Population and single genome kinetics driving the evolution of multiple linked multiclass drug resistance mutations in the viral protease and reverse transcriptase of HIV-1 subtype C in children receiving early protease inhibitor based combination therapy

This thesis examines the evolution of HIV-1 subtype C multiple linked multi-class antiretroviral resistance mutations in the viral protease (PR) and reverse transcriptase (RT) genes of vertically infected children. Emergence of PI resistance on the backdrop of pre-existing non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance could compromise long-term treatment options in such children. We characterised multi-class drug resistance using single genome sequencing (SGS) in children with viraemia while receiving PI-based ART. We applied SGS of HIV-1 protease (PR) and reverse transcriptase (RT) to longitudinal samples from a cohort of the Children with HIV Early Antiretroviral Therapy (CHER) trial with viral loads >1000c/ml after 40 weeks of early ART. Bulk sequencing revealed NVP-selected resistance in 50% of these children while SGS revealed NVP-selected resistance in 70%. Two children had baseline NRTI and PI mutations, suggesting previous maternal ART. Linked multi-class drug resistance following PI-based ART was detected by SGS in 2/10 children. In one child, the majority species contained M184V in RT linked to L10F, M46I/L, I54V and V82A in PR and a triple-class drug resistant variant with these mutations linked to the NNRTI mutation V108I. In the second child, the majority species contained M184V and V82A linked within viral genomes. I correlated nucleotide variation of PR-RT with the number of single genomes obtained at each time point and ART status and used maximum likelihood trees, recombination analysis, positive selection analysis and co-evolution analysis to describe the evolution of PR-RT of the viral populations. Six children who received early ART for 40/96 weeks only or received continuous ART for the duration of the CHER trial had clusters of identical sequences from baseline and week 40 of ART. These sequences did not harbour known drug resistance mutations. Therefore one could hypothesize viral replication from a persisting viral reservoir that was established from infection that occurred prior to the initiation of ART. The rooted ML trees of 2 children who developed drug resistance during ART had clusters of identical sequences harbouring common drug resistance mutations from multiple time points which is characteristic of the selection of drug resistant viral populations that cause virological failure during ART. When drug resistant viral populations developed during treatment failure, M184V single mutated viruses were selected from multiple wildtype viral populations but only one population became the major contributor to drug resistant viraemia in both children. Triple-class drug resistant sequences that had common DRMs (M184V, V108I in RT and M46I in PR) did not cluster together. I found no evidence of recombination or coevolving sites in PR-RT for any of these children. I used a luciferase based single replication cycle assay to examine drug susceptibility and replication capacity (RC) conferred by multi-class drug resistant PR-RT from the 2 children who developed such drug resistant variants. I tested the susceptibility of pseudoviruses to the components of early ART (AZT, 3TC and LPV), the components of second-line therapy for these children (Abacavir (ABC), Didanosine (ddI), Efavirenz (EFV) and (NVP), the PIs Nelfinavir (NFV) and Saquinavir (SQV), which are also approved for use in children and Darunavir (DRV), which has been identified as a PI option needed in paediatric co-formulation. Pseudoviruses with known PI resistance conferring mutations showed reduced susceptibility to all PIs except DRV. Those with known NNRTI resistance conferring mutations showed reduced susceptibility to EFV and NVP. M184V mutated pseudoviruses conferred high-level resistance to 3TC. In one child, a combination or one of the RT mutations V35T, E36D, T39R, S48T, T165I, K173A, D177E, T200A, Q207D, R211K, V245Q, E248N, D250N, A272P, K277R, E291D, I293V, T296N may be associated with high-level ABC and ddI resistance when genetically linked with M184V. Population sequence analysis was used to characterize the viral gag genes that encoded matrix, capsid, nucleocapsid, p6, and spacer peptides 1 and 2 along with PR-RT as a single amplicon. I determined the presence of compensatory PI-resistance mutations in gag, drug resistance mutations in PR and RT and other amino acid changes that occurred during ART. To determining the polymorphic nature of these sites, I compared them to a position-specific scoring matrix for gag that was derived from HIV-1 subtype C sequences from children from Sub-Saharan Africa. P453L in the p1/p6 cleavage site of Gag emerged in the viral population of one child during PI-based ART. It was the only amino acid change in Gag that emerged among all children in the study cohort that has been characterised as a compensatory mutation that is selected by and enhances PI-resistance. This project is the first to identify multi-class drug resistance mutations in PR and RT that were linked on the same genome as well as characterise their development during early PI-based ART in children. Triple class drug resistant viruses detected in the minority species of the viral population of one child demonstrated significant levels of resistance to LPV, SQV, NFV, 3TC and NVP, and established that such variants could compromise future ART regimes if they became the dominant species of the viral population. I note that the small convenience sample (n = 10) chosen for this project limited the power of this study so that findings could not be generalized

