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1.
J Exp Clin Cancer Res ; 38(1): 446, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676012

RESUMO

BACKGROUND: NOTCH1 gene mutations in mantle cell lymphoma (MCL) have been described in about 5-10% of cases and are associated with significantly shorter survival rates. The present study aimed to investigate the biological impact of this mutation in MCL and its potential as a therapeutic target. METHODS: Activation of Notch1 signaling upon ligand-stimulation and inhibitory effects of the monoclonal anti-Notch1 antibody OMP-52M51 in NOTCH1-mutated and -unmutated MCL cells were assessed by Western Blot and gene expression profiling. Effects of OMP-52M51 treatment on tumor cell migration and tumor angiogenesis were evaluated with chemotaxis and HUVEC tube formation assays. The expression of Delta-like ligand 4 (DLL4) in MCL lymph nodes was analyzed by immunofluorescence staining and confocal microscopy. A MCL mouse model was used to assess the activity of OMP-52M51 in vivo. RESULTS: Notch1 expression can be effectively stimulated in NOTCH1-mutated Mino cells by DLL4, whereas in the NOTCH1-unmutated cell line JeKo-1, less effect was observed upon any ligand-stimulation. DLL4 was expressed by histiocytes in both, NOTCH1-mutated and -unmutated MCL lymph nodes. Treatment of NOTCH1-mutated MCL cells with the monoclonal anti-Notch1 antibody OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed the induction of numerous direct Notch target genes involved in lymphoid biology, lymphomagenesis and disease progression. Importantly, in lymph nodes from primary MCL cases with NOTCH1/2 mutations, we detected an upregulation of the same gene sets as observed in DLL4-stimulated Mino cells. Furthermore, DLL4 stimulation of NOTCH1-mutated Mino cells enhanced tumor cell migration and angiogenesis, which could be abolished by treatment with OMP-52M51. Importantly, the effects observed were specific for NOTCH1-mutated cells as they did not occur in the NOTCH1-wt cell line JeKo-1. Finally, we confirmed the potential activity of OMP-52M51 to inhibit DLL4-induced Notch1-Signaling in vivo in a xenograft mouse model of MCL. CONCLUSION: DLL4 effectively stimulates Notch1 signaling in NOTCH1-mutated MCL and is expressed by the microenvironment in MCL lymph nodes. Our results indicate that specific inhibition of the Notch1-ligand-receptor interaction might provide a therapeutic alternative for a subset of MCL patients.

2.
Oncogene ; 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31616059

RESUMO

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

3.
Blood ; 134(24): 2171-2182, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31530562

RESUMO

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the dysregulation of signaling pathways that control intrathymic T-cell development. Relapse rates are still significant, and prognosis is particularly bleak for relapsed patients. Therefore, development of novel therapies specifically targeting pathways controlling leukemia-initiating cell (LIC) activity is mandatory for fighting refractory T-ALL. The interleukin-7 receptor (IL-7R) is a crucial T-cell developmental pathway that is commonly expressed in T-ALL and has been implicated in leukemia progression; however, the significance of IL-7R/IL-7 signaling in T-ALL pathogenesis and its contribution to disease relapse remain unknown. To directly explore whether IL-7R targeting may be therapeutically efficient against T-ALL relapse, we focused on a known Notch1-induced T-ALL model, because a majority of T-ALL patients harbor activating mutations in NOTCH1, which is a transcriptional regulator of IL-7R expression. Using loss-of-function approaches, we show that Il7r-deficient, but not wild-type, mouse hematopoietic progenitors transduced with constitutively active Notch1 failed to generate leukemia upon transplantation into immunodeficient mice, thus providing formal evidence that IL-7R function is essential for Notch1-induced T-cell leukemogenesis. Moreover, we demonstrate that IL-7R expression is an early functional biomarker of T-ALL cells with LIC potential and report that impaired IL-7R signaling hampers engraftment and progression of patient-derived T-ALL xenografts. Notably, we show that IL-7R-dependent LIC activity and leukemia progression can be extended to human B-cell acute lymphoblastic leukemia (B-ALL). These results have important therapeutic implications, highlighting the relevance that targeting normal IL-7R signaling may have in future therapeutic interventions, particularly for preventing T-ALL (and B-ALL) relapse.

