Correlation between compartmental tenofovir concentrations and an ex vivo rectal biopsy model of tissue infectibility in the RMP-02/MTN-006 phase 1 study

Design: Phase 1, randomized, two-site (US), double-blind, placebo-controlled study of sexually-abstinent males and females

Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials

The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method

Association between free testosterone levels and anal human papillomavirus Types 16/18 infections in a cohort of men who have sex with men

Background Human papillomavirus (HPV) types 16 and 18 cause invasive cervical cancer and most invasive anal cancers (IACs). Overall, IAC rates are highest among men who have sex with men (MSM), especially MSM with HIV infection. Testosterone is prescribed for men showing hypogonadism and HIV-related wasting. While there are direct and indirect physiological effects of testosterone in males, its role in anal HPV16/18 infections in men is unknown. Methods Free testosterone (FT) was measured in serum from 340 Multicenter AIDS Cohort Study (MACS) participants who were tested for anal HPV16/18-DNA approximately 36 months later. The effect of log10-transformed current FT level on anal HPV16/18 prevalence was modeled using Poisson regression with robust error variance. Multivariate models controlled for other HPV types, cumulative years of exogenous testosterone use, race, age, lifetime number of receptive anal intercourse partnerships, body mass index, tobacco smoking, HIV-infection and CD4+ T-cell counts among HIV-infected, and blood draw timing. Results Participants were, on average, 60 (+5.4) years of age, White (86%), and HIV-uninfected (56%); Twenty-four percent tested positive for anal HPV16 and/or 18-DNA (HPV16 prevalence= 17.1%, HPV18=9.1%). In adjusted analysis, each half-log10 increase of FT was associated with a 1.9-fold (95% Confidence Interval: 1.11, 3.24) higher HPV16/18 prevalence. Additionally, other Group 1 high-risk HPVs were associated with a 1.56-fold (1.03, 2.37) higher HPV16/18 prevalence. Traditional risk factors for HPV16/18 infection (age, tobacco smoking; lifetime number of sexual partners, including the number of receptive anal intercourse partnerships within 24 months preceding HPV testing) were poorly correlated with one another and not statistically significantly associated with higher prevalence of HPV16/ 18 infection in unadjusted and adjusted analyses. Conclusions Higher free testosterone was associated with increased HPV16/18 prevalence measured approximately three years later, independent of sexual behavior and other potential confounders. The mechanisms underlying this association remain unclear and warrant further study

Evaluation and Management of Anal Intraepithelial Neoplasia in HIV-Negative and HIV-Positive Men Who Have Sex with Men

The incidence of human papillomavirus (HPV)–associated anal cancer in men who have sex with men (MSM) is striking and has not been mitigated by the use of highly active antiretroviral therapy. Detection and treatment of high-grade anal intraepithelial neoplasia (HGAIN) may reduce the incidence of anal cancer. Anal cytology is a useful tool to detect HGAIN; annual screening of HIV-positive MSM and biennial screening of HIV-negative MSM appears to be cost-effective. MSM with abnormal cytology should be referred for high-resolution anoscopy and biopsy. Individuals with HGAIN should receive treatment; treatment modalities for HGAIN demonstrate moderate efficacy and are usually well tolerated, but greater study is required to determine which treatment is optimal. Large prospective studies are needed to document the efficacy of screening and treatment of HGAIN on anal cancer incidence. The HPV vaccine holds promise for primary prevention of anal cancer in MSM, but significant implementation challenges remain

A phase 1 randomized, open label, rectal safety, acceptability, pharmacokinetic, and pharmacodynamic study of three formulations of tenofovir 1% Gel (the CHARM-01 study)

Objectives: The CHARM-01 study characterized the safety, acceptability, pharmacokinetics (PK), and pharmacodynamics (PD) of three tenofovir (TFV) gels for rectal application. The vaginal formulation (VF) gel was previously used in the CAPRISA 004 and VOICE vaginal microbicide Phase 2B trials and the RMP-02/MTN-006 Phase 1 rectal safety study. The reduced glycerin VF (RGVF) gel was used in the MTN-007 Phase 1 rectal microbicide trial and is currently being evaluated in the MTN-017 Phase 2 rectal microbicide trial. A third rectal specific formulation (RF) gel was also evaluated in the CHARM-01 study. Methods: Participants received 4 mL of the three TFV gels in a blinded, crossover design: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. Safety, acceptability, compartmental PK, and explant PD were monitored throughout the trial. Results: All three gels were found to be safe and acceptable. RF and RGVF PK were not significantly different. Median mucosal mononuclear cell (MMC) TFV-DP trended toward higher values for RF compared to RGVF (1136 and 320 fmol/106 cells respectively). Use of each gel in vivo was associated with significant inhibition of ex vivo colorectal tissue HIV infection. There was also a significant negative correlation between the tissue levels of TFV, tissue TFV-DP, MMC TFV-DP, rectal fluid TFV, and explant HIV-1 infection. Conclusions: All three formulations were found to be safe and acceptable. However, the safety profile of the VF gel was only based on exposure to one dose whereas participants received seven doses of the RGVF and RF gels. There was a trend towards higher tissue MMC levels of TFV-DP associated with use of the RF gel. Use of all gels was associated with significant inhibition of ex vivo tissue HIV infection. Trial Registration: ClinicalTrials.gov NCT01575405

