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1.
J Allergy Clin Immunol ; 141(5): 1844-1853.e2, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-28859974

RESUMEN

BACKGROUND: Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin-related modifier (SUMO) and comprises an important regulator of protein function. OBJECTIVE: We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1). METHODS: STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1-deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs. RESULTS: We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (702IKTE705) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild-type (WT) STAT1 cells, as well as the loss-of-function mutants L706S and Y701C. Furthermore, stimulation with IFN-γ led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V-transfected compared with WT-transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss-of-function and gain-of-function variants. CONCLUSION: This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain-of-function STAT1 disease in human subjects.


Asunto(s)
Mutación con Ganancia de Función/inmunología , Mutación/genética , Factor de Transcripción STAT1/genética , Ubiquitina/genética , Animales , Células COS , Candidiasis Mucocutánea Crónica/genética , Línea Celular , Chlorocebus aethiops , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Expresión Génica/genética , Humanos , Fosforilación/genética , Proteína SUMO-1/genética , Sumoilación/genética , Transcripción Genética/genética , Activación Transcripcional/genética , Transfección/métodos
2.
Am J Respir Cell Mol Biol ; 58(2): 241-252, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-28915071

RESUMEN

The incidence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing, but host responses in respiratory epithelium infected with NTM are not fully understood. In this work, we aimed to identify infection-relevant gene expression signatures of NTM infection of the respiratory epithelium. We infected air-liquid interface (ALI) primary respiratory epithelial cell cultures with Mycobacterium avium subsp. avium (MAC) or Mycobacterium abscessus subsp. abscessus (MAB). We used cells from four different donors to obtain generalizable data. Differentiated respiratory epithelial cells at the ALI were infected with MAC or MAB at a multiplicity of infection of 100:1 or 1,000:1, and RNA sequencing was performed at Days 1 and 3 after infection. In response to infection, we found down-regulation of ciliary genes but upregulation of genes associated with cytokines/chemokines, such as IL-32, and cholesterol biosynthesis. Inflammatory response genes tended to be more upregulated by MAB than by MAC infection. Primary respiratory epithelial cell infection with NTM at the ALI identified ciliary function, cholesterol biosynthesis, and cytokine/chemokine production as major host responses to infection. Some of these pathways may be amenable to therapeutic manipulation.


Asunto(s)
Colesterol/biosíntesis , Células Epiteliales/metabolismo , Infecciones por Mycobacterium no Tuberculosas/inmunología , Micobacterias no Tuberculosas/inmunología , Mucosa Respiratoria/metabolismo , Adulto , Anciano , Adhesión Bacteriana/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Células Epiteliales/microbiología , Femenino , Perfilación de la Expresión Génica , Humanos , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Interleucinas/biosíntesis , Interleucinas/inmunología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mucosa Respiratoria/citología , Mucosa Respiratoria/microbiología
3.
Proc Natl Acad Sci U S A ; 112(30): 9394-9, 2015 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-26170288

RESUMEN

IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4(+) T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4(+) T cells. RNA-Seq analysis of CD4(+) T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4(+) T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4(+) T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.


Asunto(s)
Linfocitos T CD4-Positivos/citología , Interleucinas/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Citocinas/inmunología , Citometría de Flujo , Regulación de la Expresión Génica , Inmunoglobulina E/inmunología , Interferón gamma/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Fosforilación , Análisis de Secuencia de ARN , Transducción de Señal , Proteínas de Dominio T Box/metabolismo
4.
J Immunol ; 195(6): 2763-73, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26268658

RESUMEN

Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients.