4.
J Clin Microbiol ; 57(2)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30429257

RESUMO

Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/ml, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.

5.
Int J Mycobacteriol ; 7(3): 275-281, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30198510

RESUMO

Introduction: Tuberculosis (TB) is a significant cause of morbidity and mortality worldwide. The patient compliance with the long treatment regimens is essential for successful eradication. Pyrazinamide (PZA) shortens these regimens from 9 to 6 months, and therefore, improves treatment completion rates. Although PZA is a first-line medication for the treatment of TB, no simple or reliable assay to determine PZA resistance is yet available. In the presence of PZA, only susceptible Mycobacterium tuberculosis strains release pyrazinoic acid (POA). Therefore, the measurement and quantification of released POA is an indicator of PZA resistance. Methods: Two electrochemical sensors were constructed and tested with alternative working electrodes in conjunction with a portable potentiostat to measure the current produced when a potential difference of 2 V is applied to varying concentrations of POA in controlled solutions. Results: The large (13.2 mm) electrochemical sensor was able to detect POA at a minimum concentration of 40 µM to a statistically significant level (P = 0.0190). Similar graphical trends were obtained when testing the electrochemical sensor in the supernatant of a negative microscopic observation drug susceptibility assay culture, irrespective of the presence of PZA. Conclusion: Inexpensive and reusable electrochemical sensors with a portable potentiostat are a promising tool for the detection of POA, a biomarker of PZA susceptible M. Tuberculosis.


Assuntos
Farmacorresistência Bacteriana , Técnicas Eletroquímicas , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Antituberculosos/farmacologia , Meios de Cultura , Eletrodos , Humanos , Testes de Sensibilidade Microbiana , Potenciometria , Pirazinamida/análogos & derivados , Pirazinamida/isolamento & purificação , Tuberculose/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
6.
Development ; 145(16)2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30042180

RESUMO

T-cell development is a complex dynamic process that relies on ordered stromal signals delivered to thymus-seeding progenitors that migrate throughout different thymus microenvironments (TMEs). Particularly, Notch signaling provided by thymic epithelial cells (TECs) is crucial for T-cell fate specification and generation of mature T cells. Four canonical Notch ligands (Dll1, Dll4, Jag1 and Jag2) are expressed in the thymus, but their spatial distribution in functional TMEs is largely unknown, especially in humans, and their impact on Notch1 activation during T-lymphopoiesis remains undefined. Based on immunohistochemistry and quantitative confocal microscopy of fetal, postnatal and adult human and mouse thymus samples, we show that spatial regulation of Notch ligand expression defines discrete Notch signaling niches and dynamic species-specific TMEs. We further show that Notch ligand expression, particularly DLL4, is tightly regulated in cortical TECs during human thymus ontogeny and involution. Also, we provide the first evidence that NOTCH1 activation is induced in vivo in CD34+ progenitors and developing thymocytes at particular cortical niches of the human fetal and postnatal thymus. Collectively, our results show that human thymopoiesis involves complex spatiotemporal regulation of Notch ligand expression, which ensures the coordinated delivery of niche-specific NOTCH1 signals required for dynamic T-cell development.


Assuntos
Receptor Notch1/metabolismo , Timo/crescimento & desenvolvimento , Timo/metabolismo , Adolescente , Adulto , Envelhecimento/metabolismo , Animais , Antígenos CD34/metabolismo , Criança , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Recém-Nascido , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , Proteínas Serrate-Jagged/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Timo/citologia , Timo/embriologia
7.
J Clin Invest ; 128(7): 2802-2818, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29781813

RESUMO

NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse.


