RAGE ligation affects T cell activation and controls T cell differentiation

The pattern recognition receptor, RAGE, has been shown to be involved in adaptive immune responses but its role on the components of these responses is not well understood. We have studied the effects of a small molecule inhibitor of RAGE and the deletion of the receptor (RAGE-/- mice) on T cell responses involved in autoimmunity and allograft rejection. Syngeneic islet graft and islet allograft rejection was reduced in NOD and B6 mice treated with TTP488, a small molecule RAGE inhibitor (p < 0.001). RAGE-/- mice with streptozotocin-induced diabetes showed delayed rejection of islet allografts compared with wild type (WT) mice (p < 0.02). This response in vivo correlated with reduced proliferative responses of RAGE-/- T cells in MLRs and in WT T cells cultured with TTP488. Overall T cell proliferation following activation with anti-CD3 and anti-CD28 mAbs were similar in RAGE-/- and WT cells, but RAGE-/- T cells did not respond to costimulation with anti-CD28 mAb. Furthermore, culture supernatants from cultures with anti-CD3 and anti-CD28 mAbs showed higher levels of IL-10, IL-5, and TNF-alpha with RAGE-/- compared with WT T cells, and WT T cells showed reduced production of IFN-gamma in the presence of TTP488, suggesting that RAGE may be important in the differentiation of T cell subjects. Indeed, by real-time PCR, we found higher levels of RAGE mRNA expression on clonal T cells activated under Th1 differentiating conditions. We conclude that activation of RAGE on T cells is involved in early events that lead to differentiation of Th1(+) T cells.     

Denaturing Gradient Gel Electrophoresis as a Tool To Determine Batch Similarity of Probiotic Cultures of Porcine Cecal Bacteria

A continuous-flow porcine cecal bacterial culture has been used experimentally as treatment against enterotoxigenic Escherichia coli in weanling pigs. Periodically, the cultures must be started from frozen stock. Our results indicate that denaturing gradient gel electrophoresis can be applied as an indirect indication of culture similarity for each new batch generated from frozen stock.     

Research in the ranks: vulnerable subjects, coercible collaboration, and the hepatitis E vaccine trial in Nepal

Concern over the diminished autonomy of members of the armed forces has resulted in the classification of these groups as "vulnerable" in many international codes of research ethics, a designation that places the onus on researchers to provide special justification for the inclusion of these persons in research. This paper examines the application of these ethical requirements to a recent trial carried out by U.S. Army researchers among soldiers of the Royal Nepal Army (RNA) and concludes that the requirements to justify conducting research in this population were not met. Furthermore, noting the human rights abuses rampant in the RNA, it is appropriate to question the choice of this institution as both a partner and a subject pool for U.S. state-sponsored research. This case study raises another important ethical question about the vulnerability to coerced collaboration of groups or institutions. In response, I propose the idea of "institutional vulnerability" as an extension of the idea of individual "juridic vulnerability." The recent military and financial assistance that the RNA received from the U.S. Army, in light of their partnership in this biomedical research trial, constitutes an appropriate and revealing context in which to ground this discussion.     

Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast

Discovering target and off-target effects of specific compounds is critical to drug discovery and development. We generated a compendium of "chemical-genetic interaction" profiles by testing the collection of viable yeast haploid deletion mutants for hypersensitivity to 82 compounds and natural product extracts. To cluster compounds with a similar mode-of-action and to reveal insights into the cellular pathways and proteins affected, we applied both a hierarchical clustering and a factorgram method, which allows a gene or compound to be associated with more than one group. In particular, tamoxifen, a breast cancer therapeutic, was found to disrupt calcium homeostasis and phosphatidylserine (PS) was recognized as a target for papuamide B, a cytotoxic lipopeptide with anti-HIV activity. Further, the profile of crude extracts resembled that of its constituent purified natural product, enabling detailed classification of extract activity prior to purification. This compendium should serve as a valuable key for interpreting cellular effects of novel compounds with similar activities.     

Long-term Effects of Free Air CO2 Enrichment (FACE) on Soil Respiration

Emissions of CO₂ from soils make up one of the largest fluxes in the global C cycle, thus small changes in soil respiration may have large impacts on global C cycling. Anthropogenic additions of CO₂ to the atmosphere are expected to alter soil carbon cycling, an important component of the global carbon budget. As part of the Duke Forest Free-Air CO₂ Enrichment (FACE) experiment, we examined how forest growth at elevated (+200 ppmv) atmospheric CO₂ concentration affects soil CO₂ dynamics over 7 years of continuous enrichment. Soil respiration, soil CO₂ concentrations, and the isotopic signature of soil CO₂ were measured monthly throughout the 7 years of treatment. Estimated annual rates of soil CO₂ efflux have been significantly higher in the elevated plots in every year of the study, but over the last 5 years the magnitude of the CO₂ enrichment effect on soil CO₂ efflux has declined. Gas well samples indicate that over 7 years fumigation has led to sustained increases in soil CO₂ concentrations and depletion in the δ¹³C of soil CO₂ at all but the shallowest soil depths.     

