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      Efficacy of pyrazinoic acid dry powder aerosols in resolving necrotic and non-necrotic granulomas in a guinea pig model of tuberculosis

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          Abstract

          New therapeutic strategies are needed to treat drug resistant tuberculosis (TB) and to improve treatment for drug sensitive TB. Pyrazinamide (PZA) is a critical component of current first-line TB therapy. However, the rise in PZA-resistant TB cases jeopardizes the future utility of PZA. To address this problem, we used the guinea pig model of TB and tested the efficacy of an inhaled dry powder combination, referred to as Pyrazinoic acid/ester Dry Powder (PDP), which is comprised of pyrazinoic acid (POA), the active moiety of PZA, and pyrazinoic acid ester (PAE), which is a PZA analog. Both POA and PAE have the advantage of being able to act on PZA-resistant Mycobacterium tuberculosis. When used in combination with oral rifampicin (R), inhaled PDP had striking effects on tissue pathology. Effects were observed in lungs, the site of delivery, but also in the spleen and liver indicating both local and systemic effects of inhaled PDP. Tissue granulomas that harbor M. tuberculosis in a persistent state are a hallmark of TB and they pose a challenge for therapy. Compared to other treatments, which preferentially cleared non-necrotic granulomas, R+PDP reduced necrotic granulomas more effectively. The increased ability of R+PDP to act on more recalcitrant necrotic granulomas suggests a novel mechanism of action. The results presented in this report reveal the potential for developing therapies involving POA that are optimized to target necrotic as well as non-necrotic granulomas as a means of achieving more complete sterilization of M. tuberculosis bacilli and preventing disease relapse when therapy ends.

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          The association between sterilizing activity and drug distribution into tuberculosis lesions

          Finding new treatment-shortening antibiotics to improve cure rates and curb the alarming emergence of drug resistance is the major objective of tuberculosis (TB) drug development. Using a MALDI mass spectrometry imaging suite in a biosafety containment facility, we show that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside 1 . In contrast, moxifloxacin which is active in vitro against persisters, a sub-population of Mycobacterium tuberculosis that persists in specific niches under drug pressure, and achieved treatment shortening in mice 2 , does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials. We also suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug resistant TB. We propose an alternative working model to prioritize new antibiotic regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs. The finding that lesion penetration contributes to treatment outcome has wide implications for TB.
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            Tuberculosis: what we don't know can, and does, hurt us.

            Mycobacterium tuberculosis has a penetrance of its host population that would be the envy of most human pathogens. About one-third of the human population would have a positive skin test for the infection and is thus thought to harbor the bacterium. Globally, 22 "high-burden" countries account for more than 80% of the active tuberculosis cases in the world, which shows the inequitable distribution of the disease. There is no effective vaccine against infection, and current drug therapies are fraught with problems, predominantly because of the protracted nature of the treatment and the increasing occurrence of drug resistance. Here we focus on the biology of the host-pathogen interaction and discuss new and evolving strategies for intervention.
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              Host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action.

              The current standard of treatment against tuberculosis consists of a cocktail of first-line drugs, including isoniazid and pyrazinamide. Although these drugs are known to be bactericidal, contribution of host cell responses in the context of antimycobacterial chemotherapy, if any, remains unknown. We demonstrate that isoniazid and pyrazinamide promote autophagy activation and phagosomal maturation in Mycobacterium tuberculosis (Mtb)-infected host cells. Treatment of Mtb-infected macrophages with isoniazid or pyrazinamide caused significant activation of cellular and mitochondrial reactive oxygen species and autophagy, which was triggered by bacterial hydroxyl radical generation. Mycobacterium marinum-infected autophagy-defective, atg7 mutant Drosophila exhibited decreased survival rates, which could not be rescued by antimycobacterial treatment, indicating that autophagy is required for effective antimycobacterial drug action in vivo. Moreover, activation of autophagy by antibiotic treatment dampened Mtb-induced proinflammatory responses in macrophages. Together, these findings underscore the importance of host autophagy in orchestrating successful antimicrobial responses to mycobacteria during chemotherapy. Copyright © 2012 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Methodology
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Methodology
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Methodology
                Role: Formal analysisRole: Methodology
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                27 September 2018
                2018
                : 13
                : 9
                : e0204495
                Affiliations
                [1 ] Department of Pathology and Laboratory Medicine and Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America
                [2 ] Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America
                [3 ] RTI International, Research Triangle Park, North Carolina, United States of America
                [4 ] Department of Biostatistics and North Carolina Translational and Clinical Sciences Institute, University of North Carolina, Chapel Hill, North Carolina, United States of America
                [5 ] Department of Chemistry, University at Albany, Albany, New York, United States of America
                Public Health England, UNITED KINGDOM
                Author notes

                Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: MB, AJH, PGD and EY have filed a provisional patent PCT/US2017/061544 "Tuberculosis therapeutic compounds and methods”. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0003-1180-0030
                Article
                PONE-D-18-01227
                10.1371/journal.pone.0204495
                6160074
                30261007
                af930da7-dc99-4812-b74c-57a9c29a11a7
                © 2018 Montgomery et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 12 January 2018
                : 10 September 2018
                Page count
                Figures: 4, Tables: 4, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100006108, National Center for Advancing Translational Sciences;
                Award ID: ULTR001111
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: CHE-1337594
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100006108, National Center for Advancing Translational Sciences;
                Award ID: ULTR001111
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: 5P30CA016086-41
                Funded by: funder-id http://dx.doi.org/10.13039/100006108, National Center for Advancing Translational Sciences;
                Award ID: UL1TR002489
                Award Recipient :
                This work was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Award 1UL1TR001111 and NC TraCS 4DR31502 awards to MB and AJH and through National Science Foundation grant award CHE-1337594 to JTW. Histopathology was performed by the Animal Histopathology & Laboratory Medicine Core at the University of North Carolina, UNC Lineberger Comprehensive Cancer Center, which is supported in part by an NCI Center Core Support Grant (5P30CA016086-41). National Center for Advancing Translational Sciences (UL1TR002489) to FCL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Immune Cells
                Granulomas
                Biology and Life Sciences
                Immunology
                Immune Cells
                Granulomas
                Medicine and Health Sciences
                Immunology
                Immune Cells
                Granulomas
                Biology and Life Sciences
                Organisms
                Bacteria
                Actinobacteria
                Mycobacterium Tuberculosis
                Biology and Life Sciences
                Physiology
                Immune Physiology
                Spleen
                Medicine and Health Sciences
                Physiology
                Immune Physiology
                Spleen
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
                Tuberculosis
                Medicine and Health Sciences
                Tropical Diseases
                Tuberculosis
                Research and Analysis Methods
                Experimental Organism Systems
                Animal Models
                Guinea Pigs
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Mammals
                Rodents
                Guinea Pigs
                Physical Sciences
                Materials Science
                Materials by Structure
                Powders
                Medicine and Health Sciences
                Pharmaceutics
                Drug Therapy
                Medicine and Health Sciences
                Diagnostic Medicine
                Signs and Symptoms
                Necrosis
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Signs and Symptoms
                Necrosis
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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