HIV Treatment as Prevention: Considerations in the Design, Conduct, and Analysis of Cluster Randomized Controlled Trials of Combination HIV Prevention

The rigorous evaluation of the impact of combination HIV prevention packages at the population level will be critical for the future of HIV prevention. In this review, we discuss important considerations for the design and interpretation of cluster randomized controlled trials (C-RCTs) of combination prevention interventions. We focus on three large C-RCTs that will start soon and are designed to test the hypothesis that combination prevention packages, including expanded access to antiretroviral therapy, can substantially reduce HIV incidence. Using a general framework to integrate mathematical modelling analysis into the design, conduct, and analysis of C-RCTs will complement traditional statistical analyses and strengthen the evaluation of the interventions. Importantly, even with combination interventions, it may be challenging to substantially reduce HIV incidence over the 2- to 3-y duration of a C-RCT, unless interventions are scaled up rapidly and key populations are reached. Thus, we propose the innovative use of mathematical modelling to conduct interim analyses, when interim HIV incidence data are not available, to allow the ongoing trials to be modified or adapted to reduce the likelihood of inconclusive outcomes. The preplanned, interactive use of mathematical models during C-RCTs will also provide a valuable opportunity to validate and refine model projections.     

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.     

Epstein-Barr Virus LMP2A Reduces Hyperactivation Induced by LMP1 to Restore Normal B Cell Phenotype in Transgenic Mice

Epstein-Barr virus (EBV) latently infects most of the human population and is strongly associated with lymphoproliferative disorders. EBV encodes several latency proteins affecting B cell proliferation and survival, including latent membrane protein 2A (LMP2A) and the EBV oncoprotein LMP1. LMP1 and LMP2A signaling mimics CD40 and BCR signaling, respectively, and has been proposed to alter B cell functions including the ability of latently-infected B cells to access and transit the germinal center. In addition, several studies suggested a role for LMP2A modulation of LMP1 signaling in cell lines by alteration of TRAFs, signaling molecules used by LMP1. In this study, we investigated whether LMP1 and LMP2A co-expression in a transgenic mouse model alters B cell maturation and the response to antigen, and whether LMP2A modulates LMP1 function. Naïve LMP1/2A mice had similar lymphocyte populations and antibody production by flow cytometry and ELISA compared to controls. In the response to antigen, LMP2A expression in LMP1/2A animals rescued the impairment in germinal center generation promoted by LMP1. LMP1/2A animals produced high-affinity, class-switched antibody and plasma cells at levels similar to controls. In vitro, LMP1 upregulated activation markers and promoted B cell hyperproliferation, and co-expression of LMP2A restored a wild-type phenotype. By RT-PCR and immunoblot, LMP1 B cells demonstrated TRAF2 levels four-fold higher than non-transgenic controls, and co-expression of LMP2A restored TRAF2 levels to wild-type levels. No difference in TRAF3 levels was detected. While modulation of other TRAF family members remains to be assessed, normalization of the LMP1-induced B cell phenotype through LMP2A modulation of TRAF2 may be a pathway by which LMP2A controls B cell function. These findings identify an advance in the understanding of how Epstein-Barr virus can access the germinal center in vivo, a site critical for both the genesis of immunological memory and of virus-associated tumors.     

Discovery of novel 5-(ethyl or hydroxymethyl) analogs of 2'-'up' fluoro (or hydroxyl) pyrimidine nucleosides as a new class of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium inhibitors

Discovery of novel antimycobacterial compounds that work on distinctive targets and by diverse mechanisms of action is urgently required for the treatment of mycobacterial infections due to the emerging global health threat of tuberculosis. We have identified a new class of 5-ethyl or hydroxy (or methoxy) methyl-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium bovis, Mycobacterium tuberculosis (H37Ra, H37Rv) and Mycobacterium avium. A series of 2'-'up' fluoro (or hydroxy) nucleosides (1, 2, 4-6, 9, 10, 13, 16, 18, 21, 24) was synthesized and evaluated for antimycobacterial activity. Among 2'-fluorinated compounds, 1-(3-bromo-2,3-dideoxy-2-fluoro-β-d-arabinofuranosyl)-5-ethyluracil (13) exhibited promising activity against M. bovis and Mtb alone, and showed synergism when combined with isoniazid. The most active compound emerging from these studies, 1-(β-d-arabinofuranosyl)-4-thio-5-hydroxymethyluracil (21) inhibited Mtb (H37Ra) (MIC(50)=0.5 μg/mL) and M. bovis (MIC(50)=0.5 μg/mL) at low concentrations, and was ten times more potent against Mtb (H37Ra) than cycloserine (MIC(50)=5.0 μg/mL), a second line drug. It also showed an additive effect when combined with isoniazid. Compound 21 retained sensitivity against a rifampicin-resistant (H37Rv) strain of Mtb (MIC(50)=1 μg/mL) at concentrations similar to that for a rifampicin-sensitive (H37Rv) strain, suggesting that it has no cross-resistance to a first-line anti-TB drug. In addition, the replication of M. avium was also inhibited by 21 (MIC(50)=10 μg/mL). No cellular toxicity of 13 or 21 was observed up to the highest concentration tested (CC(50)>100 μg/mL). These observations offer promise for a new drug treatment regimen to augment and complement the current chemotherapy of TB.     