Assuntos
Receptores de Hialuronatos/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Receptor Notch1/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Transplante de Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mutação , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/genética , Transdução de Sinais
8.
Salud ment ; 38(5): 347-351, sep.-oct. 2015. graf
Artigo em Espanhol | LILACS-Express | ID: lil-778950

RESUMO

ANTECEDENTES: Los efectos del estrés dependen de la percepción de los estresores y de las habilidades para afrontar situaciones estresantes. OBJETIVO: Evaluar una intervención interactiva para desarrollar recursos y habilidades para un manejo adecuado del estrés en profesionales de la salud. MÉTODO: Esta intervención se probó utilizando un Cédula diseñada para evaluar componentes de Conocimientos, Habilidades y Actitudes sobre manejo del estrés, conformada por 17 afirmaciones para dimensión de Conocimientos, 11 sobre Habilidades para manejo del estrés; y 20 sobre Actitudes para reaccionar ante el estrés. Participaron 165 profesionales y pasantes de carreras afines. Se hicieron análisis estadísticos para comparar los puntajes de los tres componentes antes y después de la intervención utilizando la prueba t de Student, se aplicó la prueba de McNemar para comparar los resultados. RESULTADOS: Sobre la efectividad de la intervención, en los análisis de comparación de los puntajes, se encontraron diferencias estadísticamente significativas a favor de la intervención en los tres componentes: Conocimientos (t = -9.77, gl = 164, p = .000), Habilidades (t = -10.19, gl = 164, p = .000) y Actitudes (t = -4.80, gl = 164, p = .000).Los análisis por reactivo mostraron incrementos estadísticamente significativos en el número de respuestas correctas después de la intervención en los tres componentes. DISCUSIÓN Y CONCLUSIÓN: Los resultados de esta intervención interactiva fueron estadísticamente significativos en Conocimientos, Habilidades y Actitudes para manejo del estrés. Además de ser una intervención innovadora, breve, basada en la traslación del conocimiento (4 semanas), y disponible en Internet, la evidencia de su efectividad hace que se perfile como una herramienta válida para el manejo adecuado del estrés.


BACKGROUND: The effects of stress depend on the perception of stressors and a person's skills for coping with stressful situations. OBJECTIVE: Evaluate an interactive intervention to develop resources and skills for proper stress management among healthcare professionals. METHOD: This intervention was tested using a chart designed to evaluate the knowledge, skills and attitudes components of stress management, consisting of 17 statements about Knowledge, 11 about Stress management; 20 about Attitudes for reacting to stress. A total of 165 professionals and interns from related careers participated. Statistical analyses were conducted to compare the scores of the three components before and after the intervention using the Student's t test. McNemar's test was used to compare the results. RESULTS: In regard to the effectiveness of the intervention, an analysis of the comparison of scores found statistically significant differences in favor of intervention in the three components: Knowledge (t = -9.77, df = 164, p = .000), Skills (t = -10.19, df = 164, p = .000) and Attitudes (t = -4.80, df = 164, p = .000). The item analysis showed statistically significant increases in the number of correct answers after the intervention in the three components. DISCUSSION AND CONCLUSION: The results of this interactive intervention were statistically significant in Knowledge, Skills and Attitudes for stress management. In addition to being a brief, innovative intervention, based on knowledge translation (4 weeks) and available on the Internet, the evidence of its effectiveness suggests it is a valid tool for proper stress management.

9.
Nucleic Acids Res ; 43(2): 760-74, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25539926

RESUMO

Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.


Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Linfócitos T/imunologia , Transcrição Genética , Diferenciação Celular/genética , Expressão Gênica , Humanos , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timo/citologia , Timo/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Salud ment ; 37(4): 355-360, jul.-ago. 2014. tab
Artigo em Espanhol | LILACS-Express | ID: lil-729733

RESUMO

Objetivo Conocer la violencia que viven las trabajadoras sexuales en el ejercicio de su actividad y si ésta se asocia con su consumo de alcohol y otras sustancias. Método Es un estudio de campo tipo no experimental, descriptivo, con una muestra no probabilística de 103 trabajadoras sexuales seleccionadas por conveniencia. Se utilizó un instrumento diseñado ex profeso, consistente en una entrevista semi-estructurada. Resultados El 50.8% trabajaba en un bar, el 39.0% en la calle y el 10.2% en un cabaret. Más de la mitad (52.4%) refirió haber vivido experiencias violentas en el lugar de trabajo; de éstas, el 95.8% sufrió violencia física, el 26.0% violencia sexual y el 24.0% violencia emocional. Respecto a los actores involucrados en episodios violentos, en el 97.9% de los casos fueron compañeras de trabajo, en el 77.2%, clientes; en el 30.2%, propietarios o empleados de los bares y en el 25.0%, la policía. El 98% de las trabajadoras sexuales reportaron haber consumido alcohol en el último mes, el 23.3% cocaína y el 11.7% mariguana. Cabe señalar que 40% fuma tabaco. El 75% de las trabajadoras sexuales se reporta como bebedora alta. El 97% informó que generalmente los clientes las presionan para beber alcohol y un porcentaje similar refirió que consumen por las exigencias de los encargados de los lugares de trabajo. Las experiencias de violencia en el trabajo se asociaron con el consumo de cocaína [χ²= 6.417, (100, 1) gl p<.05] y de tabaco [2=7.486 (100,1) gl p<.01]. Esta asociación se mantuvo si la violencia había ocurrido en el último mes. El consumo de alcohol se relacionó con las experiencias de violencia física [χ²=5.180 (100,1)gl p<.05], y de violencia emocional [χ²=4.514 (100,1)gl p<.05]. Discusión Los resultados muestran que la violencia es una experiencia frecuente en su ambiente de trabajo, y es ejercida por múltiples actores; destaca el que se mencione a otras trabajadoras sexuales como quienes la ejercen, lo que no se reporta en la bibliografía. El consumo de alcohol y otras drogas se asocia con esta violencia, por lo que debe explorarse más este vínculo en futuros estudios. Se discuten los hallazgos, haciendo recomendaciones para la atención de esta problemática en las mujeres.


Objective The aim of this paper is to explore in women sex workers the violence they experience in the exercise of their activity, and if it is associated with alcohol use and the consumption of other substances such as cocaine, mariguana and tobacco. Method This is a descriptive, non-experimental study, developed with a non-random sample of 103 sex workers selected for convenience type field. The selection criteria were being between 18 and 65 years of age, being a sex worker, and literate. An expressly designed instrument consisting of a semi-structured interview was used. Results Regarding the workplace, 50.8% of them worked in bars, 39.0% on the streets, and 10.2% in cabarets. More than a half reported having suffered violent experiences in the workplace; of these, 95.8% experienced physical violence, 26.0%, sexual violence, and 24.0%, emotional violence. Regarding the actors involved in violent episodes, they were other sex workers (97%), customers (77.2%), owners or employees of bars (30.2%), and police (25.0%). Regarding substance use, 98% of the sex workers reported having consumed alcohol in the past month; 23.3%, cocaine; and 11.7%, marijuana. Likewise, 40% smoked tobacco. Seventy-five percent of the sex workers reported being a high drinker. Most of the women reported that customers generally pressed them in to drinking alcohol and a high percentage reported that they consumed it because of the demands of bar or cabaret managers. The experiences of workplace violence were associated with cocaine use (χ2 = 6.417 [100, 1] df p <.05) and tobacco (χ2 = 7.486 [100.1] gl p <.01). This association remained if violence had occurred in the last month. Alcohol consumption was associated with experiences of physical violence (χ2 = 5.180 [100.1] gl p <.05), and emotional abuse (χ2 = 4.514 [100.1] gl p <.05). Discussion The results show that violence is a common experience in the work environment of these women, and that this is carried out by multiple actors. Findings highlight that sex workers point out to other sex workers as those who exercise more violence. High alcohol use and the use of other drugs are associated with this violence, requiring future studies to explore in-depth this link. The findings are discussed, with recommendations for the care of these problems in female sex workers.

