin vakavia sivuvaikutuksia, jotka lisäävät potilaiden riskiä sairastua mm. diabetekseen ja sepelvaltimotautiin. Yksi leimaavimpia sivuvaikutuksia on lipodystrofia eli ihonalaisen rasvakudoksen häviäminen (lipoatrofia) kasvoista, raajoista ja vatsalta samalla kun rasvaa kertyy ylen määrin vatsaonteloon ja niskaan. Ilmiön syyt ovat epäselvät. Useiden HIV:ta vastaan suunnattujen lääkeaineiden on epäilty aiheuttavan lipodystrofiaa mm. tuhoamalla mitokondrioita, solujen energiatehtaita . Lipodystrofiaan ei ole tehokasta hoitoa, ellei HIV-lääkitystä muuteta, mutta esim. uridiini on ollut lupaava apu solumallitutkimusten valossa. Tavoitteet: Tutkia liittyykö yhdistelmähoitoon tai sen käyttöön liittyvään lipodystrofiaan verisuonien jäykistymistä, onko lipoatrofinen rasvakudos tulehtunutta verrattuna ei-lipoatrofiseen rasvakudokseen, eroaako lipoatrofinen ei-lipoatrofisesta rasvakudoksesta mm. mitokondriomäärän ja aineenvaihduntaan vaikuttavien geenien ilmentymisen suhteen sekä poikkeaako lipodystrofiassa paremmin säilyvä niskan rasva häviävästä vatsan ihonalaisrasvasta ja onko se mahdollisesti ruskeata rasvaa. Lisäksi tutkimme, voiko ravintolisänä käytetty uridiini parantaa lipoatrofiaa ja siihen liittyviä aineenvaihduntahäiriöitä, kuten rasvamaksaa ja heikentynyttä insuliiniherkkyyttä. Menetelmät: Tutkimuksiin osallistui 64 HIV-positiivista yhdistelmähoidettua potilasta, joista 45:lla oli ja 19:lla ei ollut kehittynyt lääkitykseen liittyviä rasvakudoksen muutoksia. Verisuonijäykkyys tutkittiin pulssiaaltoanalyysilla, kehon koostumus mitattiin kaksienergisella röntgenabsorptiometria- sekä magneettikuvaantamisella ja maksan rasvapitoisuus protonispektroskopialla. Rasvakudosnäytteet otettiin potilaiden vatsan ja niskan ihoalaisrasvasta ja niistä mitattiin eri geenien ilmentymistä sekä mitokondrioiden ja tulehdussolujen määrää mm. DNA:n monistustekniikalla ja kudosleikevärjäyksin. Uridiinin tehoa lipoatrofian hoidossa arvioitiin 3kk satunnaistetussa lumelääkekontrolloidussa tutkimuksessa, johon osallistui 20 HIV-positiivista yhdistelmähoidettua lipoatrofista henkilöä. Tulokset: HIV-lääkityksen kesto, mutta ei lipodystrofia, altistaa verisuonien jäykistymiselle iästä ja verenpainetasosta riippumatta. Lipoatrofisessa rasvakudoksessa tulehdukseen liittyvien geenien ilmentyminen ja tulehdussolujen määrä ovat lisääntyneet, kun taas mitokondriomäärä sekä rasvasolujen muodostumiseen ja toimintaan liittyvien geenien ilmentyminen vähentyneet verrattuna ei-lipoatrofiseen rasvakudokseen. Lipodystrofiassa säilyvä/lisääntyvä niskan rasva on vähemmän tulehtunutta kuin herkemmin häviävä vatsan ihonalaisrasva eikä se ole ruskeata rasvaa. Lipodystrofisten henkilöiden niskan rasvassa on vähemmän mitokondrioita kuin ei-lipodystrofisten henkilöiden niskan rasvassa, vaikka kudokset ovat ulkoisesti samannäköisiä. Niskan ja vatsan alueen ihonalaisrasva eroaa eniten ns. homeobox-geenien ilmentymisessä eli sellaisten geenien, jotka määrittelevät kudosten sijainnin ja ominaisuudet sikiökehityksen varhaisvaiheessa. Uridiini lisää ihonalaisrasvan määrää lipoatrofisilla potilailla, mutta ei vaikuta maksan rasvapitoisuuteen tai insuliiniherkkyyteen. Johtopäätökset: HIV:n hoitoon käytettyjen lääkkeiden pitkäaikaiskäyttö lisää verisuonien jäykkyyttä ja siten potilaiden riskiä sairastua sydän- ja verisuonitauteihin. Lipoatrofinen rasva on tulehtunut ja sen mitokondriovarannot vähentyneet. Koska mitokondrioiden vähyys on todettavissa niskarasvassa myös sellaisilla lipodystrofisilla henkilöillä, joilla se on säilynyt atrofialta, mitokondriokatoa ei voida pitää lipoatrofiaa suoraan aiheuttavana tekijänä. Niskan ja vatsan ihonalaisrasvan merkittävin ero on elinkehitystä ohjaavissa geeneissä, mikä voi selittää kudosten erilaisen alttiuden lääkkeiden sivuvaikutuksille. Uridiini on tehokas hoito HIV-potilaiden lipodystrofiaan muuttumattoman yhdistelmähoidon aikana