Evaluation of blood-brain barrier transport and CNS drug metabolism in diseased and control brain after intravenous L-DOPA in a unilateral rat model of Parkinson's disease

Abstract Background Changes in blood-brain barrier (BBB) functionality have been implicated in Parkinson's disease. This study aimed to investigate BBB transport of L-DOPA transport in conjunction with its intra-brain conversion, in both control and diseased cerebral hemispheres in the unilateral rat rotenone model of Parkinson's disease. Methods In Lewis rats, at 14 days after unilateral infusion of rotenone into the medial forebrain bundle, L-DOPA was administered intravenously (10, 25 or 50 mg/kg). Serial blood samples and brain striatal microdialysates were analysed for L-DOPA, and the dopamine metabolites DOPAC and HVA. Ex-vivo brain tissue was analyzed for changes in tyrosine hydroxylase staining as a biomarker for Parkinson's disease severity. Data were analysed by population pharmacokinetic analysis (NONMEM) to compare BBB transport of L-DOPA in conjunction with the conversion of L-DOPA into DOPAC and HVA, in control and diseased cerebral hemisphere. Results Plasma pharmacokinetics of L-DOPA could be described by a 3-compartmental model. In rotenone responders (71%), no difference in L-DOPA BBB transport was found between diseased and control cerebral hemisphere. However, in the diseased compared with the control side, basal microdialysate levels of DOPAC and HVA were substantially lower, whereas following L-DOPA administration their elimination rates were higher. Conclusions Parkinson's disease-like pathology, indicated by a huge reduction of tyrosine hydroxylase as well as by substantially reduced levels and higher elimination rates of DOPAC and HVA, does not result in changes in BBB transport of L-DOPA. Taking the results of this study and that of previous ones, it can be concluded that changes in BBB functionality are not a specific characteristic of Parkinson's disease, and cannot account for the decreased benefit of L-DOPA at later stages of Parkinson's disease.</p

Activation Mobilizes the Cholesterol in the Late Endosomes-Lysosomes of Niemann Pick Type C Cells

A variety of intercalating amphipaths increase the chemical activity of plasma membrane cholesterol. To test whether intracellular cholesterol can be similarly activated, we examined NPC1 and NPC2 fibroblasts, since they accumulate large amounts of cholesterol in their late endosomes and lysosomes (LE/L). We gauged the mobility of intracellular sterol from its appearance at the surface of the intact cells, as determined by its susceptibility to cholesterol oxidase and its isotope exchange with extracellular 2-(hydroxypropyl)-β-cyclodextrin-cholesterol. The entire cytoplasmic cholesterol pool in these cells was mobile, exchanging with the plasma membrane with an apparent half-time of ∼3–4 hours, ∼4–5 times slower than that for wild type human fibroblasts (half-time ∼0.75 hours). The mobility of the intracellular cholesterol was increased by the membrane-intercalating amphipaths chlorpromazine and 1-octanol. Chlorpromazine also promoted the net transfer of LE/L cholesterol to serum and cyclodextrin. Surprisingly, the mobility of LE/L cholesterol was greatly stimulated by treating intact NPC cells with glutaraldehyde or formaldehyde. Similar effects were seen with wild type fibroblasts in which the LE/L cholesterol pool had been expanded using U18666A. We also showed that the cholesterol in the intracellular membranes of fixed wild-type fibroblasts was mobile; it was rapidly oxidized by cholesterol oxidase and was rapidly replenished by exogenous sterol. We conclude that a) the cholesterol in NPC cells can exit the LE/L (and the extensive membranous inclusions therein) over a few hours; b) this mobility is stimulated by the activation of the cholesterol with intercalating amphipaths; c) intracellular cholesterol is even more mobile in fixed cells; and d) amphipaths that activate cholesterol might be useful in treating NPC disease