Multiscale Modeling of Red Blood Cell Mechanics and Blood Flow in Malaria

Red blood cells (RBCs) infected by a Plasmodium parasite in malaria may lose their membrane deformability with a relative membrane stiffening more than ten-fold in comparison with healthy RBCs leading to potential capillary occlusions. Moreover, infected RBCs are able to adhere to other healthy and parasitized cells and to the vascular endothelium resulting in a substantial disruption of normal blood circulation. In the present work, we simulate infected RBCs in malaria using a multiscale RBC model based on the dissipative particle dynamics method, coupling scales at the sub-cellular level with scales at the vessel size. Our objective is to conduct a full validation of the RBC model with a diverse set of experimental data, including temperature dependence, and to identify the limitations of this purely mechanistic model. The simulated elastic deformations of parasitized RBCs match those obtained in optical-tweezers experiments for different stages of intra-erythrocytic parasite development. The rheological properties of RBCs in malaria are compared with those obtained by optical magnetic twisting cytometry and by monitoring membrane fluctuations at room, physiological, and febrile temperatures. We also study the dynamics of infected RBCs in Poiseuille flow in comparison with healthy cells and present validated bulk viscosity predictions of malaria-infected blood for a wide range of parasitemia levels (percentage of infected RBCs with respect to the total number of cells in a unit volume).United States. National Institutes of Health (Grant R01HL094270)National Science Foundation (U.S.). (Grant CBET-0852948)Singapore-MIT Alliance for Research and Technology Cente

AST: An Automated Sequence-Sampling Method for Improving the Taxonomic Diversity of Gene Phylogenetic Trees

A challenge in phylogenetic inference of gene trees is how to properly sample a large pool of homologous sequences to derive a good representative subset of sequences. Such a need arises in various applications, e.g. when (1) accuracy-oriented phylogenetic reconstruction methods may not be able to deal with a large pool of sequences due to their high demand in computing resources; (2) applications analyzing a collection of gene trees may prefer to use trees with fewer operational taxonomic units (OTUs), for instance for the detection of horizontal gene transfer events by identifying phylogenetic conflicts; and (3) the pool of available sequences is biased towards extensively studied species. In the past, the creation of subsamples often relied on manual selection. Here we present an Automated sequence-Sampling method for improving the Taxonomic diversity of gene phylogenetic trees, AST, to obtain representative sequences that maximize the taxonomic diversity of the sampled sequences. To demonstrate the effectiveness of AST, we have tested it to solve four problems, namely, inference of the evolutionary histories of the small ribosomal subunit protein S5 of E. coli, 16 S ribosomal RNAs and glycosyl-transferase gene family 8, and a study of ancient horizontal gene transfers from bacteria to plants. Our results show that the resolution of our computational results is almost as good as that of manual inference by domain experts, hence making the tool generally useful to phylogenetic studies by non-phylogeny specialists. The program is available at http://csbl.bmb.uga.edu/~zhouchan/AST.php

Molecular Systematics of the Deep-Sea Hydrothermal Vent Endemic Brachyuran Family Bythograeidae: A Comparison of Three Bayesian Species Tree Methods

Brachyuran crabs of the family Bythograeidae are endemic to deep-sea hydrothermal vents and represent one of the most successful groups of macroinvertebrates that have colonized this extreme environment. Occurring worldwide, the family includes six genera (Allograea, Austinograea, Bythograea, Cyanagraea, Gandalfus, and Segonzacia) and fourteen formally described species. To investigate their evolutionary relationships, we conducted Maximum Likelihood and Bayesian molecular phylogenetic analyses, based on DNA sequences from fragments of three mitochondrial genes (16S rDNA, Cytochrome oxidase I, and Cytochrome b) and three nuclear genes (28S rDNA, the sodium–potassium ATPase a-subunit ‘NaK’, and Histone H3A). We employed traditional concatenated (i.e., supermatrix) phylogenetic methods, as well as three recently developed Bayesian multilocus methods aimed at inferring species trees from potentially discordant gene trees. We found strong support for two main clades within Bythograeidae: one comprising the members of the genus Bythograea; and the other comprising the remaining genera. Relationships within each of these two clades were partially resolved. We compare our results with an earlier hypothesis on the phylogenetic relationships among bythograeid genera based on morphology. We also discuss the biogeography of the family in the light of our results. Our species tree analyses reveal differences in how each of the three methods weighs conflicting phylogenetic signal from different gene partitions and how limits on the number of outgroup taxa may affect the results