Cost-Effective Trap qPCR Approach to Evaluate Telomerase Activity: an Important Tool for Aging, Cancer, and Chronic Disease Research

OBJECTIVES: Telomeres are a terminal "DNA cap" that prevent chromosomal fusion and degradation. However, aging is inherent to life, and so is the loss of terminal sequences. Telomerase is a specialized reverse transcriptase encoded by self-splicing introns that counteract chromosome erosion. Telomerase activity is observed during early embryonic development, but after the blastocyst stage, the expression of telomerase reduces. The consequences of either insufficient or unrestrained telomerase activity underscore the importance of ongoing studies aimed at elucidating the regulation of telomerase activity in humans. In the present study, we aimed to standardize a simplified telomerase repeat-amplification protocol (TRAP) assay to detect telomerase activity in unstimulated and PHA-stimulated mononuclear cells. METHODS and RESULTS: Our optimized qPCR-based can efficiently evaluate telomerase activity. Quantification of protein and DNA between unstimulated and PHA-stimulated peripheral blood mononuclear cells revealed cellular activation and cell-cycle entry. The assay also showed that relative telomerase activity is significantly different between these two conditions, supporting the applicability of the assay. Furthermore, our findings corroborated that telomerase activity decreases with age. CONCLUSIONS: Telomeres and telomerase are implicated in aging and development of chronic diseases and cancer; however, difficulty in accessing commercial kits to investigate these aspects is a critical constraint in health surveillance studies. Our optimized assay was successfully used to differentiate telomerase activity between unstimulated and stimulated cells, clearly showing the reactivation of telomerase upon cell activation. This assay is affordable, reproducible, and can be executed in resource-limited settings.

Moderate levels of physical fitness maintain telomere length in non-senescent T CD8+ cells of aged men

OBJECTIVES: Immunosenescence is an age-associated change characterized by a decreased immune response. Although physical activity has been described as fundamental for maintaining the quality of life, few studies have evaluated the effects of different levels of exercise on telomere length in aged populations. The present study aimed to analyze the effects of different levels of physical activity, classified by the Maximal oxygen consumption (VO2 max) values, on the telomere length of memory Cluster of differentiation (CD) CD4+(CD45ROneg and CD45RO+), effector CD8+CD28neg, and CD8+CD28+ T cells in aged individuals. METHODS: Fifty-three healthy elderly men (aged 65-85 years) were included in this study. Their fitness level was classified according to the American College of Sports Medicine (ACSM) for VO2 max (mL/kg/min). Blood samples were obtained from all participants to analyze the percentage of CD3, CD4, CD8, CD28+, naïve, and subpopulations of memory T cells by using flow cytometry. Furthermore, using the Flow-FISH methodology, the CD4+CD45RO+, CD4+CD45ROneg, CD8+CD28+, and CD8+CD28negT cell telomere lengths were measured. RESULTS: There was a greater proportion of effector memory T CD4+ cells and longer telomeres in CD8+CD28+ T cells in the moderate physical fitness group than in the other groups. There was a higher proportion of terminally differentiated memory effector T cells in the low physical fitness group. CONCLUSION: A moderate physical activity may positively influence the telomere shortening of CD28+CD8+T cells. However, additional studies are necessary to evaluate the importance of this finding with regard to immune function responses in older men.

Lung cysts in chronic paracoccidioidomycosis / Cistos pulmonares na paracoccidioidomicose crônica

On HRCT scans, lung cysts are characterized by rounded areas of low attenuation in the lung parenchyma and a well-defined interface with the normal adjacent lung. The most common cystic lung diseases are lymphangioleiomyomatosis, Langerhans cell histiocytosis, and lymphocytic interstitial pneumonia. In a retrospective analysis of the HRCT findings in 50 patients diagnosed with chronic paracoccidioidomycosis, we found lung cysts in 5 cases (10%), indicating that patients with paracoccidioidomycosis can present with lung cysts on HRCT scans. Therefore, paracoccidioidomycosis should be included in the differential diagnosis of cystic lung diseases. .

Os cistos pulmonares na TCAR são caracterizados por áreas arredondadas de baixo coeficiente de atenuação no parênquima pulmonar com uma interface bem definida com o pulmão adjacente normal. As doenças pulmonares císticas mais comuns são linfangioleiomiomatose, histiocitose de células de Langerhans e pneumonia intersticial linfocítica. Em uma análise retrospectiva de achados de TCAR em 50 pacientes com diagnóstico de paracoccidioidomicose crônica residual, observou-se a presença de cistos pulmonares em 5 casos (10%), mostrando que pacientes com paracoccidioidomicose podem apresentar cistos pulmonares na TCAR. Portanto, essa infecção deve entrar no diagnóstico diferencial das doenças císticas pulmonares. .

The lung in paracoccidioidomycosis: new insights into old problems

OBJECTIVES: Chronic paracoccidioidomycosis can diffusely affect the lungs. Even after antifungal therapy, patients may present with residual respiratory abnormalities due to fungus-induced lung fibrosis. METHODS: A cross-sectional analysis of 50 consecutive inactive, chronic paracoccidioidomycosis patients was performed using high resolution computed tomography, pulmonary function tests, ergospirometry, the six-minute walk test and health-related quality of life questionnaires. RESULTS: Radiological abnormalities were present in 98% of cases, the most frequent of which were architectural distortion (90%), reticulate and septal thickening (88%), centrilobular and paraseptal emphysema (84%) and parenchymal bands (74%). Patients typically presented with a mild obstructive disorder and a mild reduction in diffusion capacity with preserved exercise capacity, including VO2max and six-minute walking distance. Patient evaluation with the Saint-George Respiratory Questionnaire showed low impairment in the health-related quality of life, and the Medical Research Council questionnaire indicated a low dyspnea index. There were, however, patients with significant oxygen desaturation upon exercise that was associated with respiratory distress compared with the non-desaturated patients. The initial counterimmunoelectrophoresis of these patients was higher and lung emphysema was more prominent; however, there were no differences in the interstitial fibrotic tomographic abnormalities, tobacco exposure, functional responses, exercise capacity or quality of life. CONCLUSIONS: Inactive, chronic paracoccidioidomycosis patients show persistent and disseminated radiological abnormalities by high resolution computed tomography, short impairments in pulmonary function and low impacts on aerobic capacity and quality of life. However, there was a subset of individuals whose functional impairment was more severe. These patients present with higher initial ...