Asunto(s)
Hemo-Oxigenasa 1/sangre , Metaloproteinasa 1 de la Matriz/sangre , Estrés Oxidativo/fisiología , Tuberculosis Pulmonar/patología , Adulto , Anciano , Biomarcadores/sangre , Brasil , Femenino , Hemo-Oxigenasa 1/metabolismo , Humanos , India , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de Unión a TGF-beta Latente/sangre , Pulmón/microbiología , Pulmón/patología , Macrófagos/microbiología , Macrófagos/patología , Masculino , Metaloproteinasa 1 de la Matriz/biosíntesis , Persona de Mediana Edad , Mycobacterium tuberculosis/inmunología , Factor de Transcripción AP-1/metabolismo , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Estados Unidos , Adulto Joven
5.
Clin Infect Dis ; 62(8): 986-94, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26743090

RESUMEN

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a rare, severe, otherwise fatal viral infection of the white matter of the brain caused by the polyomavirus JC virus, which typically occurs only in immunocompromised patients. One patient with dominant gain-of-function (GOF) mutation in signal transducer and activator of transcription 1 (STAT1) with chronic mucocutaneous candidiasis and PML was reported previously. We aim to identify the molecular defect in 3 patients with PML and to review the literature on PML in primary immune defects (PIDs). METHODS: STAT1 was sequenced in 3 patients with PML. U3C cell lines were transfected with STAT1 and assays to search for STAT1 phosphorylation, transcriptional response, and target gene expression were performed. RESULTS: We identified 3 new unrelated cases of PML in patients with GOF STAT1 mutations, including the novel STAT1 mutation, L400Q. These STAT1 mutations caused delayed STAT1 dephosphorylation and enhanced interferon-gamma-driven responses. In our review of the literature regarding PML in primary immune deficiencies we found 26 cases, only 54% of which were molecularly characterized, the remainder being syndromically diagnosed only. CONCLUSIONS: The occurrence of PML in 4 cases of STAT1 GOF suggests that STAT1 plays a critical role in the control of JC virus in the central nervous system.


Asunto(s)
Síndromes de Inmunodeficiencia/genética , Leucoencefalopatía Multifocal Progresiva/genética , Mutación , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/fisiología , Adulto , Encéfalo/diagnóstico por imagen , Línea Celular Tumoral , Femenino , Regulación de la Expresión Génica , Humanos , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/diagnóstico por imagen , Interferón gamma/farmacología , Virus JC/crecimiento & desarrollo , Leucoencefalopatía Multifocal Progresiva/complicaciones , Leucoencefalopatía Multifocal Progresiva/diagnóstico por imagen , Leucoencefalopatía Multifocal Progresiva/inmunología , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Activación Transcripcional , Carga Viral , Adulto Joven
6.
N Engl J Med ; 367(8): 725-34, 2012 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-22913682

RESUMEN

BACKGROUND: Autoantibodies against interferon-γ are associated with severe disseminated opportunistic infection, but their importance and prevalence are unknown. METHODS: We enrolled 203 persons from sites in Thailand and Taiwan in five groups: 52 patients with disseminated, rapidly or slowly growing, nontuberculous mycobacterial infection (group 1); 45 patients with another opportunistic infection, with or without nontuberculous mycobacterial infection (group 2); 9 patients with disseminated tuberculosis (group 3); 49 patients with pulmonary tuberculosis (group 4); and 48 healthy controls (group 5). Clinical histories were recorded, and blood specimens were obtained. RESULTS: Patients in groups 1 and 2 had CD4+ T-lymphocyte counts that were similar to those in patients in groups 4 and 5, and they were not infected with the human immunodeficiency virus (HIV). Washed cells obtained from patients in groups 1 and 2 had intact cytokine production and a response to cytokine stimulation. In contrast, plasma obtained from these patients inhibited the activity of interferon-γ in normal cells. High-titer anti-interferon-γ autoantibodies were detected in 81% of patients in group 1, 96% of patients in group 2, 11% of patients in group 3, 2% of patients in group 4, and 2% of controls (group 5). Forty other anticytokine autoantibodies were assayed. One patient with cryptococcal meningitis had autoantibodies only against granulocyte-macrophage colony-stimulating factor. No other anticytokine autoantibodies or genetic defects correlated with infections. There was no familial clustering. CONCLUSIONS: Neutralizing anti-interferon-γ autoantibodies were detected in 88% of Asian adults with multiple opportunistic infections and were associated with an adult-onset immunodeficiency akin to that of advanced HIV infection. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research; ClinicalTrials.gov number, NCT00814827.).


