28 results on '"Timothy J. Break"'
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2. Abstract 4351: Multiplex detection of G12/G13 KRAS mutations with an electrochemiluminescent hybridization assay
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Annamaria Szabolcs, Timothy J. Break, Isaac H. Shin, Seth B. Harkins, and Jacob N. Wohlstadter
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Cancer Research ,Oncology - Abstract
Purpose: Oncogenic Kirsten rat sarcoma virus (KRAS) mutations are the most prevalent cancer mutations in all human tumors. Also, genotyping the KRAS gene has become increasingly important with the emergence of new evidence highlighting differences in downstream signaling pathways, tumor microenvironment composition, treatment responses and prognoses linked to specific single nucleotide polymorphisms (SNPs) in the G12 or G13 codons. FDA approval of G12C-specific anti-KRAS drugs highlights the importance of the development of reliable SNP assays to interrogate the mutation status in a tumor sample. Currently available genotyping assays are based on NGS, PCR, or ddPCR. These methods can be time-consuming, costly, suffer from amplification bias, or require follow-up testing or bioinformatics skills to analyze. Building on Meso Scale Discovery’s (MSD) existing technology, we aimed to develop a method for the identification of eight KRAS SNPs located on the G12-G13 codons in a single reaction. Methods: Ten-spot, 96-well N-PLEX plates were used for the study. SNP-specific upstream probes carrying unique 5′ leader sequences complementary to spot-specific captures on the N-PLEX plates were designed for eight KRAS genotypes (WT, G12R, G12C, G12S, G12A, G12D, G12V, G13D). Locus-specific downstream probes were 5’ phosphorylated for ligation and 3’ biotinylated for detection. Synthetic DNA targets were used as assay calibrators, and commercially available FFPE reference samples were tested for validation. DNA (10 ng) was PCR amplified with primers flanking the KRAS mutation sites. A multiplexed mixture of upstream and downstream probes was hybridized to the amplicons, and 30 cycles of ligation was performed using DNA ligase in a thermal cycler. Samples were subsequently hybridized to spot-specific capture oligonucleotides on the surface of the assay plates and detected with SULFO-TAG labeled streptavidin. Electrochemiluminescence readout was collected on an MSD imager. Results: Optimization of probe concentrations and ligation temperature was performed to maximize signal-to-background ratios and assay specificity. Spike recovery experiments using artificial target DNA showed successful identification of KRAS mutant genotypes at spike levels as low as 0.1% over wild-type background. Validation experiments with gDNA extracted from FFPE reference samples confirmed that the multiplex SNP assay can accurately differentiate eight KRAS genotypes in samples with >0.4% tumor burden. Conclusion: These data highlight a novel approach to simultaneously identify eight KRAS genotypes from 10 ng of DNA input in a single reaction within 4-5 hours. The assay is capable of identifying double mutants and can provide a semiquantitative assessment of tumor burden without the need for follow-up testing. Citation Format: Annamaria Szabolcs, Timothy J. Break, Isaac H. Shin, Seth B. Harkins, Jacob N. Wohlstadter. Multiplex detection of G12/G13 KRAS mutations with an electrochemiluminescent hybridization assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4351.
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- 2023
3. Response to Comments on 'Aberrant type 1 immunity drives susceptibility to mucosal fungal infections'
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Heather D. Hickman, Chyi-Chia Richard Lee, Peter Olbrich, Jean K. Lim, Daniel Chauss, Vasileios Oikonomou, Daniel L. Barber, Philip M. Murphy, Monica M. Schmitt, Tilo Freiwald, Marc Swidergall, Stefania Pittaluga, Steven M. Holland, Daniel Martin, Luigi D. Notarangelo, Oliver J. Harrison, Scott G. Filler, Lindsey B. Rosen, Ian A. Myles, Wint Lwin, Sergio M. Pontejo, Behdad Afzali, Timothy J. Break, Niki M. Moutsopoulos, Vincent M. Bruno, Julie Alejo, Jigar V. Desai, Nicolas Bouladoux, Elise M. N. Ferré, Norma V. Solis, Mitsuru Matsumoto, David V. Serreze, Yasmine Belkaid, Andy Renteria, John P. Shannon, Nicolas Dutzan, Teresa Greenwell-Wild, Michail S. Lionakis, Drake W. Williams, Kevin W. Hoffman, Muthulekha Swamydas, and Mark S. Anderson
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Multidisciplinary ,Mucous Membrane ,Type 1 immunity ,Mycoses ,business.industry ,Immunology ,Autoantibody ,Medicine ,Humans ,business ,Immunity, Mucosal ,Article - Abstract
Puel and Casanova and Kisand et al . challenge our conclusions that interferonopathy and not IL-17/IL-22 autoantibodies promote candidiasis in autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy. We acknowledge that conclusive evidence for causation is difficult to obtain in complex human diseases. However, our studies clearly document interferonopathy driving mucosal candidiasis with intact IL-17/IL-22 responses in Aire -deficient mice, with strong corroborative evidence in patients.
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- 2021
4. Infections in the monogenic autoimmune syndrome APECED
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Vasileios Oikonomou, Niki M. Moutsopoulos, Timothy J. Break, Sarah L. Gaffen, and Michail S. Lionakis
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animal structures ,T-Lymphocytes ,Immunology ,Autoimmunity ,Candidiasis, Cutaneous ,medicine.disease_cause ,Article ,Sepsis ,Immune Tolerance ,medicine ,Animals ,Humans ,Immunology and Allergy ,Chronic mucocutaneous candidiasis ,Clonal Selection, Antigen-Mediated ,Polyendocrinopathies, Autoimmune ,business.industry ,Autoantibody ,Bacterial pneumonia ,COVID-19 ,medicine.disease ,Autoimmune regulator ,Bronchiectasis ,Pneumonia ,Disease Susceptibility ,Central tolerance ,business ,Transcription Factors - Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by mutations in the Autoimmune Regulator (AIRE) gene, which impair the thymic negative selection of self-reactive T-cells and underlie the development of autoimmunity that targets multiple endocrine and non-endocrine tissues. Beyond autoimmunity, APECED features heightened susceptibility to certain specific infections, which is mediated by anti-cytokine autoantibodies and/or T-cell driven autoimmune tissue injury. These include the 'signature' APECED infection chronic mucocutaneous candidiasis (CMC), but also life-threatening coronavirus disease 2019 (COVID-19) pneumonia, bronchiectasis-associated bacterial pneumonia, and sepsis by encapsulated bacteria. Here we discuss the expanding understanding of the immunological mechanisms that contribute to infection susceptibility in this prototypic syndrome of impaired central tolerance, which provide the foundation for devising improved diagnostic and therapeutic strategies for affected patients.
