RESUMO
OBJECTIVES: The aim of the present study was to validate a commercially available automated assay for the measurement of total adenosine deaminase (tADA) and its isoenzymes (ADA1 and ADA2) in saliva in a fast and accurate way, and evaluate the possible changes of these analytes in individuals with SARS-CoV-2 infection. METHODS: The validation, in addition to the evaluation of precision and accuracy, included the analysis of the effects of the main procedures that are currently being used for SARS-CoV-2 inactivation in saliva and a pilot study to evaluate the possible changes in salivary tADA and isoenzymes in individuals infected with SARS-CoV-2. RESULTS: The automated assay proved to be accurate and precise, with intra- and inter-assay coefficients of variation below 8.2%, linearity under dilution linear regression with R2 close to 1, and recovery percentage between 80 and 120% in all cases. This assay was affected when the sample is treated with heat or SDS for virus inactivation but tolerated Triton X-100 and NP-40. Individuals with SARS-CoV-2 infection (n=71) and who recovered from infection (n=11) had higher mean values of activity of tADA and its isoenzymes than healthy individuals (n=35). CONCLUSIONS: tADA and its isoenzymes ADA1 and ADA2 can be measured accurately and precisely in saliva samples in a rapid, economical, and reproducible way and can be analyzed after chemical inactivation with Triton X-100 and NP-40. Besides, the changes observed in tADA and isoenzymes in individuals with COVID-19 open the possibility of their potential use as non-invasive biomarkers in this disease.
Assuntos
Adenosina Desaminase/metabolismo , Bioensaio/métodos , Biomarcadores/metabolismo , COVID-19/diagnóstico , SARS-CoV-2/enzimologia , Saliva/enzimologia , Adulto , COVID-19/virologia , Estudos de Casos e Controles , Feminino , Humanos , Isoenzimas , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto JovemRESUMO
Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.
Assuntos
Orelha Média/metabolismo , Perda Auditiva/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Otite Média com Derrame/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Vesícula/metabolismo , Vesícula/patologia , Líquidos Corporais/metabolismo , Hipóxia Celular/genética , Modelos Animais de Doenças , Orelha Média/efeitos dos fármacos , Orelha Média/patologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Perda Auditiva/etiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes/genética , Nitroimidazóis/análise , Otite Média com Derrame/complicações , Ftalazinas/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais , Sunitinibe , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVES: To evaluate four sample treatments in a safe and straightforward procedure to detect SARS-CoV-2 in saliva. METHODS: Four sample treatments were evaluated in a 3-step procedure to detect SARS-CoV-2 in saliva: 1) heating at 95 °C for 5 min for sample inactivation; 2) sample treatment; 3) analysis by reverse-transcription loop-mediated isothermal amplification (LAMP). Saliva samples used were from infected individuals or were spiked with known quantities of viral particles. RESULTS: Three treatments had a limit of detection (LOD) of 500.000 viral particles per ml of saliva and could be used to detect individuals with potential to transmit the disease. The treatment of phosphate buffer, dithiothreitol, ethylenediaminetetraacetic acid and proteinase K, with an additional 95 °C heating step, yielded a lower LOD of 95; its sensitivity ranged from 100% in patients with nasopharyngeal swab reverse-transcriptase polymerase chain reaction cycle threshold values <20 to 47.8% for values >30. CONCLUSIONS: This report highlights the importance of an adequate sample treatment for saliva to detect SARS-CoV-2 and describes a flexible procedure that can be adapted to point-of-care. Although its sensitivity when LAMP is used is lower than reverse-transcriptase polymerase chain reaction, this procedure can contribute to COVID-19 control by detecting individuals able to transmit the disease.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Saliva , Sensibilidade e EspecificidadeRESUMO
High ferritin serum levels can be found in patients with macrophage activation syndrome, and increased serum ferritin due to cytokine storm have been reported in severe COVID-19 patients. Saliva is being increasingly used in COVID-19 tests as a diagnostic sample for virus detection and quantification. This study aimed to evaluate the possible changes in ferritin in saliva in COVID-19 patients. In addition, the effects of different inactivation SARS-CoV-2 treatments in ferritin measurements in saliva, the correlation between ferritin in saliva and serum, and the possible effects of correction of ferritin values by total protein were assessed. Ferritin was measured in saliva from healthy (n = 30) and COVID-19 (n = 65) patients with severe, (n = 18) or mild (n = 47) disease, depending on the need for nasal flow oxygen or assisted respiration. Ferritin was also measured in paired serum and saliva samples (n = 32) from healthy and COVID-19 patients. The evaluated inactivation protocols did not affect the assay's results except the addition of 0.5% SDS. Significantly higher ferritin was found in the saliva of COVID-19 patients (median; 25-75th percentile) (27.75; 9.77-52.2 µg/L), compared with healthy controls (4.21; 2.6-8.08 µg/L). Individuals with severe COVID-19 showed higher ferritin values in saliva (48.7; 18.7-53.9) than mild ones (15.5; 5.28-41.3 µg/L). Significant correlation (r = 0.425; p < 0.001) was found between serum and saliva in ferritin. Ferritin levels were higher in COVID-19 patients in serum and saliva, and the highest values were found in those patients presenting severe symptomatology. In conclusion, ferritin in saliva has the potential to be a biomarker to evaluate severity in patients with COVID-19.
