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ABSTRACT: Invasive aspergillosis is a major threat to patients suffering from chronic granulomatous disease (CGD). Fungal pathogenesis is the result of a diminished antifungal capacity and dysregulated inflammation. A deficient NADPH-oxidase results in defective phagolysosomal alkalization. This study investigates the contribution of defective pH-regulation of the CGD phagocyte in fungal pathogenesis. We therefore evaluated the effect of the acidotropic, antimalarial drug chloroquine (CQ) for its antifungal capacity of polymorphonuclear cells (PMN), and on the inflammatory response of peripheral blood mononuclear cells (PBMC). Chloroquine exerts a direct pH-dependent antifungal effect on Aspergillus fumigatus and A. nidulans, it increases the antifungal activity of CGD PMN at a significantly lower concentration compared to healthy PMN, and decreases the hyperinflammatory state of CGD PBMC as observed by decreased TNF-α and IL-1β release. CQ targets both limbs of fungal pathogenesis and might be of great value in the clearance of invasive aspergillosis in the CGD host.
The Journal of Infectious Diseases 03/2013; · 6.41 Impact Factor
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Charles E Rose,
Sandra Romero-Steiner,
Robert L Burton,
George M Carlone,
David Goldblatt,
Moon H Nahm,
Lindsey Ashton,
Mitch Haston,
Nina Ekström,
Raili Haikala, [......],
Nathalie Durant,
Jan T Poolman,
Phil Fernsten,
Xinhong Yu,
Branda T Hu,
Kathrin U Jansen,
Milan Blake, Elles R Simonetti,
Peter W M Hermans,
Brian D Plikaytis
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ABSTRACT: Antibody-mediated killing of Streptococcus pneumoniae (pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies by use of a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and required for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis-of-variance (ANOVA) mixed-effects model, provided a common reference for assessing agreement among these laboratories. Agreement was evaluated in terms of assay accuracy, reproducibility, repeatability, precision, and bias. We also reviewed four acceptance criterion intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy, and concordance results among laboratories and the consensus titers revealed acceptable agreement. The results of this study indicate that the bioassays evaluated in this study are robust, and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template for evaluating future multilaboratory studies.
Clinical and vaccine immunology: CVI 11/2010; 18(1):135-42. · 2.37 Impact Factor
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ABSTRACT: Invasive aspergillosis is a major threat for patients suffering from chronic granulomatous disease (CGD). Although Aspergillus fumigatus is the most commonly encountered Aspergillus species, the presence of A. nidulans appears to be disproportionately high in CGD patients. The purpose of this study was to investigate the involvement of the NADPH oxidase and the resulting reactive oxygen species (ROS) in host defense against fungi and to clarify their relationship toward A. nidulans. Murine CGD alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) from healthy controls and CGD patients were challenged with either A. fumigatus or A. nidulans. Analysis of the antifungal effects of ROS revealed that A. nidulans, in contrast to A. fumigatus, is not susceptible to ROS. In addition, infection with live A. nidulans did not result in any measurable ROS release. Remarkably, human CGD PMN and PBMC and murine CGD AM were at least equipotent at arresting conidial germination compared to healthy controls. Blocking of the NADPH oxidase resulted in significantly reduced damage of A. fumigatus but did not affect A. nidulans hyphae. Furthermore, the microbicidal activity of CGD PMN was maintained toward A. nidulans but not A. fumigatus. In summary, antifungal resistance to A. nidulans is not directly ROS related. The etiology of A. nidulans infections in CGD cannot be explained by the simple absence of the direct microbicidal effect of ROS. In vivo, the NADPH oxidase is a critical regulator of innate immunity whose unraveling will improve our understanding of fungal pathogenesis in CGD.
Infection and immunity 11/2010; 79(2):767-73. · 4.21 Impact Factor
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ABSTRACT: The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO(2)) it encounters during transmission, colonization, and infection. Since CO(2) is important for various cellular processes, factors that allow optimal CO(2) sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO(2)-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO(2) to bicarbonate (HCO(3)(-)), an essential step to prevent the cellular release of CO(2). The addition of unsaturated fatty acids (UFAs) reversed the CO(2)-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Deltapca), indicating that PCA-mediated CO(2) fixation is at least associated with HCO(3)(-)-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO(2) fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO(2)-independent and naturally circulating CO(2)-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO(2) fixation pathways for pneumococcal growth and survival.
Journal of bacteriology 08/2010; 192(15):4054-62. · 3.94 Impact Factor