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- 2021
5. Aberrant type 1 immunity drives susceptibility to mucosal fungal infections
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Julie Alejo, Jigar V. Desai, Ian A. Myles, Niki M. Moutsopoulos, Peter Olbrich, Michail S. Lionakis, Vasileios Oikonomou, Philip M. Murphy, Norma V. Solis, Tilo Freiwald, Scott G. Filler, Heather D. Hickman, Steven M. Holland, Stefania Pittaluga, Sergio M. Pontejo, Teresa Greenwell-Wild, Marc Swidergall, Lindsey B. Rosen, Kevin W. Hoffman, Mark S. Anderson, Timothy J. Break, Vincent M. Bruno, Daniel Chauss, Wint Lwin, Muthulekha Swamydas, Behdad Afzali, Chyi-Chia Richard Lee, David V. Serreze, Daniel Martin, Oliver J. Harrison, Yasmine Belkaid, Jean K. Lim, Andy Renteria, John P. Shannon, Nicolas Dutzan, Monica M. Schmitt, Daniel L. Barber, Luigi D. Notarangelo, Drake W. Williams, Elise M. N. Ferré, Mitsuru Matsumoto, and Nicolas Bouladoux
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Male ,T-Lymphocytes ,medicine.disease_cause ,Autoimmunity ,Mice ,Interferon ,Candida albicans ,Receptors ,2.1 Biological and endogenous factors ,STAT1 ,Aetiology ,Immunologic Surveillance ,Inbred BALB C ,Mucosal ,Multidisciplinary ,Interleukin-17 ,Candidiasis ,Middle Aged ,STAT1 Transcription Factor ,Infectious Diseases ,Female ,Infection ,Genomics and Computational Biology Core ,medicine.drug ,Adult ,Adolescent ,General Science & Technology ,Biology ,Chronic Mucocutaneous ,Autoimmune Disease ,Article ,Interferon-gamma ,Young Adult ,Immune system ,Immunity ,medicine ,Animals ,Humans ,Aged ,Janus Kinases ,Animal ,Interleukins ,Inflammatory and immune system ,Mouth Mucosa ,Emerging Infectious Diseases ,Polyendocrinopathies ,Mucosal immunology ,Disease Models ,Immunology ,STAT protein ,biology.protein ,Janus kinase ,Autoimmune - Abstract
INTRODUCTION: Studies of monogenic diseases have uncovered the importance of immune pathways in human tissue-specific immunity and antimicrobial defense. In particular, human inborn errors of the interleukin-17 (IL-17) receptor signaling pathway and corroborating mouse studies have established the critical contribution of type 17 responses inmucosa-specific fungal surveillance. The yeast Candida albicans is the signature pathogen in autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED), an inherited autoimmune disease caused by loss-of-function mutations in the autoimmune regulator (AIRE) gene. Fungal disease in APECED is limited to chronic mucocutaneous candidiasis (CMC) without dissemination, suggesting a central defect in barrier immunity. RATIONALE: AIRE deficiency impairs central immune tolerance, resulting in the generation of pathogenic autoreactive T cells and autoantibodies directed against many tissue-specific antigens and certain cytokines, including “type 17” cytokines. However, although a majority of APECED patients with CMC have type 17 cytokine–targeted autoantibodies, whether type 17 or other localmucosal immune responses are affected in AIRE deficiency has not been determined. Here, we broadly investigated oral mucosal immune responses both in a model of oropharyngeal candidiasis in Aire(−/−) mice and in a large cohort of APECED patients. RESULTS: Type 17 immune responses at the oralmucosa were unexpectedly intact in mice and humans with AIRE deficiency. To define alternative mechanisms of fungal susceptibility, we investigated Aire(−/−) mice, which exhibited oralmucosa-specific susceptibility to candidiasis without dissemination and controlled experimental challenges with viruses and bacteria normally, thereby phenocopying the infection predisposition observed in APECED patients. Notably, Aire(−/−) CD4(+) and CD8(+) T cells accumulated in increased numbers and displayed an activated and proliferative phenotype within the oral mucosa and were both necessary and sufficient to drive mucosal fungal infection in Aire deficiency. Enhanced production of interferon-γ (IFN-γ) by Aire(−/−) mucosal CD4(+) and CD8(+) T cells resulted in exacerbated IFN-γ/STAT1-mediated responses in the oral mucosa, which promoted IFN-γ–dependent epithelial barrier disruption and mucosal fungal susceptibility. Genetic and pharmacologic inhibition of IFN-γ or JAK-STAT signaling ameliorated mucosal fungal disease in Aire(−/−) mice. Aberrant type 1 responseswere also observed in the oral mucosa of APECED patients. CONCLUSION: We identify a T cell–dependent interferonopathy as a critical local mucosal mechanism underlying CMC in APECED. Although type 17 mucosal immunity is critical for host defense against barrier infection, mucosal type 17 responses were intact in patients with APECED and in a mouse model of the disease. These findings show that, in contrast to the known protective roles of T cells in antifungal host defense, aberrant type 1–associated T cell responses can be detrimental to antifungal mucosal immunity. They also support a paradigm by which exaggerated immunopathology may facilitate susceptibility to mucosal fungal infection by impairing the integrity of the epithelial barrier. Finally, they pave the way for investigating type 1 mucosal responses in other CMC-manifesting diseases and for the prevention and treatment of CMC in APECED patients using FDA-approved therapies that target IFN-γ or JAK-STAT signaling.
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- 2021
6. IL-22 production is regulated by IL-23 during Listeria monocytogenes infection but is not required for bacterial clearance or tissue protection.
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Amy C Graham, Karen D Carr, Amy N Sieve, Mohanalaxmi Indramohan, Timothy J Break, and Rance E Berg
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Medicine ,Science - Abstract
Listeria monocytogenes (LM) is a gram-positive bacterium that is a common contaminant of processed meats and dairy products. In humans, ingestion of LM can result in intracellular infection of the spleen and liver, which can ultimately lead to septicemia, meningitis, and spontaneous abortion. Interleukin (IL)-23 is a cytokine that regulates innate and adaptive immune responses by inducing the production of IL-17A, IL-17F, and IL-22. We have recently demonstrated that the IL-23/IL-17 axis is required for optimal recruitment of neutrophils to the liver, but not the spleen, during LM infection. Furthermore, these cytokines are required for the clearance of LM during systemic infection. In other infectious models, IL-22 induces the secretion of anti-microbial peptides and protects tissues from damage by preventing apoptosis. However, the role of IL-22 has not been thoroughly investigated during LM infection. In the present study, we show that LM induces the production of IL-22 in vivo. Interestingly, IL-23 is required for the production of IL-22 during primary, but not secondary, LM infection. Our findings suggest that IL-22 is not required for clearance of LM during primary or secondary infection, using both systemic and mucosal models of infection. IL-22 is also not required for the protection of LM infected spleens and livers from organ damage. Collectively, these data indicate that IL-22 produced during LM infection must play a role other than clearance of LM or protection of tissues from pathogen- or immune-mediated damage.
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- 2011
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7. PD-L1 up-regulation restrains Th17 cell differentiation in STAT3 loss- and STAT1 gain-of-function patients
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Michail S. Lionakis, Jonathan J. Lyons, Diego B. Lopez, Timothy J. Break, Alexandra F. Freeman, Michael P. O'Connell, John S. Barber, Yongge Zhao, Yuan Zhang, Daniel L. Barber, Monica G. Lawrence, Steven M. Holland, Chi Ma, and Joshua D. Milner
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0301 basic medicine ,Cellular differentiation ,medicine.medical_treatment ,Immunology ,Biology ,03 medical and health sciences ,030104 developmental biology ,Immune system ,Cytokine ,Downregulation and upregulation ,PD-L1 ,Knockout mouse ,biology.protein ,medicine ,Cancer research ,Immunology and Allergy ,Phosphorylation ,SOCS3 - Abstract
Patients with hypomorphic mutations in STAT3 and patients with hypermorphic mutations in STAT1 share several clinical and cellular phenotypes suggesting overlapping pathophysiologic mechanisms. We, therefore, examined cytokine signaling and CD4+ T cell differentiation in these cohorts to characterize common pathways. As expected, differentiation of Th17 cells was impaired in both cohorts. We found that STAT1 was hyperphosphorylated in response to cytokine stimulation in both cohorts and that STAT1-dependent PD-L1 up-regulation—known to inhibit Th17 differentiation in mouse models—was markedly enhanced as well. Overexpression of SOCS3 strongly inhibited phosphorylation of STAT1 and PD-L1 up-regulation, suggesting that diminished SOCS3 expression may lead to the observed effects. Defects in Th17 differentiation could be partially overcome in vitro via PD-L1 inhibition and in a mouse model of STAT3 loss-of-function by crossing them with PD-1 knockout mice. PD-L1 may be a potential therapeutic target in several genetic diseases of immune deficiency affecting cytokine signaling.
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- 2017
8. On-going Mechanical Damage from Mastication Drives Homeostatic Th17 Cell Responses at the Oral Barrier
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Nicolas, Dutzan, Loreto, Abusleme, Hayley, Bridgeman, Teresa, Greenwell-Wild, Tamsin, Zangerle-Murray, Mark E, Fife, Nicolas, Bouladoux, Holly, Linley, Laurie, Brenchley, Kelly, Wemyss, Gloria, Calderon, Bo-Young, Hong, Timothy J, Break, Dawn M E, Bowdish, Michail S, Lionakis, Simon A, Jones, Giorgio, Trinchieri, Patricia I, Diaz, Yasmine, Belkaid, Joanne E, Konkel, and Niki M, Moutsopoulos
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Mice, Knockout ,Microbiota ,Immunology ,Gingiva ,Mouth Mucosa ,Flow Cytometry ,Real-Time Polymerase Chain Reaction ,Mice, Inbred C57BL ,Mice ,Infectious Diseases ,Animals ,Humans ,Mastication ,Th17 Cells ,Immunology and Allergy ,Immunity, Mucosal ,Immunologic Surveillance - Abstract
Immuno-surveillance networks operating at barrier sites are tuned by local tissue cues to ensure effective immunity. Site-specific commensal bacteria provide key signals ensuring host defense in the skin and gut. However, how the oral microbiome and tissue-specific signals balance immunity and regulation at the gingiva, a key oral barrier, remains minimally explored. In contrast to the skin and gut, we demonstrate that gingiva-resident T helper 17 (Th17) cells developed via a commensal colonization-independent mechanism. Accumulation of Th17 cells at the gingiva was driven in response to the physiological barrier damage that occurs during mastication. Physiological mechanical damage, via induction of interleukin 6 (IL-6) from epithelial cells, tailored effector T cell function, promoting increases in gingival Th17 cell numbers. These data highlight that diverse tissue-specific mechanisms govern education of Th17 cell responses and demonstrate that mechanical damage helps define the immune tone of this important oral barrier.