Assuntos
COVID-19 , Ferritinas/análise , Saliva/química , Biomarcadores , COVID-19/diagnóstico , HumanosRESUMO
Desde el 70 al 80 Aniversario de la fundación del Instituto de Medicina Tropical "Pedro Kourí", los investigadores, profesionales y técnicos de este centro, han laborado en cooperación con diversas instituciones académicas y de los servicios de salud de Cuba (en equipos multidisciplinarios) en las tareas de desarrollo científico, innovación tecnológica y construcción de capacidades. Este artículo resume las principales investigaciones realizadas sobre la tuberculosis desde 2008-2017; como contribuciones al Programa Nacional de Control y Eliminación de la Tuberculosis con la perspectiva de aumentar la calidad, efectividad y eficiencia de las acciones y acelerar la solución de la incidencia y la mortalidad por tuberculosis, para la investigación, la vigilancia y control de la tuberculosis. Se presentan evidencias que fundamentan las innovaciones en el manejo clínico terapéutico de pacientes inmunocompetentes e inmunocomprometidos, en niños y adolescentes, sobre las indicaciones para el control de la infección tuberculosa latente, la vigilancia de los desenlaces del tratamiento anti-tuberculosis, la investigación de contactos de casos, los cambios en el sistema de registro e información, el monitoreo y evaluación de la calidad de la detección de casos de tuberculosis, el impacto sobre la morbilidad y la mortalidad.
During the period extending from the 70th to the 80th anniversary of the founding of Pedro Kouri Tropical Medicine Institute, researchers, professionals and technicians from the center, in joint cooperation with a variety of Cuban academic and health care institutions, have integrated into multidisciplinary teams engaged in tasks related to scientific development, technological innovation and capacity building. The paper summarizes the main research studies about tuberculosis conducted from 2008 to 2017 as contributions to the National Tuberculosis Control and Elimination Program, with a view to enhancing the quality, effectiveness and efficiency of actions, swiftly finding a solution to tuberculosis incidence and mortality, and fostering tuberculosis research, surveillance and control. Evidence is presented which substantiates innovations in the clinical therapeutic management of both immunocompetent and immunocompromised patients, children and adolescents, and indications are provided for latent tuberculosis infection control, surveillance of anti-tuberculosis treatment outcomes, investigation of case contacts, changes in the record and information system, quality monitoring and evaluation of the detection of tuberculosis cases, and impact on morbidity and mortality.
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BACKGROUND: Jeff is a dominant mouse mutant displaying chronic otitis media. The gene underlying Jeff is Fbxo11, a member of the large F-box family, which are specificity factors for the SCF E3 ubiquitin ligase complex. Jeff homozygotes die shortly after birth displaying a number of developmental abnormalities including cleft palate and eyes open at birth. TGF-beta signalling is involved in a number of epithelial developmental processes and we have investigated the impact of the Jeff mutation on the expression of this pathway. RESULTS: Phospho-Smad2 (pSmad2) is significantly upregulated in epithelia of Jeff homozygotes. Moreover, there was a significant increase in nuclear localization of pSmad2 in contrast to wild type. Mice heterozygous for both Jeff and Smad2 mutations recapitulate many of the features of the Jeff homozygous phenotype. However, tissue immunoprecipitations failed to detect any interaction between Fbxo11 and Smad2. Fbxo11 is known to neddylate p53, a co-factor of pSmad2, but we did not find any evidence of genetic interactions between Jeff and p53 mutants. Nevertheless, p53 levels are substantially reduced in Jeff mice suggesting that Fbxo11 plays a role in stabilizing p53. CONCLUSION: Overall, our findings support a model whereby Fbxo11, possibly via stabilization of p53, is required to limit the accumulation of pSmad2 in the nucleus of epithelial cells of palatal shelves, eyelids and airways of the lungs. The finding that Fbxo11 impacts upon TGF-beta signalling has important implications for our understanding of the underlying disease mechanisms of middle ear inflammatory disease.