11.
Tuberculosis (Edinb) ; 93(5): 515-22, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23867321

RESUMO

Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance.


Assuntos
Amidoidrolases/genética , Antituberculosos/farmacologia , Mycobacterium tuberculosis/metabolismo , Pirazinamida/farmacologia , Amidoidrolases/biossíntese , Amidoidrolases/metabolismo , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana/fisiologia , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas/genética , Pirazinamida/análogos & derivados , Pirazinamida/metabolismo , RNA Bacteriano/genética , RNA Mensageiro/genética
12.
Am J Trop Med Hyg ; 87(1): 153-61, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22764307

RESUMO

Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co(2+), Mn(2+), and Zn(2+) restored pyrazinamidase activity, only Co(2+) enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu(2+), Fe(2+), Fe(3+), and Mg(2+) did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn(2+). However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.


Assuntos
Amidoidrolases/metabolismo , Metais/farmacologia , Mycobacterium tuberculosis/enzimologia , Amidoidrolases/química , Dicroísmo Circular , Cinética , Modelos Moleculares , Espectrofotometria Atômica
13.
Microb Drug Resist ; 18(4): 372-5, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22372927

RESUMO

BACKGROUND: Pyrazinamide (PZA) is an important drug in the treatment of tuberculosis. Microbiological methods of PZA susceptibility testing are controversial and have low reproducibility. After conversion of PZA into pyrazinoic acid (POA) by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. OBJECTIVE: To evaluate the rate of POA extrusion from Mycobacterium tuberculosis as a parameter to detect PZA resistance. METHODS: The rate of POA extrusion and PZA susceptibility determined by BACTEC 460 were measured for 34 strains in a previous study. PZA resistance was modeled in a logistic regression with the pyrazinoic efflux rate. RESULT: POA efflux rate predicted PZA resistance with 70.83%-92.85% sensitivity and 100% specificity compared with BACTEC 460. CONCLUSION: POA efflux rate could be a useful tool for predicting PZA resistance in M. tuberculosis. Further exploration of this approach may lead to the development of new tools for diagnosing PZA resistance, which may be of public health importance.


Assuntos
Antituberculosos/metabolismo , Farmacorresistência Bacteriana/fisiologia , Mycobacterium tuberculosis/metabolismo , Pirazinamida/análogos & derivados , Pirazinamida/metabolismo , Antituberculosos/farmacologia , Transporte Biológico/fisiologia , Biotransformação , Humanos , Cinética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Pirazinamida/farmacologia , Análise de Regressão , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tuberculose Pulmonar/microbiologia
14.
Tuberculosis (Edinb) ; 92(1): 84-91, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22004792