Discovery of Sexual Dimorphisms in Metabolic and Genetic Biomarkers

Metabolomic profiling and the integration of whole-genome genetic association data has proven to be a powerful tool to comprehensively explore gene regulatory networks and to investigate the effects of genetic variation at the molecular level. Serum metabolite concentrations allow a direct readout of biological processes, and association of specific metabolomic signatures with complex diseases such as Alzheimer's disease and cardiovascular and metabolic disorders has been shown. There are well-known correlations between sex and the incidence, prevalence, age of onset, symptoms, and severity of a disease, as well as the reaction to drugs. However, most of the studies published so far did not consider the role of sexual dimorphism and did not analyse their data stratified by gender. This study investigated sex-specific differences of serum metabolite concentrations and their underlying genetic determination. For discovery and replication we used more than 3,300 independent individuals from KORA F3 and F4 with metabolite measurements of 131 metabolites, including amino acids, phosphatidylcholines, sphingomyelins, acylcarnitines, and C6-sugars. A linear regression approach revealed significant concentration differences between males and females for 102 out of 131 metabolites (p-values<3.8 x 10(-4); Bonferroni-corrected threshold). Sex-specific genome-wide association studies (GWAS) showed genome-wide significant differences in beta-estimates for SNPs in the CPS1 locus (carbamoyl-phosphate synthase 1, significance level: p<3.8 x 10(-10); Bonferroni-corrected threshold) for glycine. We showed that the metabolite profiles of males and females are significantly different and, furthermore, that specific genetic variants in metabolism-related genes depict sexual dimorphism. Our study provides new important insights into sex-specific differences of cell regulatory processes and underscores that studies should consider sex-specific effects in design and interpretation

Pediculosis and the Pediatrician

Head lice commonly evoke feelings of disgust, revulsion, anger, and shame among parents and patients. There should, however, be no great cause for such alarm if a physician suspects pediculosis capitis. The recent introduction of several new pediculicidal drugs now allows a choice among four distinct therapeutic agents, which should substantially improve control of isolated cases and epidemics. Physicians must be aware that consumer groups are pressing public health authorities and drug manufacturers to establish proper treatment standards and safety warnings for the use of these agents. In addition, some controversy surrounds the use of lindane in children. This paper reviews the epidemiology and clinical appearance of pediculosis capitis in children, with emphasis on these recent developments. Pubic lice ( Phthirus pubis ) and body lice ( Pedicutus humanus corporis ), both of which are much less common pediatric infestations, are mentioned only briefly.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72607/1/j.1525-1470.1984.tb00447.x.pd

A Simple Iterative Algorithm for Parsimonious Binary Kernel Fisher Discrimination

By applying recent results in optimization theory variously known as optimization transfer or majorize/minimize algorithms, an algorithm for binary, kernel, Fisher discriminant analysis is introduced that makes use of a non-smooth penalty on the coefficients to provide a parsimonious solution. The problem is converted into a smooth optimization that can be solved iteratively with no greater overhead than iteratively re-weighted least-squares. The result is simple, easily programmed and is shown to perform, in terms of both accuracy and parsimony, as well as or better than a number of leading machine learning algorithms on two well-studied and substantial benchmarks

Task-Dependent Interaction between Parietal and Contralateral Primary Motor Cortex during Explicit versus Implicit Motor Imagery

Both mental rotation (MR) and motor imagery (MI) involve an internalization of movement within motor and parietal cortex. Transcranial magnetic stimulation (TMS) techniques allow for a task-dependent investigation of the interhemispheric interaction between these areas. We used image-guided dual-coil TMS to investigate interactions between right inferior parietal lobe (rIPL) and left primary motor cortex (M1) in 11 healthy participants. They performed MI (right index-thumb pinching in time with a 1 Hz metronome) or hand MR tasks, while motor evoked potentials (MEPs) were recorded from right first dorsal interosseous. At rest, rIPL conditioning 6 ms prior to M1 stimulation facilitated MEPs in all participants, whereas this facilitation was abolished during MR. While rIPL conditioning 12 ms prior to M1 stimulation had no effect on MEPs at rest, it suppressed corticomotor excitability during MI. These results support the idea that rIPL forms part of a distinct inhibitory network that may prevent unwanted movement during imagery tasks

Host genetic factors associated with hepatocellular carcinoma in patients with hepatitis C virus infection: a systematic review

Hepatitis C virus (HCV)-infected patients are at risk of developing hepatocellular carcinoma (HCC). Individuals at heightened riskcould be targeted by intensive follow-up surveillance. We have conducted a systematic review of the literature to identify host genetic predisposition to HCC in HCV-infected patients. A comprehensive search of Medline and Embase databases was performed and the strength of evidence of associations for each gene on development of HCC was evaluated. We identified 166 relevant studies, relating to 137 different genes, or combinations thereof. 17 genes were classified as having “good” evidence of an association, a significant association was observed for 37 genes but this finding had not yet been replicated, 56 genes had mixed or limited evidence of an association, and 27 genes showed no association. IFNL3/4, TNF-α and PNPLA3 genes had the most evidence of an association. There was, however, considerable heterogeneity in study design and data quality. In conclusion, we identified a number of genes with evidence of association with HCC, but also a need for more standardised approaches to address this clinically critical question. It is important to consider the underlying mechanism of these relationships and which are confounded by the presence of other HCC risk factors and response to therapy. We also identified many genes where the evidence of association is contradictory or requires replication, as well as a number where associations have been studied but no evidence found. These findings should help to direct future studies on host genetic predisposition to HCC in patients with HCV infection