Innovative trial designs are practical solutions for improving the treatment of tuberculosis

A growing number of new drugs for the treatment of tuberculosis are in clinical development. Confirmatory phase 3 trials are expensive and time-consuming and the question of whether one particular drug combination can be used to treat tuberculosis is less important from a public health perspective than the question of which are the shortest, simplest, most effective, and safest regimens. While preclinical and phase 1 studies provide some guidance in the selection of combinations for clinical evaluation, a large number of combinations will require phase 2 testing to ensure that only the best regimens advance to phase 3. The multi-arm multi-stage trial design is an example of a treatment selection-adaptive design where multiple experimental arms are each simultaneously compared with a common control and interim analyses allow for poor performing arms to be dropped early. Such designs, if designed and implemented correctly, require fewer patients, can be completed in a shorter time frame, and answer more relevant questions without any loss in statistical validity or scientific integrity. There are, however, practical issues that must be considered in applying this in tuberculosis treatment trials. More innovative trials designs should be considered to speed drug and regimen development for the treatment of tuberculosis.     

A method for quantitative characterization of growth in the 3-D structure of rat pulmonary arteries

Understanding mechanisms causing pulmonary vascular disease (PVD) frequently requires a thorough understanding of the underlying structural changes in the pulmonary circulation. Animal models have been used extensively to study different forms of PVD but conventional experimental techniques are limited in their ability to allow the study of the whole pulmonary vasculature at once. In this study, we introduce novel techniques of arterial casting, high-resolution imaging and tree analysis to study the pulmonary circulation in rodent models. Male Sprague-Dawley rats were used at 20, 36, 52, 100 and 160 days of age. A technique involving arterial casting with Microfil silicone polymer, high-resolution micro-computed tomography (micro-CT) at 12.5 μm resolution and image data analysis involving segmentation and skeletonization was developed to both qualitatively and quantitatively describe the changes in the pulmonary vasculature with increasing age. Parameters identified to affect the quality of pulmonary arterial casting included polymer flow rate, total injected volume, polymer viscosity and polymerization time. By optimizing these parameters, we successfully created arterial casts of the pulmonary circulation in rats of different ages and demonstrated the feasibility of quantitatively characterizing the changes in the number of vessels with postnatal growth. These preliminary data suggest that the number of vessels with lumen diameters of 50-200 μm increases rapidly in both lungs between 52 and 100 days of age. With this new technique, the pulmonary vasculature can now be studied in a whole lung animal model to better understand the global effects of disease on vascular structure.     

Apolipoprotein E predicts incident cardiovascular disease risk in women but not in men with concurrently high levels of high-density lipoprotein cholesterol and C-reactive protein

Although there is great interest in the notion that dysfunctional transformation of high-density lipoprotein (HDL) facilitates development of atherosclerosis and cardiovascular disease (CVD), few studies in human populations directly address this issue. As apolipoprotein E (apoE) is a constituent of HDL thought to be important for HDL antiatherogenic function, we sought to assess the role of apoE in CVD risk in subjects likely to display dysfunctional transformation of HDL. Association of apoE levels with incident CVD risk was investigated using Cox multivariable proportional hazards modeling. Analyses were performed in subgroups of women and men likely to display dysfunctional transformation of HDL deriving from previous subgroup identification based upon defining characteristics of concurrently high levels of HDL cholesterol and systemic inflammation as reflected by high C-reactive protein levels. Results revealed apoE levels (dichotomized as highest quartile vs combined 3 lowest quartiles) as predicting subgroup risk in women (hazard ratio, 4.52; 95% confidence interval, 1.07-19.12; P = .040) but not in men. Further sex differences were manifested in terms of the relationship of apoE levels with age. Analysis revealed positive correlation of apoE levels with age in women (r = 0.47, P < .0001) but not in men (r = 0.04, P = .43). Apolipoprotein E levels predict incident CVD risk in women with high levels of HDL cholesterol and C-reactive protein but not in men. Future studies should be oriented toward investigations of apoE as related to multiplicity of HDL functionality and toward assessment of potential roles for apoE in dysfunctional transformation of HDL.     

The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue

The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.     

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

Hepatitis C virus (HCV) infects ～2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.     

Regulatory element copy number differences shape primate expression profiles

Gene expression differences are shaped by selective pressures and contribute to phenotypic differences between species. We identified 964 copy number differences (CNDs) of conserved sequences across three primate species and examined their potential effects on gene expression profiles. Samples with copy number different genes had significantly different expression than samples with neutral copy number. Genes encoding regulatory molecules differed in copy number and were associated with significant expression differences. Additionally, we identified 127 CNDs that were processed pseudogenes and some of which were expressed. Furthermore, there were copy number-different regulatory regions such as ultraconserved elements and long intergenic noncoding RNAs with the potential to affect expression. We postulate that CNDs of these conserved sequences fine-tune developmental pathways by altering the levels of RNA.     

IL-38 binds to the IL-36 receptor and has biological effects on immune cells similar to IL-36 receptor antagonist

The functional role of IL-1 family member 10, recently renamed IL-38, remains unknown. In the present study we aimed to elucidate the biological function of IL-38 and to identify its receptor. Heat-killed Candida albicans was used to stimulate memory T-lymphocyte cytokine production in freshly obtained human peripheral blood mononuclear cells from healthy subjects. The addition of recombinant IL-38 (152 amino acids) inhibited the production of T-cell cytokines IL-22 (37% decrease) and IL-17 (39% decrease). The reduction in IL-22 and IL-17 caused by IL-38 was similar to that caused by the naturally occurring IL-36 receptor antagonist (IL-36Ra) in the same peripheral blood mononuclear cells cultures. IL-8 production induced by IL-36γ was reduced by IL-38 (42% decrease) and also was reduced by IL-36Ra (73% decrease). When human blood monocyte-derived dendritic cells were used, IL-38 as well as IL-36Ra increased LPS-induced IL-6 by twofold. We screened immobilized extracellular domains of each member of the IL-1 receptor family, including the IL-36 receptor (also known as "IL-1 receptor-related protein 2") and observed that IL-38 bound only to the IL-36 receptor, as did IL-36Ra. The dose-response suppression of IL-38 as well as that of IL-36Ra of Candida-induced IL-22 and IL-17 was not that of the classic IL-1 receptor antagonist (anakinra), because low concentrations were optimal for inhibiting IL-22 production, whereas higher concentrations modestly increased IL-22. These data provide evidence that IL-38 binds to the IL-36R, as does IL-36Ra, and that IL-38 and IL-36Ra have similar biological effects on immune cells by engaging the IL-36 receptor.