RESUMO

Pyrazinamide is one of the most important drugs in the treatment of latent Mycobacterium tuberculosis infection. The emergence of strains resistant to pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include pyrazinamide. The accepted mechanism of action states that after the conversion of pyrazinamide into pyrazinoic acid by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. The pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of pyrazinamidase activity significantly contribute to pyrazinamide resistance, the mechanism of resistance is not completely understood. The pyrazinoic acid efflux rate may depend on multiple factors, including pyrazinamidase activity, intracellular pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the pyrazinoic acid efflux rate to the susceptibility to pyrazinamide is recognized, its quantitative effect remains unknown. Thirty-four M. tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which pyrazinoic acid is released from the bacteria and the initial velocity at which pyrazinamide enters the bacteria were estimated. The data indicated that pyrazinoic acid efflux rates for pyrazinamide-susceptible M. tuberculosis strains fell within a specific range, and M. tuberculosis strains with a pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high pyrazinoic acid efflux rate for M. smegmatis, which is innately resistant to pyrazinamide: its pyrazinoic acid efflux rate was found to be 900 fold higher than the average efflux rate for M. tuberculosis strains. No significant variability was observed in the pyrazinamide flux rate. The pyrazinoic acid efflux rate explained 61% of the variability in Bactec pyrazinamide susceptibility, 24% of Wayne activity, and 51% of the Bactec 460TB growth index. In contrast, pyrazinamidase activity accounted for only 27% of the Bactec pyrazinamide susceptibility. This finding suggests that mechanisms other than pncA mutations (reduction of pyrazinamidase activity) are also implicated in pyrazinamide resistance, and that pyrazinoic acid efflux rate acts as a better proxy for pyrazinamide resistance than the presence of pncA mutations. This is relevant to the design of molecular diagnostics for pyrazinamide susceptibility, which currently rely on pncA gene mutation detection.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/análogos & derivados , Pirazinamida/farmacologia , Amidoidrolases/genética , Humanos , Mutação/efeitos dos fármacos , Saúde Pública
15.
BMC Infect Dis ; 11: 269, 2011 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-21985457

RESUMO

BACKGROUND: Bleach-sedimentation may improve microscopy for diagnosing tuberculosis by sterilising sputum and concentrating Mycobacterium tuberculosis. We studied gravity bleach-sedimentation effects on safety, sensitivity, speed and reliability of smear-microscopy. METHODS: This blinded, controlled study used sputum specimens (n = 72) from tuberculosis patients. Bleach concentrations and exposure times required to sterilise sputum (n = 31) were determined. In the light of these results, the performance of 5 gravity bleach-sedimentation techniques that sterilise sputum specimens (n = 16) were compared. The best-performing of these bleach-sedimentation techniques involved adding 1 volume of 5% bleach to 1 volume of sputum, shaking for 10-minutes, diluting in 8 volumes distilled water and sedimenting overnight before microscopy. This technique was further evaluated by comparing numbers of visible acid-fast bacilli, slide-reading speed and reliability for triplicate smears before versus after bleach-sedimentation of sputum specimens (n = 25). Triplicate smears were made to increase precision and were stained using the Ziehl-Neelsen method. RESULTS: M. tuberculosis in sputum was successfully sterilised by adding equal volumes of 15% bleach for 1-minute, 6% for 5-minutes or 3% for 20-minutes. Bleach-sedimentation significantly decreased the number of acid-fast bacilli visualised compared with conventional smears (geometric mean of acid-fast bacilli per 100 microscopy fields 166, 95%CI 68-406, versus 346, 95%CI 139-862, respectively; p = 0.02). Bleach-sedimentation diluted paucibacillary specimens less than specimens with higher concentrations of visible acid-fast bacilli (p = 0.02). Smears made from bleach-sedimented sputum were read more rapidly than conventional smears (9.6 versus 11.2 minutes, respectively, p = 0.03). Counting conventional acid-fast bacilli had high reliability (inter-observer agreement, r = 0.991) that was significantly reduced (p = 0.03) by bleach-sedimentation (to r = 0.707) because occasional strongly positive bleach-sedimented smears were misread as negative. CONCLUSIONS: Gravity bleach-sedimentation improved laboratory safety by sterilising sputum but decreased the concentration of acid-fast bacilli visible on microscopy, especially for sputum specimens containing high concentrations of M. tuberculosis. Bleach-sedimentation allowed examination of more of each specimen in the time available but decreased the inter-observer reliability with which slides were read. Thus bleach-sedimentation effects vary depending upon specimen characteristics and whether microscopy was done for a specified time, or until a specified number of microscopy fields had been read. These findings provide an explanation for the contradictory results of previous studies.