Parameters identification of HIV dynamic models for HAART treated patients: A comparative study

We present a comparative study of parameters identification of HIV dynamic models for naive patients that are treated with two different HAART (Highly Active Anti-Retroviral Therapy) protocols during a period of 48 weeks. Three HIV models of increasing complexity (in terms of number of state variables and parameters) have been chosen, and for each one the model parameters are computed by solving a nonlinear optimization problem via sequential quadratic programming (SQP). Model parameters are divided into “group dependent”, common to all patients treated with same HAART protocol, and “patient dependent”, specific for each patient, and are estimated in a way that an overall cost function comprising the fitting error of CD4+ concentration and viral load measurements. A preliminary parameter space grid search algorithm is performed in order to find a suitable initial guess for the SQP algorithm. Numerical results indicate that all considered models can give a good matching despite the scarcity of available measurements for each patient, and in this limited situation the minimal model appears to be (slightly) more effective than the other models

Highlights on HIV eradication in 2013

Almost 20 years after the introduction of HAART, scientific community, doctors and patients are still struggling with the absence of effective strategies aimed at eradicating HIV infection, or at preventing it through a vaccin

HIV-Related Stigmatization in Treatment Settings: Effects on Patient Comfort, Risk Disclosure, and Treatment Decisions

The major focus for the present study was to examine the effects of provider stigmatization on the medical care of HIV+ patients, by using an experimental paradigm and examining a conceptual framework to clarify the relationship between provider stigmatization and negative treatment outcomes. Initial qualitative findings from focus groups (n = 18) indicated that several key elements of stigmatizing treatment experiences included judgmental and condescending language, patient avoidance, increased physical distance between patient and provider during conversations and procedures, and use of extra, unnecessary precautions (e.g. use of extra gloves, masks). These provider behaviors were experimentally manipulated and incorporated into computerized vignettes containing audio and visual stimuli depicting typical medical appointments. In the experimental phase, participants (n = 90) were randomly assigned to view either a highly stigmatizing or a non-stigmatizing treatment vignette and then subsequently rate their willingness to engage in HIV care. Findings indicated that patients assigned to the highly stigmatizing condition were the most unwilling to engage in HIV care as demonstrated in lower intentions to remain in care, disclose sexual and substance use risk behaviors, and discuss medication adherence difficulties. As hypothesized, the effect of the experimental stigma condition on patients\u27 willingness to engage in care was mediated by patients\u27 feelings of comfort and their perceptions of stigma within the patient-provider interaction. Findings from the present study may help to inform the development of interventions to assist healthcare providers in creating more positive treatment experiences for their HIV+ patients to improve implementation of self care and reduction of risk behaviors

Physiological Cybernetics: An Old-Novel Approach for Students in Biomedical Systems