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- 2017
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9. Lymphocyte-driven regional immunopathology in pneumonitis caused by impaired central immune tolerance
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Juan C. Martinez, Kevin P. Fennelly, Ziyue Xu, Sally Hunsberger, Les R. Folio, Kenneth N. Olivier, Chyi-Chia Richard Lee, Michael Allgäuer, Douglas B. Kuhns, Julie E. Niemela, Theo Heller, Michail S. Lionakis, Gary Kleiner, Peter D. Burbelo, Kerry Dobbs, Elise M. N. Ferré, Dirk Darnell, Jean K. Lim, Thomas A. Fleisher, Arun Rajan, Amy P. Hsu, Martin Bozzola, Kevin W. Hoffman, Megan A. Cooper, Muthulekha Swamydas, Dakai Jin, Laquita N. Snow, Robin R. Deterding, Anamaria Bondici, Karen K. Winer, Timothy J. Break, Daniel J. Mollura, Samar Ojaimi, David E. Kleiner, Wenjuan Gu, Luigi D. Notarangelo, and Steven M. Holland
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Adult ,Male ,animal structures ,Adolescent ,T-Lymphocytes ,Autoimmunity ,medicine.disease_cause ,Article ,Autoimmune Diseases ,Pulmonary function testing ,Immune tolerance ,Young Adult ,Immunopathology ,Humans ,Medicine ,Lymphocytes ,Prospective Studies ,Child ,Autoantibodies ,Pneumonitis ,B-Lymphocytes ,Lung ,medicine.diagnostic_test ,business.industry ,Infant, Newborn ,Autoantibody ,Infant ,Pneumonia ,General Medicine ,Middle Aged ,respiratory system ,medicine.disease ,Bronchoalveolar lavage ,medicine.anatomical_structure ,Child, Preschool ,Immunology ,Female ,business - Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a monogenic disorder caused by AIRE mutations, presents with several autoimmune diseases. Among these, endocrine organ failure is widely recognized, but the prevalence, immunopathogenesis, and treatment of non-endocrine manifestations such as pneumonitis remain poorly characterized. We enrolled 50 patients with APECED in a prospective observational study and comprehensively examined their clinical and radiographic findings, performed pulmonary function tests, and analyzed immunological characteristics in blood, bronchoalveolar lavage fluid, and endobronchial and lung biopsies. Pneumonitis was found in >40% of our patients, presented early in life, was misdiagnosed despite chronic respiratory symptoms and accompanying radiographic and pulmonary function abnormalities, and caused hypoxemic respiratory failure and death. Autoantibodies against BPIFB1 and KCNRG and the homozygous c.967_979del13 AIRE mutation are associated with pneumonitis development. APECED pneumonitis features compartmentalized immunopathology, with accumulation of activated neutrophils in the airways and lymphocytic infiltration in intraepithelial, submucosal, peribronchiolar, and interstitial areas. Beyond APECED, we extend these observations to lung disease seen in other conditions with secondary AIRE deficiency (thymoma and RAG deficiency). Aire-deficient mice had similar compartmentalized cellular immune responses in the airways and lung tissue, which was ameliorated by deficiency of T and B lymphocytes. Accordingly, T and B lymphocyte-directed immunomodulation controlled symptoms and radiographic abnormalities and improved pulmonary function in patients with APECED pneumonitis. Collectively, our findings unveil lung autoimmunity as a common, early, and unrecognized manifestation of APECED and provide insights into the immunopathogenesis and treatment of pulmonary autoimmunity associated with impaired central immune tolerance.
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- 2019
10. Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis
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Daniel H. Kaplan, Partha S. Biswas, Felix E. Y. Aggor, Michail S. Lionakis, Natasha Whibley, Wei Shan, Jay K. Kolls, Vincent M. Bruno, Rachel D. Bailey, Bianca M. Coleman, Amol C. Shetty, Scott K. Durum, Carrie McCracken, Sarah L. Gaffen, Julian R. Naglik, Giraldina Trevejo-Nunez, and Timothy J. Break
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0301 basic medicine ,Male ,STAT3 Transcription Factor ,Immunology ,Biology ,Oropharyngeal Candidiasis ,Article ,Interleukin 22 ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Immunity ,Candidiasis, Oral ,Candida albicans ,medicine ,Animals ,Immunology and Allergy ,Oral mucosa ,STAT3 ,Mice, Knockout ,Interleukins ,Interleukin-17 ,Mouth Mucosa ,Epithelial Cells ,General Medicine ,biology.organism_classification ,Epithelium ,Mice, Inbred C57BL ,stomatognathic diseases ,030104 developmental biology ,medicine.anatomical_structure ,STAT protein ,Cancer research ,biology.protein ,Female ,030215 immunology ,Signal Transduction - Abstract
Oropharyngeal candidiasis (OPC) is an opportunistic infection of the oral mucosa caused by the commensal fungus Candida albicans. IL-17 and IL-22 both mediate antifungal immunity yet activate distinct downstream signaling pathways. While much is known about IL-17-dependent immunity in OPC, the activities of IL-22 are less well delineated. We show that induction of Il22 is independent of Dectin-1, CARD9 and aryl hydrocarbon receptor (AhR) and is driven by IL-23 and the C. albicans pore forming peptide candidalysin. Despite similar induction requirements and cellular sources, IL-22 and IL-17 function non-redundantly during OPC and exert opposing roles in neutrophil recruitment. The IL-22 and IL-17 receptors are required in anatomically distinct locations; loss of IL-22RA1 in the oral basal epithelial layer (BEL) but not the suprabasal epithelial layer (SEL) causes susceptibility to OPC, whereas IL-17RA is needed in the SEL. Our data reveal that IL-22 is a major activator of STAT3 in the BEL during OPC. Moreover, loss of STAT3 in the BEL but not the SEL renders mice susceptible to OPC. Transcriptional profiling of RNASeq data linked IL-22/STAT3 to oral epithelial cell proliferation and survival, but also, unexpectedly, to driving an IL-17 gene signature. We show that IL-22 acts on the BEL to replenish the IL-17RA-expressing SEL, thereby restoring the ability of the oral epithelium to respond to IL-17. Consequently, IL-22 signaling in BEL ‘licenses’ IL-17R signaling in the oral epithelium, revealing spatially distinct yet cooperative activities of IL-22 and IL-17 in oral candidiasis. This work also suggests that oral thrush in Jobs’ syndrome patients may be caused by STAT3 impairments in the oral epithelium, not just Th17 cells.
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- 2020
11. Critical Adverse Impact of IL-6 in Acute Pneumovirus Infection
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Timothy J. Break, Michelle Ma, Helene F. Rosenberg, Caroline M. Percopo, Todd A. Brenner, Karen Laky, and Julia O. Krumholz
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medicine.medical_treatment ,Immunology ,Respiratory System ,Inflammation ,Virus ,Article ,Proinflammatory cytokine ,Pathogenesis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,parasitic diseases ,Macrophages, Alveolar ,medicine ,Immunology and Allergy ,Animals ,Pneumovirus Infections ,Interleukin 6 ,Pathogen ,Lung ,Mice, Knockout ,Mice, Inbred BALB C ,biology ,business.industry ,Interleukin-6 ,Probiotics ,Recombinant Proteins ,Mice, Inbred C57BL ,Cytokine ,biology.protein ,Respiratory virus ,Murine pneumonia virus ,medicine.symptom ,business ,030215 immunology ,Lactobacillus plantarum - Abstract
Severe respiratory virus infections feature robust local host responses that contribute to disease severity. Immunomodulatory strategies that limit virus-induced inflammation may be of critical importance, notably in the absence of antiviral vaccines. In this study, we examined the role of the pleiotropic cytokine IL-6 in acute infection with pneumonia virus of mice (PVM), a natural rodent pathogen that is related to respiratory syncytial virus and that generates local inflammation as a feature of severe infection. In contrast to Influenza A, PVM is substantially less lethal in IL-6−/− mice than it is in wild-type, a finding associated with diminished neutrophil recruitment and reduced fluid accumulation in lung tissue. Ly6Chi proinflammatory monocytes are recruited in response to PVM via a CCR2-dependent mechanism, but they are not a major source of IL-6 nor do they contribute to lethal sequelae of infection. By contrast, alveolar macrophages are readily infected with PVM in vivo; ablation of alveolar macrophages results in prolonged survival in association with a reduction in virus-induced IL-6. Finally, as shown previously, administration of immunobiotic Lactobacillus plantarum to the respiratory tracts of PVM-infected mice promoted survival in association with diminished levels of IL-6. We demonstrated in this study that IL-6 suppression is a critical feature of the protective mechanism; PVM-infected IL-6−/− mice responded to low doses of L. plantarum, and administration of IL-6 overcame L. plantarum–mediated protection in PVM-infected wild-type mice. Taken together, these results connect the actions of IL-6 to PVM pathogenesis and suggest cytokine blockade as a potential therapeutic modality in severe infection.