Assuntos
Técnicas Bacteriológicas/métodos , Centrifugação/métodos , Desinfecção/métodos , Mycobacterium tuberculosis/isolamento & purificação , Manejo de Espécimes/métodos , Escarro/microbiologia , Tuberculose/diagnóstico , Desinfetantes/farmacologia , Humanos , Microscopia/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Hipoclorito de Sódio/farmacologia , Fatores de Tempo , Tuberculose/microbiologia
16.
PLoS One ; 5(3): e9577, 2010 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-20351778

RESUMO

BACKGROUND: The microscopic observation drug susceptibility (MODS) assay for rapid, low-cost detection of tuberculosis and multidrug resistant tuberculosis depends upon visualization of the characteristic cording colonies of Mycobacterium tuberculosis in liquid media. This has conventionally required an inverted light microscope in order to inspect the MODS culture plates from below. Few tuberculosis laboratories have this item and the capital cost of $5,000 for a high-end microscope could be a significant obstacle to MODS roll-out. METHODOLOGY: We hypothesized that the precise definition provided by costly high-specification inverted light microscopes might not be necessary for pattern recognition. SIGNIFICANCE: In this work we describe the development of a low-cost artesenal inverted microscope that can operate in both a standard or digital mode to effectively replace the expensive commercial inverted light microscope, and an integrated system that could permit a local and remote diagnosis of tuberculosis.


Assuntos
Testes de Sensibilidade Microbiana/métodos , Técnicas Microbiológicas/métodos , Microscopia/métodos , Mycobacterium tuberculosis/metabolismo , Desenho de Equipamento , Humanos , Testes de Sensibilidade Microbiana/economia , Técnicas Microbiológicas/economia , Microscopia/economia , Microscopia/instrumentação , Tuberculose/diagnóstico , Tuberculose/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
17.
Quito; Fondo de Desarrollo de la Naciones Unidas para la Mujer (UNIFEM). Oficina Regional Países Andinos; 1 ed; Mar. 2010. 80 p. ilus.(Serie: En la Economía Contamos Todas y Todos. Cuaderno de trabajo, 10).
Monografia em Espanhol | PERNAL | ID: pnc-6378

RESUMO

El presente documento actualiza los hallazgos encontrados en el estudio realizado en Perú, durante la primera fase del programa. En él se sintetiza los principales avances identificados en la ayuda al desarrollo y en el presupuesto nacional, que podrían servir para reforzar la eventual institucionalización de los Presupuestos Sensibles al Género (PSG). Se presenta brevemente un diagnóstico de la cooperación al desarrollo (Políticas de donantes, flujos de la ayuda, principales instrumentos que se emplean para la gestión de la ayuda en el país y, los instrumentos de monitoreo) y también, se sintetiza la estructura de planificación, de evaluación y del presupuesto nacional, para luego mostrar el grado de avance de la incorporación del enfoque de género en estos elementos(AU)


Assuntos
Gênero e Saúde , Identidade de Gênero , Orçamentos , Desenvolvimento Econômico , Desenvolvimento Sustentável , Política de Inovação e Desenvolvimento , Peru
18.
Tuberculosis (Edinb) ; 89(2): 109-13, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19249243

RESUMO

Resistance of Mycobacterium tuberculosis to pyrazinamide is associated with mutations in the pncA gene, which codes for pyrazinamidase. The association between the enzymatic activity of mutated pyrazinamidases and the level of pyrazinamide resistance remains poorly understood. Twelve M. tuberculosis clinical isolates resistant to pyrazinamide were selected based on Wayne activity and localization of pyrazinamidase mutation. Recombinant pyrazinamidases were expressed and tested for their kinetic parameters (activity, k(cat), K(m), and efficiency). Pyrazinamide resistance level was measured by Bactec-460TB and 7H9 culture. The linear correlation between the resistance level and the kinetic parameters of the corresponding mutated pyrazinamidase was calculated. The enzymatic activity and efficiency of the mutated pyrazinamidases varied with the site of mutation and ranged widely from low to high levels close to the corresponding of the wild type enzyme. The level of resistance was significantly associated with pyrazinamidase activity and efficiency, but only 27.3% of its statistical variability was explained. Although pyrazinamidase mutations are indeed associated with resistance, the loss of pyrazinamidase activity and efficiency as assessed in the recombinant mutated enzymes is not sufficient to explain a high variability of the level of pyrazinamide resistance, suggesting that complementary mechanisms for pyrazinamide resistance in M. tuberculosis with mutations in pncA are more important than currently thought.


