Lentiviral gene therapy rescues p47phox chronic granulomatous disease and the ability to fight Salmonella infection in mice

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder characterised by recurrent and often life-threatening infections and hyperinflammation. It is caused by defects of the phagocytic NADPH oxidase, a multicomponent enzyme system responsible for effective pathogen killing. A phase I/II clinical trial of lentiviral gene therapy is underway for the most common form of CGD, X-linked, caused by mutations in the gp91phox subunit of the NADPH oxidase. We propose to use a similar strategy to tackle p47phox-deficient CGD, caused by mutations in NCF1, which encodes the p47phox cytosolic component of the enzymatic complex. We generated a pCCLCHIM-p47phox lentiviral vector, containing the chimeric Cathepsin G/FES myeloid promoter and a codon-optimised version of the human NCF1 cDNA. Here we show that transduction with the pCCLCHIM-p47phox vector efficiently restores p47phox expression and biochemical NADPH oxidase function in p47phox-deficient human and murine cells. We also tested the ability of our gene therapy approach to control infection by challenging p47phox-null mice with Salmonella Typhimurium, a leading cause of sepsis in CGD patients, and found that mice reconstituted with lentivirus-transduced hematopoietic stem cells had a reduced bacterial load compared with untreated mice. Overall, our results potentially support the clinical development of a gene therapy approach using the pCCLCHIM-p47phox vector

Scientific Opportunities to Reduce Risk in Nuclear Process Science

Cleaning up the nation’s nuclear weapons complex remains as one of the most technologically challenging and financially costly problems facing the U.S. Department of Energy (DOE). Safety, cost, and technological challenges have often delayed progress in retrieval, processing, and final disposition of high-level waste, spent nuclear fuel, and challenging materials. Some of the issues result from the difficulty and complexity of the technological issues; others have programmatic bases, such as contracting strategies that may provide undue focus on near-term, specific clean-up goals or difficulty in developing and maintaining stakeholder confidence in the proposed solutions. We propose that independent basic fundamental science research focused on the full cleanup life-cycle offers an opportunity to help address these challenges by providing 1) scientific insight into the fundamental mechanisms involved in currently selected processing and disposal options, 2) a rational path to the development of alternative technologies should the primary options fail, 3) confidence that models that predict long-term performance of different disposal options are based upon the best available science, 4) fundamental science discovery that enables transformational solutions to revolutionize the current baseline processes

Basic Research Needs for Geosciences: Facilitating 21st Century Energy Systems

To identify research areas in geosciences, such as behavior of multiphase fluid-solid systems on a variety of scales, chemical migration processes in geologic media, characterization of geologic systems, and modeling and simulation of geologic systems, needed for improved energy systems

NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent, TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium.

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn's disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb (-/-)) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm(avir); invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb (-/-) mice are efficiently infected by S.Tm(avir) and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb (-/-) Myd88 (-/-) mice indicated that the S.Tm(avir)-inflicted disease in Cybb (-/-) mice hinges on CD11c(+)CX3CR1(+) monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm(avir) in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface

Propensity for selfing varies within a population of hermaphroditic snails: coexistence of selfers, outcrossers and mixed-mating individuals

To understand mating-system evolution in self-compatible hermaphrodites, variation in selfing rates is highly relevant. Empirical studies are rarely designed to capture variation between individuals, instead often comparing species and populations. Yet, evolution primarily occurs within populations, rendering among-individual variation essential. Observed individual selfing rates depend on the environment (e.g. differences in mate availability) and individuals' propensity for selfing. We quantified individual variation in selfing propensity in the snail Radix balthica by conducting laboratory mating trials that manipulated mate availability (low versus moderate) and estimating selfing rates from progeny arrays. We also measured female lifetime fitness. We found substantial among-individual variation in selfing propensity, including pure selfers (32%), pure outcrossers (31%) and mixed-mating individuals that selfed and outcrossed (37%). Experimental levels of mate availability did not significantly affect selfing rates. Selfers had reduced female liftetime fitness. Our results show that the propensity for selfing can differ considerably among individuals, with similar proportions of selfers, outcrossers and mixed maters. As mate availability did not affect selfing, our ‘moderate’ experimental level of mate availability might still have been too low to prompt selfers to outcross. This and the observed fitness differences also cautiously suggest that investigating the heritability of selfing propensities might be worthwhile in this population

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