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- 2018
12. VT-1598 inhibits the in vitro growth of mucosal Candida strains and protects against fluconazole-susceptible and -resistant oral candidiasis in IL-17 signalling-deficient mice
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Christopher M. Yates, Christina M. Henderson, Michail S. Lionakis, David S. Perlin, Robert J. Schotzinger, Elise M. N. Ferré, Kelley R. Healey, Edward P. Garvey, Mukil Natarajan, Oren J Cohen, Adrian M. Zelazny, Ulrich Siebenlist, Jigar V. Desai, and Timothy J. Break
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0301 basic medicine ,Microbiology (medical) ,Antifungal Agents ,Pyridines ,030106 microbiology ,Administration, Oral ,Tetrazoles ,Drug resistance ,Microbial Sensitivity Tests ,Microbiology ,03 medical and health sciences ,Mice ,Tongue ,Oral administration ,In vivo ,Candidiasis, Oral ,Drug Resistance, Fungal ,medicine ,Animals ,Humans ,Pharmacology (medical) ,Chronic mucocutaneous candidiasis ,Fluconazole ,Original Research ,Candida ,Pharmacology ,Mice, Knockout ,business.industry ,Interleukin-17 ,Mucous membrane ,medicine.disease ,Corpus albicans ,030104 developmental biology ,Infectious Diseases ,medicine.anatomical_structure ,business ,medicine.drug - Abstract
Background Chronic mucocutaneous candidiasis (CMC) treatment often induces drug resistance, posing long-term challenges. A novel broad-spectrum fungal CYP51 inhibitor, VT-1598, specifically targets fungal CYP51, but not human CYP enzymes. Objectives To determine the efficacy of VT-1598 in the treatment of oral Candida infection caused by fluconazole-susceptible and -resistant clinical isolates. Methods The MICs of VT-1598 and fluconazole for 28 Candida isolates recovered from patients with inherited CMC were determined using CLSI M27-A3 and M27-S4 guidelines. Plasma and tongue VT-1598 or fluconazole concentrations were measured in mice following oral administration to determine tissue distribution. Tongue fungal load was determined in IL-17 signalling-deficient Act1-/- mice following sublingual Candida albicans infection and oral treatment with fluconazole or VT-1598. Results Among the 28 Candida isolates, 10 (36%) had fluconazole MICs of ≥4 mg/L, whereas VT-1598 demonstrated potent in vitro activity against all isolates (MIC90, 0.125 mg/L). After oral administration, VT-1598 levels in mouse plasma and tongue were significantly greater than those of fluconazole. In vivo, VT-1598 exhibited significant efficacy against fluconazole-susceptible and -resistant C. albicans, even at low drug doses. Furthermore, after a 10 day washout period, tongue fungal burdens in fluconazole-treated mice returned to vehicle control levels, whereas, in contrast, they were undetectable in mice treated with VT-1598. Conclusions VT-1598 effectively controls in vitro growth of mucosally derived Candida clinical isolates, including fluconazole-resistant strains. In vivo, VT-1598 eliminates C. albicans, even after a long washout period or at low doses. Therefore, VT-1598 is a promising drug candidate that may significantly improve treatment options for CMC patients.
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- 2018
13. VT-1161 protects mice against oropharyngeal candidiasis caused by fluconazole-susceptible and -resistant Candida albicans
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Elise M. N. Ferré, Adrian M. Zelazny, Michail S. Lionakis, William J. Hoekstra, Timothy J. Break, Edward P. Garvey, Ulrich Siebenlist, Jigar V. Desai, Mukil Natarajan, Christina M. Henderson, and Robert J. Schotzinger
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0301 basic medicine ,Microbiology (medical) ,Antifungal Agents ,Pyridines ,030106 microbiology ,Tetrazoles ,Candida glabrata ,Drug resistance ,Microbial Sensitivity Tests ,Oropharyngeal Candidiasis ,Microbiology ,03 medical and health sciences ,Mice ,In vivo ,Immunity ,Candidiasis, Oral ,Drug Resistance, Fungal ,Candida albicans ,medicine ,Animals ,Humans ,Pharmacology (medical) ,Fluconazole ,Adaptor Proteins, Signal Transducing ,Original Research ,Pharmacology ,Mice, Knockout ,biology ,business.industry ,biology.organism_classification ,Corpus albicans ,030104 developmental biology ,Infectious Diseases ,business ,medicine.drug - Abstract
Background Candida albicans, the most common human fungal pathogen, causes chronic mucosal infections in patients with inborn errors of IL-17 immunity that rely heavily on chronic, often lifelong, azole antifungal agents for treatment. However, a rise in azole resistance has predicated a need for developing new antifungal drugs. Objectives To test the in vitro and in vivo efficacy of VT-1161 and VT-1129 in the treatment of oropharyngeal candidiasis with azole-susceptible or -resistant C. albicans strains. Methods MICs of VT-1161, VT-1129 and nine licensed antifungal drugs were determined for 31 Candida clinical isolates. The drug concentrations in mouse serum and tongues were measured following oral administration. IL-17-signalling-deficient Act1-/- mice were infected with fluconazole-susceptible or fluconazole-resistant C. albicans strains, and the amount of mucosal fungal burden was determined after fluconazole or VT-1161 treatment. Results Fourteen isolates (45%) were not fluconazole susceptible (MIC ≥4 mg/L). VT-1161 and VT-1129 showed significant in vitro activity against the majority of the 31 mucosal clinical isolates (MIC50 0.03 and 0.06 mg/L, respectively), including Candida glabrata (MIC50, 0.125 and 0.25 mg/L, respectively). After oral doses, VT-1161 and VT-1129 concentrations in mouse serum and tongues were well above their MIC50 values. VT-1161 was highly effective as treatment of both fluconazole-susceptible and -resistant oropharyngeal candidiasis in Act1-/- mice. Conclusions VT-1129 and VT-1161 exhibit significant in vitro activity against Candida strains, including fluconazole-resistant C. albicans and C. glabrata. VT-1161 administration in mice results in significant mucosal drug accumulation and eradicates infection caused by fluconazole-susceptible and -resistant Candida strains.
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- 2017
14. Inflammatory Monocyte Recruitment Is Regulated by Interleukin-23 during Systemic Bacterial Infection
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Timothy J. Break, Amy N. Sieve, Rance E. Berg, and Mohanalaxmi Indramohan
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Male ,Chemokine ,Immunology ,Spleen ,Inflammation ,Biology ,CCL2 ,Nitric Oxide ,CCL7 ,Interleukin-23 ,Microbiology ,Monocytes ,Proinflammatory cytokine ,Mice ,medicine ,Animals ,Humans ,Listeriosis ,Mice, Knockout ,Host Response and Inflammation ,Innate immune system ,Tumor Necrosis Factor-alpha ,Listeria monocytogenes ,Immunity, Innate ,Mice, Inbred C57BL ,Infectious Diseases ,medicine.anatomical_structure ,Interleukin-23 Subunit p19 ,biology.protein ,Female ,Parasitology ,Tumor necrosis factor alpha ,medicine.symptom - Abstract
Listeria monocytogenes is a Gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals and spontaneous abortion in pregnant women. The innate immune response against L. monocytogenes is primarily mediated by neutrophils and monocytes. Interleukin-23 (IL-23) is an important proinflammatory cytokine well known for its role in neutrophil recruitment in various infectious and autoimmune diseases. We have previously shown that IL-23 is required for host resistance against L. monocytogenes and for neutrophil recruitment to the liver, but not the spleen, during infection. Despite efficient neutrophil recruitment to the spleen, IL-23p19 knockout (KO) mice have an increased bacterial burden in this organ, suggesting that IL-23 may regulate the recruitment/function of another cell type to the spleen. In this study, we show that specific depletion of neutrophils abrogated the differences in bacterial burdens in the livers but not the spleens of C57BL/6 (B6) and IL-23p19 KO mice. Interestingly, L. monocytogenes -infected IL-23p19 KO mice had fewer monocytes in the spleen than B6 mice, as well as a reduction in the monocyte-recruiting chemokines CCL2 and CCL7. Additionally, the overall concentrations of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO • ), as well as the percentages and total numbers of monocytes producing TNF-α and NO • , were reduced in IL-23p19 KO mice compared to levels in B6 mice, leading to increased bacterial burdens in the spleens of L. monocytogenes -infected IL-23p19 KO mice. Collectively, our data establish that IL-23 is required for the optimal recruitment of TNF-α- and NO • -producing inflammatory monocytes, thus revealing a novel mechanism by which this proinflammatory cytokine provides protection against bacterial infection.