Assuntos
Amidoidrolases/metabolismo , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Pirazinamida/farmacologia , Amidoidrolases/genética , Clonagem Molecular , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteínas Recombinantes/metabolismo , Escarro/microbiologia
19.
Blood ; 110(13): 4331-40, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17823309

RESUMO

The T-cell receptor beta (TCRbeta)/pre-TCRalpha (pTalpha) pre-TCR complex (pre-TCR) signals the expansion and differentiation of de-veloping thymocytes. Functional pro-perties of the pre-TCR rely on its unique pTalpha chain, which suggests the participation of specific intracellular adaptors. However, pTalpha-interacting molecules remain unknown. Here, we identified a polyproline-arginine sequence in the human pTalpha cytoplasmic tail that interacted in vitro with SH3 domains of the CIN85/CMS family of adaptors, and mediated the recruitment of multiprotein complexes involving all (CMS, CIN85, and CD2BP3) members. Supporting the physiologic relevance of this interaction, we found that 1 such adaptor, CMS, interacted in vivo with human pTalpha, and its expression was selectively up-regulated during human thymopoiesis in pre-TCR-activated thymocytes. Upon activation, pre-TCR clustering was induced, and CMS and polymerized actin were simultaneously recruited to the pre-TCR activation site. CMS also associated via its C-terminal region to the actin cytoskeleton in the endocytic compartment, where it colocalized with internalized pTalpha in traffic to lysosomal degradation. Notably, deletion of the pTalpha CIN85/CMS-binding motif impaired pre-TCR-mediated Ca(2+) mobilization and NFAT transcriptional activity, and precluded activation induced by overexpression of a CMS-SH3 N-terminal mutant. These results provide the first molecular evidence for a pTalpha intracellular adaptor involved in pre-TCR function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação , Sinalização do Cálcio , Linhagem Celular , Proteínas do Citoesqueleto/genética , Citosol , Humanos , Complexos Multiproteicos , Fatores de Transcrição NFATC/metabolismo , Timo/citologia , Transfecção
20.
J Biol Chem ; 277(6): 3993-4002, 2002 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-11724779

RESUMO

The bases that support the versatility of the T cell receptor (TCR) to generate distinct T cell responses remain unclear. We have previously shown that mutant cells in the transmembrane domain of TCRbeta chain are impaired in TCR-induced apoptosis but are not affected in other functions. Here we describe the biochemical mechanisms by which this mutant receptor supports some T cell responses but fails to induce apoptosis. Extracellular signal-regulated protein kinase (ERK) is activated at higher and more sustained levels in TCRbeta-mutated than in wild type cells. Conversely, activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase is severely reduced in mutant cells. By attempting to link this unbalanced induction to altered upstream events, we found that ZAP-70 is normally activated. However, although SLP-76 phosphorylation is normally induced, TCR engagement of mutant cells results in lower tyrosine phosphorylation of LAT but in higher tyrosine phosphorylation of Vav than in wild type cells. The results suggest that an altered signaling cascade leading to an imbalance in mitogen-activated protein kinase activities is involved in the selective impairment of apoptosis in these mutant cells. Furthermore, they also provide new insights in the contribution of TCR to decipher the signals that mediate apoptosis distinctly from proliferation.


Assuntos
Apoptose , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/fisiologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Humanos , Células Jurkat , Lectinas Tipo C , Mutação , Fosforilação , Testes de Precipitina , Proteína Quinase C/metabolismo , Transporte Proteico , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70
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