Wiener in a seminal book (Wiener, 1948) associated the ancient Greek word ‘κυβερνητικος’ to the control of physiological systems. “Thus, as far back as four years ago, the group of scientists about Dr. Rosenblueth and myself had already become aware of the essential unity of the set of problems centering about communication, control and statistical mechanics, whether in the machine or in the living tissue. [...] We have decided to call the entire field [...] by the name Cybernetics, which we form from the Greek κυβερνητης or steersman. In choosing this term, we wish to recognize that the first significant paper on feed-back mechanisms is an article on governors, which was published by Clerk Maxwell in 1868 and that governor is derived from a Latin corruption of κυβερνητης. We also wish to refer to the fact that the steering engines of a ship are indeed one of the earliest and best developed forms of feed-back mechanisms.” The increasing knowledge in each sector of science led to a huge diversification of scientific research, especially in a borderline sector like cybernetics applied to physiological systems. A first problem to solve was the following: let’s suppose that two groups, one with a control engineering experience and the other one with a medical background (e.g., physicians), decide to cooperate, because they strongly believe that a joined research could be useful for developing mathematical and statistical models. Usually physicians do not have enough time to study and apply advanced modelling. Wiener approached the communication between scientists belonging to different disciplines: “If a physiologist, who knows no mathematics, works together with a mathematician, who knows no physiology, the one will be unable to state his problem in terms that the other can manipulate, and the second will be unable to put the answers in any form that the first can understand. [...] The mathematician need not have the skill to conduct a physiological experiment, but he must have the skill to understand one, to criticize one, and to suggest one. The physiologist need not be able to prove a certain mathematical theorem, but he must be able to grasp its physiological significance and to tell the mathematician for what he should look.” A correct interaction in terms of a clear communication and reciprocal comprehension of the objectives of the research activity between groups with different competences is a crucial aspect in any interdisciplinary research. In 2003 at the University of Pisa it was decided to introduce a new course for undergraduate students in biomedical engineering, based on the Wiener ‘utopia’, in order to teach a novel discipline useful for helping biomedical students to communicate and cooperate effectively with physicians. We named this new course as Physiological Cybernetics, remembering the old Wiener definition. The organization of this course was a difficult task, and it required to gain experience in order to integrate so different disciplines and to produce a common language between students in biomedical engineer and physicians. At a first glance this attempt seemed to be too ambitious, because the different approaches of biomedical engineers with respect to physicians seemed incompatible and even the languages of the two groups were so different to remember the Babel tower… A great deal of effort and attention was required to produce appealing and stimulating lectures, but after many years we can affirm that this challenge is successful, especially for the enthusiastic answers of the students: their number was increasing year after year (about seventy students per year are now attending the course). A strict and trusted cooperation between different groups of physicians is growing up and several groups of physicians belonging to different medical fields are going to join us for new interactions. The aim of this chapter is to describe how the approach to physiological cybernetics has led to integrate academic lessons with research activities. To be more specific, the basic idea of Physiological Cybernetics was to search for models able to emulate physiological systems based on the feedback theory and/or the system theory. In fact, recently, the widespread use of friendly software packages for modelling, along with the development of powerful identification and control techniques has led to a renewed interest in control (Khoo, 2011; Hoppensteadt & Peskin, 2002; Cobelli & Carson, 2008) and identification (Westwick & Kearney, 2003) of physiological systems. Unfortunately physiological systems are intrinsically time variant and highly non linear. Moreover, an effective balance of the model complexity is a difficult task: low order models are usually too simple to be useful, on the other hand high order models are too complex for simulation purposes and they have too many unknown parameters to be identified. Each model selected for investigation was studied by a group of biomedical students supervised by physicians. Each model required several iterations and reformulations, due to the continuous adjustment of the research objectives, changing their final horizon, because of the gap between experimental data and theoretical models, so that the answers to the doubts and questions were continuously moving with the obtained partial results. A final goal of the research was to apply a mathematical framework for helping medical diagnostic techniques and for testing new therapeutic protocols. The procedure of model extraction followed two main pathways: the first one (pathway A) led to a formulation of a mathematical model usually based on differential equations and on an as deep as possible insight into physiological mechanisms (Marmarelis, 2004; Ottesen et al., 2004; Edelstein-Keshet, 2005; Jones et al., 2009) via a physical description of the system. The second one (pathway B) was founded on a model description based on a black-box and data-driven identification (Westwick & Kearney, 2003; Cobelli & Carson, 2008), usually leaving to stochastic models with a parametric or non-parametric structure (Ljung, 1987), depending on the a-priori knowledge of constitutive laws governing the observed system. In this paper we will describe two examples of research activity based on the Physiological Cybernetics, both of them addressed to produce a biomedical framework for predicting the effects of therapeutic actions, but following the two different pathways. The first example follows a statistical non parametric approach, the second one a mathematical model based on differential equations

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

Systematic Identification of Synergistic Drug Pairs Targeting HIV

The systematic identification of effective drug combinations has been hindered by the unavailability of methods that can explore the large combinatorial search space of drug interactions. Here we present a multiplex screening method named MuSIC (Multiplex Screening for Interacting Compounds), which expedites the comprehensive assessment of pair-wise compound interactions. We examined ~500,000 drug pairs from 1000 FDA-approved or clinically tested drugs and identified drugs that synergize to inhibit HIV replication. Our analysis reveals an enrichment of anti-inflammatory drugs in drug combinations that synergize against HIV, indicating HIV benefits from inflammation that accompanies its infection. Multiple drug pairs identified in this study, including glucocorticoid and nitazoxanide, synergize by targeting different steps of the HIV life cycle. As inflammation accompanies HIV infection, our findings indicate that inhibiting inflammation could curb HIV propagation. MuSIC can be applied to a wide variety of disease-relevant screens to facilitate efficient identification of compound combinations

Prediction of the Containment of HIV Infection by Antiretroviral Therapy - a Variable Structure Control Approach

It is demonstrated that the reachability paradigm from variable structure control theory is a suitable framework to monitor and predict the progression of the human immunodeficiency virus (HIV) infection following initiation of antiretroviral therapy (ART). A manifold is selected which characterises the infection-free steady-state. A model of HIV infection together with an associated reachability analysis is used to formulate a dynamical condition for the containment of HIV infection on the manifold. This condition is tested using data from two different HIV clinical trials which contain measurements of the CD4+ T cell count and HIV load in the peripheral blood collected from HIV infected individuals for the six month period following initiation of ART. The biological rates of the model are estimated using the multi-point identification method and data points collected in the initial period of the trial. Using the parameter estimates and the numerical solutions of the model, the predictions of the reachability analysis are shown to be consistent with the clinical diagnosis at the conclusion of the trial. The methodology captures the dynamical characteristics of eventual successful, failed and marginal outcomes. The findings evidence that the reachability analysis is an appropriate tool to monitor and develop personalised antiretroviral treatment