- Published
- 2012
15. PD-L1 up-regulation restrains Th17 cell differentiation in
- Author
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Yuan, Zhang, Chi A, Ma, Monica G, Lawrence, Timothy J, Break, Michael P, O'Connell, Jonathan J, Lyons, Diego B, López, John S, Barber, Yongge, Zhao, Daniel L, Barber, Alexandra F, Freeman, Steven M, Holland, Michail S, Lionakis, and Joshua D, Milner
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Adult ,Male ,Mice, Knockout ,STAT3 Transcription Factor ,Adolescent ,Interleukins ,Immunologic Deficiency Syndromes ,Brief Definitive Report ,Cell Differentiation ,Middle Aged ,B7-H1 Antigen ,Up-Regulation ,Mice ,Young Adult ,STAT1 Transcription Factor ,Suppressor of Cytokine Signaling 3 Protein ,Animals ,Cytokines ,Humans ,Th17 Cells ,Female ,Child ,Research Articles ,Signal Transduction - Abstract
Zhang et al. show that hyperphosphorylated STAT1 in patients with STAT1 gain-of-function and STAT3 loss-of-function is caused by impaired SOCS3 expression and leads to upregulation of PD-L1 and defects in Th17 cell differentiation that underlie susceptibility to chronic mucocutaneous candidiasis in these patients., Patients with hypomorphic mutations in STAT3 and patients with hypermorphic mutations in STAT1 share several clinical and cellular phenotypes suggesting overlapping pathophysiologic mechanisms. We, therefore, examined cytokine signaling and CD4+ T cell differentiation in these cohorts to characterize common pathways. As expected, differentiation of Th17 cells was impaired in both cohorts. We found that STAT1 was hyperphosphorylated in response to cytokine stimulation in both cohorts and that STAT1-dependent PD-L1 up-regulation—known to inhibit Th17 differentiation in mouse models—was markedly enhanced as well. Overexpression of SOCS3 strongly inhibited phosphorylation of STAT1 and PD-L1 up-regulation, suggesting that diminished SOCS3 expression may lead to the observed effects. Defects in Th17 differentiation could be partially overcome in vitro via PD-L1 inhibition and in a mouse model of STAT3 loss-of-function by crossing them with PD-1 knockout mice. PD-L1 may be a potential therapeutic target in several genetic diseases of immune deficiency affecting cytokine signaling.
- Published
- 2016
16. Redefined clinical features and diagnostic criteria in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
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Kimberly Romito, Martha Quezado, Kelly D. Stone, Chyi-Chia Richard Lee, Mukil Natarajan, Jeffrey B. Kopp, Muthulekha Swamydas, Wenjuan Gu, Steven M. Holland, Sally Hunsberger, David M. Chascsa, Julie E. Niemela, Cathleen Frein, Rachel Bishop, Morgan Similuk, Heidi H. Kong, Sarah K. Browne, Karen K. Winer, Theo Heller, Joseph Monsale, Shakuntala Rampertaap, Timothy J. Break, David M. Lang, Thomas L. Simcox, Elise M. N. Ferré, Thomas A. Fleisher, Gulbu Uzel, Lynne Yockey, Kenneth N. Olivier, Ilias Alevizos, Ariane Soldatos, Katherine R. Calvo, Jennifer Adjemian, Amanda L. Collar, Niki M. Moutsopoulos, David E. Kleiner, Stacey R. Rose, Peter D. Burbelo, Lindsey B. Rosen, Michail S. Lionakis, Amy P. Hsu, Angela Pham, Sergio D. Rosenzweig, and Anamaria Bondici
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0301 basic medicine ,medicine.medical_specialty ,business.industry ,Autoantibody ,General Medicine ,Autoimmune polyendocrinopathy ,medicine.disease ,Compound heterozygosity ,Dermatology ,03 medical and health sciences ,030104 developmental biology ,Hypoparathyroidism ,Immunology ,Adrenal insufficiency ,medicine ,Primary immunodeficiency ,Chronic mucocutaneous candidiasis ,business ,Research Article ,Pneumonitis - Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare primary immunodeficiency disorder typically caused by homozygous AIRE mutations. It classically presents with chronic mucocutaneous candidiasis and autoimmunity that primarily targets endocrine tissues; hypoparathyroidism and adrenal insufficiency are most common. Developing any two of these classic triad manifestations establishes the diagnosis. Although widely recognized in Europe, where nonendocrine autoimmune manifestations are uncommon, APECED is less defined in patients from the Western Hemisphere. We enrolled 35 consecutive American APECED patients (33 from the US) in a prospective observational natural history study and systematically examined their genetic, clinical, autoantibody, and immunological characteristics. Most patients were compound heterozygous; the most common AIRE mutation was c.967_979del13. All but one patient had anti–IFN-ω autoantibodies, including 4 of 5 patients without biallelic AIRE mutations. Urticarial eruption, hepatitis, gastritis, intestinal dysfunction, pneumonitis, and Sjögren’s-like syndrome, uncommon entities in European APECED cohorts, affected 40%–80% of American cases. Development of a classic diagnostic dyad was delayed at mean 7.38 years. Eighty percent of patients developed a median of 3 non-triad manifestations before a diagnostic dyad. Only 20% of patients had their first two manifestations among the classic triad. Urticarial eruption, intestinal dysfunction, and enamel hypoplasia were prominent among early manifestations. Patients exhibited expanded peripheral CD4+ T cells and CD21loCD38lo B lymphocytes. In summary, American APECED patients develop a diverse syndrome, with dramatic enrichment in organ-specific nonendocrine manifestations starting early in life, compared with European patients. Incorporation of these new manifestations into American diagnostic criteria would accelerate diagnosis by approximately 4 years and potentially prevent life-threatening endocrine complications.
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- 2016
17. CD8+ T cells produce a dialyzable antigen-specific activator of dendritic cells
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Sandip K. Datta, Paul J. Gardina, Danial Saleem, Michail S. Lionakis, Charles H. Kirkpatrick, Steven M. Holland, Timothy G. Myers, Glenn Nardone, Ian A. Myles, Jensen D. Reckhow, Lisa R. Olano, Timothy J. Break, and Ming Zhao
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0301 basic medicine ,CD4-Positive T-Lymphocytes ,Cellular immunity ,Immunology ,Receptors, Antigen, T-Cell ,chemical and pharmacologic phenomena ,HIV Infections ,Transfer Factor ,CD8-Positive T-Lymphocytes ,Lymphocyte Activation ,Models, Biological ,03 medical and health sciences ,Epitopes ,Leprosy ,Immunology and Allergy ,Cytotoxic T cell ,Animals ,Humans ,Antigens ,Antigen-presenting cell ,Mice, Inbred BALB C ,CD40 ,biology ,Inflammation, Extracellular Mediators, & Effector Molecules ,T-cell receptor ,Interleukin-17 ,S100 Proteins ,Immunity ,Cell Biology ,Dendritic Cells ,Natural killer T cell ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,biology.protein ,Interleukin 12 ,Dialysis ,Immunologic Memory ,CD8 ,Spleen - Abstract
Cellular lysates from PPD+ donors have been reported to transfer tuberculin reactivity to naïve recipients, but not diphtheria reactivity, and vice versa. A historically controversial topic, the terms “transfer factor” and “DLE” were used to characterize the reactivity-transferring properties of lysates. Intrigued by these reported phenomena, we found that the cellular extract derived from antigen-specific memory CD8+ T cells induces IL-6 from antigen-matched APCs. This ultimately elicits IL-17 from bystander memory CD8+ T cells. We have identified that dialyzable peptide sequences, S100a9, and the TCR β chain from CD8+ T cells contribute to the molecular nature of this activity. We further show that extracts from antigen-targeted T cells enhance immunity to Staphylococcus aureus and Candida albicans. These effects are sensitive to immunization protocols and extraction methodology in ways that may explain past discrepancies in the reproducibility of passive cellular immunity.