Asunto(s)
Anticuerpos Neutralizantes/sangre , Autoanticuerpos/sangre , Enfermedades Autoinmunes/inmunología , Interferón gamma/inmunología , Infecciones por Mycobacterium/inmunología , Infecciones Oportunistas/inmunología , Adolescente , Adulto , Edad de Inicio , Anciano , Recuento de Linfocito CD4 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Micosis/inmunología , Taiwán , Tailandia , Tuberculosis Pulmonar/inmunología , Adulto Joven
7.
J Clin Microbiol ; 53(11): 3430-7, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26269619

RESUMEN

Members of the Mycobacterium abscessus group (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducible erm(41) gene or via acquired mutations in the rrl (23S rRNA) gene. Strains harboring a truncation or a T28C substitution in erm(41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncated erm(41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 of erm(41) and 23S rRNA rrl gene mutations at position 2058 and/or 2059. Truncated and full-size erm(41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showed rrl mutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an active erm(41), inactive erm(41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation.


Asunto(s)
Antibacterianos/farmacología , Claritromicina/farmacología , Farmacorresistencia Bacteriana/genética , Infecciones por Mycobacterium no Tuberculosas/microbiología , Micobacterias no Tuberculosas/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano/genética , Humanos , Enfermedades Pulmonares/microbiología , Metiltransferasas/genética , Pruebas de Sensibilidad Microbiana , Micobacterias no Tuberculosas/genética , Polimorfismo de Nucleótido Simple , ARN Ribosómico 23S/genética , Análisis de Secuencia de ADN , Infecciones de los Tejidos Blandos/microbiología
8.
J Clin Microbiol ; 53(11): 3438-47, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26292297

RESUMEN

The smooth-to-rough colony morphology shift in Mycobacterium abscessus has been implicated in loss of glycopeptidolipid (GPL), increased pathogenicity, and clinical decline in cystic fibrosis (CF) patients. However, the evolutionary phenotypic and genetic changes remain obscure. Serial isolates from nine non-CF patients with persistent M. abscessus infection were characterized by colony morphology, lipid profile via thin-layer chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), sequencing of eight genes in the GPL locus, and expression level of fadD23, a key gene involved in the biosynthesis of complex lipids. All 50 isolates were typed as M. abscessus subspecies abscessus and were clonally related within each patient. Rough isolates, all lacking GPL, predominated at later disease stages, some showing variation within rough morphology. While most (77%) rough isolates harbored detrimental mutations in mps1 and mps2, 13% displayed previously unreported mutations in mmpL4a and mmpS4, the latter yielding a putative GPL precursor. Two isolates showed no deleterious mutations in any of the eight genes sequenced. Mixed populations harboring different GPL locus mutations were detected in 5 patients, demonstrating clonal diversification, which was likely overlooked by conventional acid-fast bacillus (AFB) culture methods. Our work highlights applications of MALDI-TOF MS beyond identification, focusing on mycobacterial lipids relevant in virulence and adaptation. Later isolates displayed accumulation of triacylglycerol and reduced expression of fadD23, sometimes preceding rough colony onset. Our results indicate that clonal diversification and a shift in lipid metabolism, including the loss of GPL, occur during chronic lung infection with M. abscessus. GPL loss alone may not account for all traits associated with rough morphology.


Asunto(s)
Proteínas Bacterianas/genética , Ligasas/genética , Metabolismo de los Lípidos/genética , Infecciones por Mycobacterium no Tuberculosas/microbiología , Micobacterias no Tuberculosas/aislamiento & purificación , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Bronquiectasia/microbiología , Fibrosis Quística/microbiología , ADN Bacteriano/genética , Femenino , Dosificación de Gen/genética , Genoma Bacteriano/genética , Humanos , Lípidos/genética , Masculino , Persona de Mediana Edad , Tipificación de Secuencias Multilocus , Micobacterias no Tuberculosas/clasificación , Micobacterias no Tuberculosas/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
9.
Emerg Infect Dis ; 20(3): 364-71, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24565502

RESUMEN

Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.