Levels of pneumococcal conjugate vaccine coverage and indirect protection against invasive pneumococcal disease and pneumonia hospitalisations in Australia: An observational study

BackgroundThere is limited empiric evidence on the coverage of pneumococcal conjugate vaccines (PCVs) required to generate substantial indirect protection. We investigate the association between population PCV coverage and indirect protection against invasive pneumococcal disease (IPD) and pneumonia hospitalisations among undervaccinated Australian children.Methods and findingsBirth and vaccination records, IPD notifications, and hospitalisations were individually linked for children aged <5 years, born between 2001 and 2012 in 2 Australian states (New South Wales and Western Australia; 1.37 million children). Using Poisson regression models, we examined the association between PCV coverage, in small geographical units, and the incidence of (1) 7-valent PCV (PCV7)-type IPD; (2) all-cause pneumonia; and (3) pneumococcal and lobar pneumonia hospitalisation in undervaccinated children. Undervaccinated children received <2 doses of PCV at <12 months of age and no doses at ≥12 months of age. Potential confounding variables were selected for adjustment a priori with the assistance of a directed acyclic graph.There were strong inverse associations between PCV coverage and the incidence of PCV7-type IPD (adjusted incidence rate ratio [aIRR] 0.967, 95% confidence interval [CI] 0.958 to 0.975, p-value < 0.001), and pneumonia hospitalisations (all-cause pneumonia: aIRR 0.991 95% CI 0.990 to 0.994, p-value < 0.001) among undervaccinated children. Subgroup analyses for children <4 months old, urban, rural, and Indigenous populations showed similar trends, although effects were smaller for rural and Indigenous populations. Approximately 50% coverage of PCV7 among children <5 years of age was estimated to prevent up to 72.5% (95% CI 51.6 to 84.4) of PCV7-type IPD among undervaccinated children, while 90% coverage was estimated to prevent 95.2% (95% CI 89.4 to 97.8). The main limitations of this study include the potential for differential loss to follow-up, geographical misclassification of children (based on residential address at birth only), and unmeasured confounders.ConclusionsIn this study, we observed substantial indirect protection at lower levels of PCV coverage than previously described—challenging assumptions that high levels of PCV coverage (i.e., greater than 90%) are required. Understanding the association between PCV coverage and indirect protection is a priority since the control of vaccine-type pneumococcal disease is a prerequisite for reducing the number of PCV doses (from 3 to 2). Reduced dose schedules have the potential to substantially reduce program costs while maintaining vaccine impact.

In an observational study, Jocelyn Chan and colleagues investigate associations between pneumococcal conjugate vaccine coverage and incidence of invasive pneumococcal disease and pneumonia among children under 5 years in Australia.     

Development of dimeric modulators for anti-apoptotic Bcl-2 proteins

Bcl-2 family proteins can be classified into two subfamilies--anti-apoptotic members and pro-apoptotic members. Mechanistically, these two subfamilies can antagonize each other through heterodimerization while homodimerization has been proposed for each subfamily to carry out their corresponding anti-apoptotic or pro-apoptotic functions. To date, many small-molecule antagonists against anti-apoptotic Bcl-2 proteins have been developed, which are monomeric modulators. In this study, a series of BH3I-1 based dimeric modulators were developed with structure-activity relationship explored. Dimeric modulators compared to the monomeric antagonists have enhanced binding activity against anti-apoptotic Bcl-2 proteins. In addition, the acidic functional group was demonstrated to be critical for the binding interaction of the small-molecule antagonists with anti-apoptotic Bcl-2 proteins. Finally, the representative dimeric modulator revealed enhanced activity in inducing cytochrome c release from mitochondria compared to its monomeric counterpart. Taken together, dimerization of monomeric modulators is one practical approach to enhance the bioactivity of Bcl-2 antagonists.     

Design and campaign synthesis of pyridine-based histone deacetylase inhibitors

A lead benzamide, bearing a cyanopyridyl moiety (3), was identified as a potent and low molecular weight histone deacetylase (HDAC) inhibitor. Various replacements of the cyano group were explored at the C3-position, along with the exploration of solubility-enhancing groups at the C5-position. It was determined that cyano substitution at the C3-position of the pyridyl core, along with a methylazetidinyl substituent at the C5-position yielded optimal HDAC1 inhibition and anti-proliferative activity in HCT-116 cells.