- Published
- 2016
18. Extracellular Superoxide Dismutase Enhances Recruitment of Immature Neutrophils to the Liver
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Timothy J. Break, Mohanalaxmi Indramohan, Ladislav Dory, Alexandra R Witter, Rance E. Berg, and Mark E. Mummert
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0301 basic medicine ,Neutrophils ,Immunology ,Congenic ,Inflammation ,Biology ,medicine.disease_cause ,Microbiology ,Gene Expression Regulation, Enzymologic ,03 medical and health sciences ,Mice ,Listeria monocytogenes ,medicine ,Animals ,chemistry.chemical_classification ,Reactive oxygen species ,Host Response and Inflammation ,Innate immune system ,Superoxide Dismutase ,Respiratory burst ,030104 developmental biology ,Infectious Diseases ,chemistry ,Liver ,Myeloperoxidase ,biology.protein ,Parasitology ,medicine.symptom ,Chemokines ,Intracellular - Abstract
Listeria monocytogenes is a Gram-positive intracellular pathogen that causes spontaneous abortion in pregnant women, as well as septicemia, meningitis, and gastroenteritis, primarily in immunocompromised individuals. Although L. monocytogenes can usually be effectively treated with antibiotics, there is still around a 25% mortality rate with individuals who develop clinical listeriosis. Neutrophils are innate immune cells required for the clearance of pathogenic organisms, including L. monocytogenes . The diverse roles of neutrophils during both infectious and noninfectious inflammation have recently gained much attention. However, the impact of reactive oxygen species, and the enzymes that control their production, on neutrophil recruitment and function is not well understood. Using congenic mice with varying levels of extracellular superoxide dismutase (ecSOD) activity, we have recently shown that the presence of ecSOD decreases clearance of L. monocytogenes while increasing the recruitment of neutrophils that are not protective in the liver. The data presented here show that ecSOD activity does not lead to a cell-intrinsic increase in neutrophil-homing potential or a decrease in protection against L. monocytogenes . Instead, ecSOD activity enhances the production of neutrophil-attracting factors and protects hyaluronic acid (HA) from damage. Furthermore, neutrophils from the livers of ecSOD-expressing mice have decreased intracellular and surface-bound myeloperoxidase, are less capable of killing phagocytosed L. monocytogenes , and have decreased oxidative burst. Collectively, our data reveal that ecSOD activity modulates neutrophil recruitment and function in a cell-extrinsic fashion, highlighting the importance of the enzyme in protecting tissues from oxidative damage.
- Published
- 2016
19. Batf3-dependent CD103+ dendritic cell accumulation is dispensable for mucosal and systemic antifungal host defense
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Michail S. Lionakis, Kevin W. Hoffman, Chyi-Chia Richard Lee, Muthulekha Swamydas, Jean K. Lim, and Timothy J. Break
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0301 basic medicine ,Microbiology (medical) ,030106 microbiology ,Immunology ,chemical and pharmacologic phenomena ,Biology ,Microbiology ,Oropharyngeal Candidiasis ,03 medical and health sciences ,Immune system ,Immunity ,Candida albicans ,medicine ,Humans ,Innate immune system ,Virulence ,Brief Report ,Candidiasis ,hemic and immune systems ,Dendritic cell ,medicine.disease ,030104 developmental biology ,Infectious Diseases ,Integrin alpha M ,Interleukin 12 ,biology.protein ,Parasitology ,Systemic candidiasis - Abstract
Dendritic cells (DCs) are critical for defense against a variety of pathogens and the formation of adaptive immune responses. The transcription factor Batf3 is critical for the development of CD103(+)CD11b(-) DCs, which promote IL-12-dependent protective immunity during viral and parasitic infections, dampen Th2 immunity during helminthic infection, and exert detrimental effects during bacterial infection. Whether CD103(+) DCs modulate immunity during systemic or mucosal fungal disease remains unknown. Herein, we report that Batf3 is critical for accumulation of CD103(+) DCs in the kidney and tongue at steady state, for their expansion during systemic and oropharyngeal candidiasis, and for tissue-specific production of IL-12 in kidney but not tongue during systemic and oropharyngeal candidiasis, respectively. Importantly, deficiency of CD103(+) DCs does not impair survival or fungal clearance during systemic or oropharyngeal candidiasis, indicating that Batf3-dependent CD103(+) DC accumulation mediates pathogen- and tissue-specific immune effects.
- Published
- 2016
20. VT-1129 and VT-1161 have in vitro activity against Candida isolates from patients with chronic mucocutaneous candidiasis
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Adrian M. Zelazny, Michail S. Lionakis, Edward P. Garvey, William J. Hoekstra, Timothy J. Break, Robert J. Schotzinger, Mukil Natarajan, Jigar V. Desai, and Christina M. Henderson
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,business.industry ,030106 microbiology ,medicine.disease ,Dermatology ,In vitro ,03 medical and health sciences ,Infectious Diseases ,Oncology ,Medicine ,Chronic mucocutaneous candidiasis ,business - Published
- 2016
21. CXCR1-mediated neutrophil degranulation and fungal killing promote Candida clearance and host survival
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Michail S. Lionakis, Mihai G. Netea, Philip M. Murphy, Muthulekha Swamydas, Brett G. Fischer, Barbara D. Alexander, Martin Jaeger, Nathaniel M. Green, Carlos A. Rodriguez, Bart Jan Kullberg, Timothy J. Break, Melissa D. Johnson, Amanda L. Collar, John R. Perfect, Chyi-Chia Richard Lee, Jean K. Lim, and Ji-Liang Gao
- Subjects
0301 basic medicine ,Chemokine CXCL5 ,Neutrophils ,Hyphae ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,Biology ,Neutropenia ,Kidney ,Ligands ,Polymorphism, Single Nucleotide ,Cell Degranulation ,Article ,Receptors, Interleukin-8A ,Microbiology ,Mice ,03 medical and health sciences ,Immune system ,medicine ,Animals ,Humans ,CXC chemokine receptors ,Alleles ,Candida ,Microbial Viability ,Innate immune system ,Candidiasis ,Degranulation ,General Medicine ,Disseminated Candidiasis ,medicine.disease ,Survival Analysis ,Tissue Donors ,3. Good health ,Disease Models, Animal ,030104 developmental biology ,lnfectious Diseases and Global Health Radboud Institute for Health Sciences [Radboudumc 4] ,Host-Pathogen Interactions ,Immunology ,Neutrophil degranulation ,Mutant Proteins ,Systemic candidiasis - Abstract
Item does not contain fulltext Systemic Candida albicans infection causes high morbidity and mortality and is now the leading cause of nosocomial bloodstream infection in the United States. Neutropenia is a major risk factor for poor outcome in infected patients; however, the molecular factors that mediate neutrophil trafficking and effector function during infection are poorly defined. Using a mouse model of systemic candidiasis, we found that the neutrophil-selective CXC chemokine receptor Cxcr1 and its ligand, Cxcl5, are highly induced in the Candida-infected kidney, the target organ in the model. To investigate the role of Cxcr1 in antifungal host defense in vivo, we generated Cxcr1(-/-) mice and analyzed their immune response to Candida. Mice lacking Cxcr1 exhibited decreased survival with enhanced Candida growth in the kidney and renal failure. Increased susceptibility of Cxcr1(-/-) mice to systemic candidiasis was not due to impaired neutrophil trafficking from the blood into the infected kidney but was the result of defective killing of the fungus by neutrophils that exhibited a cell-intrinsic decrease in degranulation. In humans, the mutant CXCR1 allele CXCR1-T276 results in impaired neutrophil degranulation and fungal killing and was associated with increased risk of disseminated candidiasis in infected patients. Together, our data demonstrate a biological function for mouse Cxcr1 in vivo and indicate that CXCR1-dependent neutrophil effector function is a critical innate protective mechanism of fungal clearance and host survival in systemic candidiasis.