Asunto(s)
Brotes de Enfermedades , Infecciones por Mycobacterium/epidemiología , Mycobacterium/aislamiento & purificación , Brasil , Fibrosis Quística/complicaciones , Genoma Bacteriano , Humanos , Tipificación de Secuencias Multilocus , Mycobacterium/clasificación , Mycobacterium/genética , Infecciones por Mycobacterium/complicaciones , Infecciones por Mycobacterium/diagnóstico , Infecciones por Mycobacterium/microbiología , Filogenia , Polimorfismo de Nucleótido Simple , Reino Unido , Estados Unidos
10.
Blood ; 119(17): 3933-9, 2012 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-22403254

RESUMEN

Patients with anti-IFN-γ autoantibodies have impaired IFN-γ signaling, leading to severe disseminated infections with intracellular pathogens, especially nontuberculous mycobacteria. Disease may be severe and progressive, despite aggressive treatment. To address the underlying pathogenic IFN-γ autoantibodies we used the therapeutic monoclonal rituximab (anti-CD20) to target patient B cells. All subjects received between 8 and 12 doses of rituximab within the first year to maintain disease remission. Subsequent doses were given for relapsed infection. We report 4 patients with refractory disease treated with rituximab who had clinical and laboratory evidence of therapeutic response as determined by clearance of infection, resolution of inflammation, reduction of anti-IFN-γ autoantibody levels, and improved IFN-γ signaling.


Asunto(s)
Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Factores Inmunológicos/uso terapéutico , Interferón gamma/inmunología , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium/tratamiento farmacológico , Anciano , Western Blotting , Femenino , Citometría de Flujo , Humanos , Interferón gamma/farmacología , Persona de Mediana Edad , Infecciones por Mycobacterium/inmunología , Infecciones por Mycobacterium/microbiología , Fosforilación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Rituximab , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo
11.
J Allergy Clin Immunol ; 131(6): 1611-23, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23534974

RESUMEN

BACKGROUND: Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE: We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS: We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS: We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS: Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.


Asunto(s)
Factores de Transcripción Forkhead/genética , Genes Dominantes , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Intestinales/genética , Mutación , Poliendocrinopatías Autoinmunes/genética , Factor de Transcripción STAT1/genética , Adolescente , Autoanticuerpos/inmunología , Línea Celular Transformada , Niño , Preescolar , ADN/metabolismo , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Humanos , Inmunofenotipificación , Interferón-alfa/inmunología , Interferón gamma/farmacología , Interleucina-17/inmunología , Interleucinas/inmunología , Enfermedades Intestinales/diagnóstico , Enfermedades Intestinales/inmunología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Masculino , Fenotipo , Fosforilación/efectos de los fármacos , Poliendocrinopatías Autoinmunes/diagnóstico , Poliendocrinopatías Autoinmunes/inmunología , Factor de Transcripción STAT1/metabolismo , Síndrome , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Activación Transcripcional , Interleucina-22
12.
J Allergy Clin Immunol ; 131(6): 1624-34, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23541320

RESUMEN

BACKGROUND: Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. OBJECTIVE: We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. METHODS: PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. RESULTS: We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ-induced gene expression, but we found impaired responses to IFN-γ restimulation. CONCLUSION: Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ-mediated inflammation.


Asunto(s)
Coccidioidomicosis/genética , Histoplasmosis/genética , Mutación , Factor de Transcripción STAT1/genética , Adolescente , Adulto , Línea Celular Transformada , Niño , Coccidioidomicosis/diagnóstico , Coccidioidomicosis/inmunología , Citocinas/biosíntesis , Femenino , Regulación de la Expresión Génica , Histoplasmosis/diagnóstico , Histoplasmosis/inmunología , Humanos , Masculino , Fosforilación , Proteínas Inhibidoras de STAT Activados/metabolismo , Factor de Transcripción STAT1/metabolismo , Células Th17/inmunología , Activación Transcripcional , Adulto Joven
13.
Emerg Infect Dis ; 19(3): 393-9, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23745217

RESUMEN

To identify clinical and therapeutic features of pulmonary nontuberculous mycobacterial (PNTM) disease, we conducted a retrospective analysis of patients referred to the Brazilian reference center, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, who received a diagnosis of PNTM during 1993­2011 with at least 1 respiratory culture positive for NTM. Associated conditions included bronchiectasis (21.8%), chronic obstructive pulmonary disease (20.7%), cardiovascular disease (15.5%), AIDS (9.8%), diabetes (9.8%), and hepatitis C (4.6%).Two patients had Hansen disease; 1 had Marfan syndrome. Four mycobacterial species comprised 85.6% of NTM infections: Mycobacterium kansasii, 59 cases (33.9%); M. avium complex, 53 (30.4%); M. abscessus, 23 (13.2%); and M. fortuitum, 14 (8.0%). A total of 42 (24.1%) cases were associated with rapidly growing mycobacteria. In countries with a high prevalence of tuberculosis, PNTM is likely misdiagnosed as tuberculosis, thus showing the need for improved capacity to diagnose mycobacterial disease as well as greater awareness of PNTM disease prevalence.