Brief cognitive behavioral therapy in primary care: a hybrid type 2 patient-randomized effectivenessimplementation design

ABSTRACT: BACKGROUND: Despite the availability of evidence-based psychotherapies for depression and anxiety, they are underused in non-mental health specialty settings such as primary care. Hybrid effectiveness-implementation designs have the potential to evaluate clinical and implementation outcomes of evidence-based psychotherapies to improve their translation into routine clinical care practices. METHODS: This protocol article discusses the study methodology and implementation strategies employed in an ongoing, hybrid, type 2 randomized controlled trial with two primary aims: (1) to determine whether a brief, manualized cognitive behavioral therapy administered by Veterans Affairs Primary Care Mental Health Integration program clinicians is effective in treating depression and anxiety in a sample of medically ill (chronic cardiopulmonary diseases) primary care patients and (2) to examine the acceptability, feasibility, and preliminary outcomes of a focused implementation strategy on improving adoption and fidelity of brief cognitive behavioral therapy at two Primary Care-Mental Health Integration clinics. The study uses a hybrid type 2 effectiveness/implementation design to simultaneously test clinical effectiveness and to collect pilot data on a multifaceted implementation strategy that includes an online training program, audit and feedback of session content, and internal and external facilitation. Additionally, the study engages the participation of an advisory council consisting of stakeholders from Primary Care-Mental Health Integration, as well as regional and national mental health leaders within the Veterans Administration. It targets recruitment of 320 participants randomized to brief cognitive behavioral therapy (n = 200) or usual care (n = 120). Both effectiveness and implementation outcomes are being assessed using mixed methods, including quantitative evaluation (e.g., intent-to-treat analyses across multiple time points) and qualitative methods (e.g., focus interviews and surveys from patients and providers). Patient-effectiveness outcomes include measures of depression, anxiety, and physical health functioning using blinded independent evaluators. Implementation outcomes include patient engagement and adherence and clinician brief cognitive behavioral therapy adoption and fidelity. CONCLUSIONS: Hybrid designs are needed to advance clinical effectiveness and implementation knowledge to improve healthcare practices. The current article describes the rationale and challenges associated with the use of a hybrid design for the study of brief cognitive behavioral therapy in primary care. Although trade-offs exist between scientific control and external validity, hybrid designs are part of an emerging approach that has the potential to rapidly advance both science and practice. Trial registration NCT01149772 at http://www.clinicaltrials.gov/ct2/show/NCT01149772.     

The extracellular release of DNA and HMGB1 from Jurkat T cells during in vitro necrotic cell death

In innate immunity, dead and dying cells release internal constituents that can serve as damage-associated molecular patterns (DAMPs) or alarmins. This release occurs more abundantly during necrosis than apoptosis and may account for the differences in the immunologic properties of these death forms. To elucidate DAMP release in necrosis, we compared the levels of two nuclear molecules (DNA and HMGB1, a non-histone protein with alarmin activity) in media following necrosis of Jurkat T cells by freeze-thawing, ethanol, heat or hydrogen peroxide treatment. In our experiments, DNA release was measured by fluorimetry with the dye PicoGreen, while HMGB1 was measured by Western blotting. As the results of our study show, each form of necrosis is associated with a distinct pattern of DNA and HMGB1 release with respect to kinetics and amounts. Of these, freeze-thawing produced the highest and most rapid increase in HMGB1 and DNA levels, although the released DNA was subject to nuclease digestion; in addition, freeze-thawing led to the production of particles measured by flow cytometry. Together, these results indicate that experimental necrosis leads to diverse patterns of nuclear molecule release which could affect their immunologic activity.