- Published
- 2016
22. Allele-Specific Effects of ecSOD in Bacterial Infection
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Ladislav Dory, Sujung Jun, Byung Jin Kim, Rance E. Berg, Harlan P. Jones, and Timothy J. Break
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chemistry.chemical_classification ,Reactive oxygen species ,SOD3 ,Superoxide ,Nitrotyrosine ,Congenic ,Spleen ,Biology ,medicine.disease_cause ,Biochemistry ,Microbiology ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Physiology (medical) ,Extracellular ,medicine ,Oxidative stress - Abstract
Extracellular superoxide dismutase (ecSOD) is the only enzyme that scavenges superoxide specifically in the extracellular compartment. Recent studies indicate that ecSOD is important for protecting tissues from oxidative damage by dampening immune responses but imply that the immunosuppressive effect may be counterproductive during acute infections against invading microorganisms. Although many studies have revealed the importance of the reactive oxygen species (ROS) in bacterial killing, the role of ecSOD during bacterial infection is beginning to be understood. In this study, Listeria monogytogene and Streptococcus pneumoniae were used to infect mice expressing various level of ecSOD: congenic mice sod3129 (relatively high), sod3wt and sod3 KO mice (none). ecSOD expression is inversely related to bacterial clearance. On the other hand, bacterial infection significantly decreases ecSOD activity in infected tissues, while the protein levels are not affected. The induction of iNOS expression is significantly lower in the liver and spleen of sod3 KO mice when compared to those of congenic sod3129 and sod3wt mice. The NO levels in liver and spleen were independent on the level of iNOS induction. However, the NO levels in plasma were significantly induced regardless of the genotype. The oxidative stress levels measured by nitrotyrosine immunoblotting in plasma and liver were significantly higher in sod3 KO mice compared to the congenic sod3129 and sod3wt mice. In conclusion, high ecSOD expression impairs the bacterial clearance and the infection diminishes the ecSOD activity. These data provide a potential target for therapeutic intervention for acute infection.
- Published
- 2017
23. Mononuclear Phagocyte-Mediated Antifungal Immunity: The Role of Chemotactic Receptors and Ligands
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Timothy J. Break, Muthulekha Swamydas, and Michail S. Lionakis
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CD4-Positive T-Lymphocytes ,Neutrophils ,Biology ,Aspergillosis ,Article ,Monocytes ,Microbiology ,Receptors, G-Protein-Coupled ,Cellular and Molecular Neuroscience ,Immunity ,medicine ,Humans ,Receptor ,Molecular Biology ,Mononuclear Phagocyte System ,Pharmacology ,Chemotactic Factors ,Effector ,Macrophages ,Candidiasis ,Hematopoietic stem cell ,Chemotaxis ,Cell Biology ,Mononuclear phagocyte system ,Cryptococcosis ,Dendritic Cells ,medicine.disease ,Immunity, Innate ,medicine.anatomical_structure ,Mycoses ,Immunology ,Molecular Medicine - Abstract
Over the past two decades, fungal infections have emerged as significant causes of morbidity and mortality in patients with hematological malignancies, hematopoietic stem cell or solid organ transplantation and acquired immunodeficiency syndrome. Besides neutrophils and CD4(+) T lymphocytes, which have long been known to play an indispensable role in promoting protective antifungal immunity, mononuclear phagocytes are now being increasingly recognized as critical mediators of host defense against fungi. Thus, a recent surge of research studies has focused on understanding the mechanisms by which resident and recruited monocytes, macrophages and dendritic cells accumulate and become activated at the sites of fungal infection. Herein, we critically review how a variety of G-protein coupled chemoattractant receptors and their ligands mediate mononuclear phagocyte recruitment and effector function during infection by the most common human fungal pathogens.
- Published
- 2015
24. CX3CR1 is dispensable for control of mucosal Candida albicans infections in mice and humans
- Author
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Chyi-Chia Richard Lee, Timothy J. Break, Norma V. Solis, Michail S. Lionakis, Martin Jaeger, Jack D. Sobel, Mihai G. Netea, Scott G. Filler, Carlos A. Rodriguez, Jean K. Lim, and Deepe, GS
- Subjects
lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,Gene Expression ,Inbred C57BL ,Interleukin-23 ,Medical and Health Sciences ,Mice ,Candida albicans ,Receptors ,CX3CR1 ,2.1 Biological and endogenous factors ,Aetiology ,Mice, Knockout ,education.field_of_study ,Interleukin-17 ,Candidiasis ,Interleukin ,Biological Sciences ,Corpus albicans ,3. Good health ,Infectious Diseases ,Chemokine ,Vagina ,Host-Pathogen Interactions ,Receptors, Chemokine ,Female ,Infection ,Vulvovaginal ,Knockout ,Immunology ,Population ,CX3C Chemokine Receptor 1 ,Opportunistic Infections ,Biology ,Microbiology ,Immune system ,Tongue ,medicine ,Animals ,Humans ,education ,Candidiasis, Vulvovaginal ,Alleles ,Agricultural and Veterinary Sciences ,Animal ,Interleukins ,medicine.disease ,biology.organism_classification ,Mice, Inbred C57BL ,Gastrointestinal Tract ,Disease Models, Animal ,Disease Models ,Recurrent vulvovaginal candidiasis ,Parasitology ,Systemic candidiasis ,Fungal and Parasitic Infections ,Digestive Diseases - Abstract
Candida albicans is part of the normal commensal microbiota of mucosal surfaces in a large percentage of the human population. However, perturbations of the host's immune response or bacterial microbiota have been shown to predispose individuals to the development of opportunistic Candida infections. It was recently discovered that a defect in the chemokine receptor CX 3 CR1 increases susceptibility of mice and humans to systemic candidiasis. However, whether CX 3 CR1 confers protection against mucosal C. albicans infection has not been investigated. Using two different mouse models, we found that Cx3cr1 is dispensable for the induction of interleukin 17A (IL-17A), IL-22, and IL-23 in the tongue after infection, as well as for the clearance of mucosal candidiasis from the tongue or lower gastrointestinal (GI) tract colonization. Furthermore, the dysfunctional human CX 3 CR1 allele CX 3 CR1-M280 was not associated with development of recurrent vulvovaginal candidiasis (RVVC) in women. Taken together, these data indicate that CX 3 CR1 is not essential for protection of the host against mucosal candidiasis, underscoring the dependence on different mammalian immune factors for control of mucosal versus systemic Candida infections.
- Published
- 2015
25. Ccr7-dependent amelioration of renal tubular injury protects against infection-induced acute kidney injury
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Timothy J. Break, Nathaniel G. Green, Hiroshi Kojima, Peter S. T. Yuen, Chyi-chia 'Richard' Lee, Muthulekha Swamydas, Robert A. Star, and Michail S. Lionakis
- Subjects
Immunology ,Immunology and Allergy - Abstract
Systemic candidiasis (SC) is a leading cause of nosocomial bloodstream infections, with mortality of >40% despite therapy. In a mouse model of SC, the most highly infected organ is the kidney, and myeloid cells, including dendritic cells (DCs), are integral for host defense. We hypothesized that Ccr7, which mediates DC trafficking and survival, is critical for host defense during SC. Ccr7 and its ligands, Ccl19 and Ccl21, were highly up-regulated in the Candida-infected kidney. All Ccr7−/− mice rapidly succumbed during SC, whereas WT mice had 80% survival at day 14. Infected Ccr7−/− mice had significantly greater kidney fungal burden, with greater tissue damage, and more severe renal failure. Infection in Ccl19−/− mice and in plt/plt mice (that lack Ccl19 and the CCL21a isoform of Ccl21) did not phenocopy the susceptibility of Ccr7−/− mice, indicating that the CCL21b-Ccr7 axis is critical for protection. Surprisingly, bone marrow chimeras revealed that the protective effects of Ccr7 are solely mediated by non-hematopoietic cells. Studies using FACS, IHC, and mRNA from sorted renal cells showed that Ccr7 is expressed by renal tubular cells (RTCs), not other renal stromal cells. Our preliminary data show the Ccr7 is a survival factor for RTCs; indeed, Ccr7 deficiency resulted in significantly greater tubulitis and tubular necrosis during SC. The renal protective effects of Ccr7 were also evident during bacterial sepsis, indicating that Ccr7 is broadly protective against infection-induced acute kidney injury. In summary, our data are the first to implicate Ccr7 in innate control of any pathogen and to uncover the contribution of Ccr7 on non-hematopoietic cells in protection against acute kidney injury and systemic fungal infection.