Asunto(s)
Enfermedades Pulmonares/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Complejo Mycobacterium avium/aislamiento & purificación , Mycobacterium kansasii/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/uso terapéutico , Brasil , Femenino , Humanos , Enfermedades Pulmonares/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Complejo Mycobacterium avium/efectos de los fármacos , Mycobacterium kansasii/efectos de los fármacos , Resultado del Tratamiento , Adulto Joven
14.
J Clin Microbiol ; 51(9): 2943-9, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23804391

RESUMEN

Mycobacterium abscessus (M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii, and Mycobacterium massiliense. We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus, M. massiliense, and M. bolletii. Based on the M. abscessus ATCC 19977(T) genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense, and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm(41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for straightforward identification of M. abscessus, M. massiliense, and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group.


Asunto(s)
Antibacterianos/farmacología , Técnicas Bacteriológicas/métodos , Claritromicina/farmacología , Farmacorresistencia Bacteriana , Micobacterias no Tuberculosas/clasificación , Reacción en Cadena de la Polimerasa/métodos , Hibridación Genómica Comparativa , Cartilla de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Variación Genética , Humanos , Datos de Secuencia Molecular , Micobacterias no Tuberculosas/efectos de los fármacos , Micobacterias no Tuberculosas/genética , Análisis de Secuencia de ADN
15.
Blood ; 118(10): 2653-5, 2011 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-21670465

RESUMEN

The syndrome of monocytopenia, B-cell and NK-cell lymphopenia, and mycobacterial, fungal, and viral infections is associated with myelodysplasia, cytogenetic abnormalities, pulmonary alveolar proteinosis, and myeloid leukemias. Both autosomal dominant and sporadic cases occur. We identified 12 distinct mutations in GATA2 affecting 20 patients and relatives with this syndrome, including recurrent missense mutations affecting the zinc finger-2 domain (R398W and T354M), suggesting dominant interference of gene function. Four discrete insertion/deletion mutations leading to frame shifts and premature termination implicate haploinsufficiency as a possible mechanism of action as well. These mutations were found in hematopoietic and somatic tissues, and several were identified in families, indicating germline transmission. Thus, GATA2 joins RUNX1 and CEBPA not only as a familial leukemia gene but also as a cause of a complex congenital immunodeficiency that evolves over decades and combines predisposition to infection and myeloid malignancy.


Asunto(s)
Factor de Transcripción GATA2/genética , Predisposición Genética a la Enfermedad , Monocitos/patología , Mutación/genética , Infecciones por Mycobacterium/etiología , Infecciones por Mycobacterium/patología , Mycobacterium/patogenicidad , Genes Dominantes , Humanos , Síndrome
16.
Virulence ; 14(1): 2215602, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37221835

RESUMEN

BACKGROUND: Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case. RESULTS: Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains. CONCLUSIONS: We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.


Asunto(s)
Fibrosis Quística , Mycobacterium abscessus , Mycobacterium , Humanos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Fibrosis Quística/microbiología , Genómica , Glucosa , Pulmón , Mutación , Mycobacterium/genética , Mycobacterium abscessus/genética , Factor de Necrosis Tumoral alfa/genética , Porinas/genética , Porinas/metabolismo
17.
J Bacteriol ; 194(22): 6356-7, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23105085

RESUMEN

Burkholderia multivorans is a Gram-negative bacterium and a member of the Burkholderia cepacia complex, which is frequently associated with respiratory infections in people with cystic fibrosis (CF) and chronic granulomatous disease (CGD). We are reporting the genome sequences of 4 B. multivorans strains, 2 from CF patients and 2 from CGD patients.