- Published
- 2017
26. VT-1598 Inhibits the in vitro Growth of Mucosal Candida Isolates and Protects Against Oropharyngeal Candidiasis in IL-17 Deficient Mice
- Author
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Adrian M. Zelazny, Oren J Cohen, Michail S. Lionakis, Edward P. Garvey, Christopher M. Yates, Christina M. Henderson, Jigar V. Desai, Elise M. N. Ferré, Mukil Natarajan, Robert J. Schotzinger, and Timothy J. Break
- Subjects
Pathology ,medicine.medical_specialty ,business.industry ,Poster Abstract ,In vitro ,Oropharyngeal Candidiasis ,Microbiology ,Abstracts ,Infectious Diseases ,Oncology ,Deficient mouse ,Medicine ,Interleukin 17 ,business ,In vitro growth - Abstract
Background Patients with chronic mucocutaneous candidiasis (CMC) often develop azole-resistant Candida infections, making treatment difficult due to lack of oral antifungal drug options. VT-1598 is a novel broad-spectrum fungal CYP51 inhibitor designed for exquisite selectivity for the fungal target versus human CYP enzymes to circumvent classic azole side effects like drug–drug interactions. We report the efficacy of VT-1598 in the treatment of oral Candida infection (including by azole-resistant strains). Methods The in vitro MIC values of 28 Candida species isolated from patients with CMC due to AIRE mutations were tested against VT-1598 and fluconazole (FLC), using CLSI broth microdilution M27-S4. Plasma VT-1598 levels were measured using LC–MS/MS with electrospray ionization. Tongue fungal load was determined in IL-17 deficient Act1- /- mice following sublingual C. albicans infection and once-daily oral treatment for 4 days with 25 mg/kg FLC or 3.2, 8, and 20 mg/kg VT-1598 starting 18 hours post-infection. Results Among 28 Candida isolates tested (22 C. albicans, three C. glabrata, and one each of C. utilis, C. dubiliensis, and C. krusei), 10 (36%) were not susceptible to FLC, based on CLSI breakpoints (>4 mg/ml). Remarkably, all 28 isolates were highly susceptible to VT-1598 (MIC50 and MIC90, 0.06 and 0.125 mg/ml, respectively). Oral administration of VT-1598 led to mean drug levels in mouse plasma (2.0, 3.0, and 11 mg/ml at the low, mid, and high doses, respectively) that were higher than the MIC values. In vivo, VT-1598 was significantly more effective, compared with FLC, against FLC-susceptible and -resistant C. albicans, and led to elimination of fungal growth even at the lowest tested dose (3.2 mg/kg). After a 10-day washout period from the last dose, mice treated with VT-1598 did not have mucosal fungal growth, while mice treated with FLC had tongue fungal loads similar to vehicle control. Conclusion VT-1598 shows in vitro activity against mucosally derived Candida, including FLC-resistant strains. In vivo, VT-1598 achieves high plasma concentrations and eliminates viable C. albicans, even at low doses and after an extended washout period. These data indicate that VT-1598 may be a significantly improved treatment option for patients with CMC. Disclosures C. M. Yates, Viamet Pharmaceuticals, Inc.: Employee, Salary. O. J. Cohen, Viamet Pharmaceuticals, Inc.: Employee, Salary. R. J. Schotzinger, Viamet Pharmaceuticals, Inc.: Employee, Salary. E. P. Garvey, Viamet Pharmaceuticals, Inc.: Employee, Salary. M. Lionakis, Viamet Pharmaceuticals: Research support (CRADA), Research support
- Published
- 2017
27. Extracellular Superoxide Dismutase Inhibits Innate Immune Responses and Clearance of an Intracellular Bacterial Infection
- Author
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Ladislav Dory, Rance E. Berg, Timothy J. Break, Mohanalaxmi Indramohan, Amy N. Sieve, Sujung Jun, and Karen D. Carr
- Subjects
Male ,Neutropenia ,Neutrophils ,Immunology ,Apoptosis ,Biology ,Nitric Oxide ,Article ,Microbiology ,Superoxide dismutase ,chemistry.chemical_compound ,Mice ,Mice, Congenic ,Immune system ,Peroxynitrous Acid ,Extracellular ,Immunology and Allergy ,Animals ,Listeriosis ,Reactive nitrogen species ,chemistry.chemical_classification ,Mice, Knockout ,Reactive oxygen species ,Innate immune system ,Superoxide Dismutase ,Tumor Necrosis Factor-alpha ,Listeria monocytogenes ,Reactive Nitrogen Species ,Immunity, Innate ,Lymphocyte Subsets ,Mice, Inbred C57BL ,Peroxynitrous acid ,Chemotaxis, Leukocyte ,chemistry ,Liver ,Enzyme Induction ,Host-Pathogen Interactions ,biology.protein ,Tumor necrosis factor alpha ,Female ,Disease Susceptibility ,Reactive Oxygen Species ,Spleen - Abstract
Reactive oxygen species and reactive nitrogen species play important roles during immune responses to bacterial pathogens. Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species and reactive nitrogen species and contributes to tissue protection during inflammatory insults. The participation of ecSOD in immune responses seems therefore intuitive, yet is poorly understood. In the current study, we used mice with varying levels of ecSOD activity to investigate the involvement of this enzyme in immune responses against Listeria monocytogenes. Surprisingly, our data demonstrate that despite enhanced neutrophil recruitment to the liver, ecSOD activity negatively affected host survival and bacterial clearance. Increased ecSOD activity was accompanied by decreased colocalization of neutrophils with bacteria, as well as increased neutrophil apoptosis, which reduced overall and neutrophil-specific TNF-α production. Liver leukocytes from mice lacking ecSOD produced equivalent NO· compared with liver leukocytes from mice expressing ecSOD. However, during infection, there were higher levels of peroxynitrite (NO3·−) in livers from mice lacking ecSOD compared with livers from mice expressing ecSOD. Neutrophil depletion studies revealed that high levels of ecSOD activity resulted in neutrophils with limited protective capacity, whereas neutrophils from mice lacking ecSOD provided superior protection compared with neutrophils from wild-type mice. Taken together, our data demonstrate that ecSOD activity reduces innate immune responses during bacterial infection and provides a potential target for therapeutic intervention.
- Published
- 2012
28. IL-22 production is regulated by IL-23 during Listeria monocytogenes infection but is not required for bacterial clearance or tissue protection
- Author
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Mohanalaxmi Indramohan, Rance E. Berg, Timothy J. Break, Karen D. Carr, Amy C. Graham, and Amy N. Sieve
- Subjects
Male ,Bacterial Diseases ,medicine.medical_treatment ,lcsh:Medicine ,Adaptive Immunity ,medicine.disease_cause ,Interleukin-23 ,Monocytes ,Interleukin 22 ,Mice ,0302 clinical medicine ,Listeriosis ,lcsh:Science ,Pathogen ,Immune Response ,0303 health sciences ,Multidisciplinary ,Coinfection ,Interleukin ,Innate Immunity ,3. Good health ,Host-Pathogen Interaction ,medicine.anatomical_structure ,Cytokine ,Infectious Diseases ,Cytokines ,Medicine ,Female ,Research Article ,Secondary infection ,Immune Cells ,Immunology ,Spleen ,Biology ,Microbiology ,Immune Activation ,03 medical and health sciences ,Immune system ,Listeria monocytogenes ,medicine ,Animals ,Immunity to Infections ,030304 developmental biology ,Inflammation ,Mucous Membrane ,Interleukins ,lcsh:R ,Immunity ,Immunoregulation ,Immune Defense ,Survival Analysis ,Immune System ,Clinical Immunology ,lcsh:Q ,030215 immunology - Abstract
Listeria monocytogenes (LM) is a gram-positive bacterium that is a common contaminant of processed meats and dairy products. In humans, ingestion of LM can result in intracellular infection of the spleen and liver, which can ultimately lead to septicemia, meningitis, and spontaneous abortion. Interleukin (IL)-23 is a cytokine that regulates innate and adaptive immune responses by inducing the production of IL-17A, IL-17F, and IL-22. We have recently demonstrated that the IL-23/IL-17 axis is required for optimal recruitment of neutrophils to the liver, but not the spleen, during LM infection. Furthermore, these cytokines are required for the clearance of LM during systemic infection. In other infectious models, IL-22 induces the secretion of anti-microbial peptides and protects tissues from damage by preventing apoptosis. However, the role of IL-22 has not been thoroughly investigated during LM infection. In the present study, we show that LM induces the production of IL-22 in vivo. Interestingly, IL-23 is required for the production of IL-22 during primary, but not secondary, LM infection. Our findings suggest that IL-22 is not required for clearance of LM during primary or secondary infection, using both systemic and mucosal models of infection. IL-22 is also not required for the protection of LM infected spleens and livers from organ damage. Collectively, these data indicate that IL-22 produced during LM infection must play a role other than clearance of LM or protection of tissues from pathogen- or immune-mediated damage.
- Published
- 2011
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