Asunto(s)
Burkholderia/clasificación , Burkholderia/genética , Fibrosis Quística/microbiología , Genoma Bacteriano , Enfermedad Granulomatosa Crónica/microbiología , Humanos , Datos de Secuencia Molecular
18.
J Bacteriol ; 194(19): 5450, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22965080

RESUMEN

Mycobacterium massiliense (Mycobacterium abscessus group) is an emerging pathogen causing pulmonary disease and skin and soft tissue infections. We report the genome sequence of the type strain CCUG 48898.


Asunto(s)
Infecciones por Mycobacterium/microbiología , Mycobacterium/clasificación , Mycobacterium/genética , Genoma Bacteriano , Humanos , Datos de Secuencia Molecular , Filogenia , Polimorfismo de Nucleótido Simple
19.
J Clin Immunol ; 32(4): 681-689, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22437822

RESUMEN

STAT1 is a key component of Interferon (IFN)-γ and IFN-α signaling and mediates protection against mycobacteria, fungal, viral infections, and cancer. Dominant negative inhibitory as well as gain of function heterozygous STAT1 mutations demonstrate that IFN-γ driven cellular responses need to be tightly regulated to control infections. We describe an autosomal dominant mutation in the SH2 domain of STAT1 that disrupts protein phosphorylation, c.1961T>A (M654K). The mutant allele does not permit STAT1 phosphorylation, and impairs STAT1 phosphorylation of the wild type allele. Protein dimerization is preserved but DNA binding activity, IFN-γ driven GAS-luciferase activity, and expression of IFN-γ target genes are reduced. IFN-α driven ISRE response, but not IFN-α driven GAS response, are preserved when cells are co-transfected with wild type and the mutant STAT1 constructs. M654K exerts a dominant negative effect on IFN-γ related immunity and is recessive for IFN-α induced immune function.


Asunto(s)
Infecciones por Mycobacterium no Tuberculosas/genética , Complejo Mycobacterium avium/patogenicidad , Factor de Transcripción STAT1/genética , Linfocitos B , Línea Celular , Preescolar , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Interferón-alfa/genética , Interferón-alfa/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Masculino , Mutación , Infecciones por Mycobacterium no Tuberculosas/microbiología , Fosforilación , Multimerización de Proteína , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/genética
20.
Blood ; 116(23): 4848-58, 2010 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-20716769

RESUMEN

Patients with thymic malignancy have high rates of autoimmunity leading to a variety of autoimmune diseases, most commonly myasthenia gravis caused by anti-acetylcholine receptor autoantibodies. High rates of autoantibodies to cytokines have also been described, although prevalence, spectrum, and functionality of these anti-cytokine autoantibodies are poorly defined. To better understand the presence and function of anti-cytokine autoantibodies, we created a luciferase immunoprecipitation system panel to search for autoantibodies against 39 different cytokines and examined plasma from controls (n = 30) and patients with thymic neoplasia (n = 17). In this screen, our patients showed statistically elevated, but highly heterogeneous immunoreactivity against 16 of the 39 cytokines. Some patients showed autoantibodies to multiple cytokines. Functional testing proved that autoantibodies directed against interferon-α, interferon-ß, interleukin-1α (IL-1α), IL-12p35, IL-12p40, and IL-17A had biologic blocking activity in vitro. All patients with opportunistic infection showed multiple anti-cytokine autoantibodies (range 3-11), suggesting that anti-cytokine autoantibodies may be important in the pathogenesis of opportunistic infections in patients with thymic malignancy. This study was registered at http://clinicaltrials.gov as NCT00001355.


Asunto(s)
Autoanticuerpos/sangre , Citocinas/inmunología , Infecciones Oportunistas/inmunología , Neoplasias del Timo/complicaciones , Neoplasias del Timo/inmunología , Adulto , Anciano , Autoantígenos/inmunología , Femenino , Humanos , Inmunoensayo , Immunoblotting , Inmunoprecipitación , Masculino , Persona de Mediana Edad , Infecciones Oportunistas/sangre , Infecciones Oportunistas/epidemiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias del Timo/sangre , Adulto Joven
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