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Clinics ; 76: e2432, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153954


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.

Humans , Female , Pregnancy , Telomerase/genetics , Telomerase/metabolism , Neoplasms , Aging , Leukocytes, Mononuclear/metabolism , Chronic Disease , Cost-Benefit Analysis
J. appl. oral sci ; 27: e20180529, 2019. graf
Article in English | LILACS | ID: biblio-1012507


Abstract Objectives: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. Methodology: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1β, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. Results: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1β and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1β and TNF-α, whereas UDMA reduced IL-1β release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. Conclusions: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1β and TNF-α secretion by PBMC.

Humans , Polyethylene Glycols/pharmacology , Polymethacrylic Acids/pharmacology , Polyurethanes/pharmacology , Leukocytes, Mononuclear/drug effects , Cytokines/metabolism , Bisphenol A-Glycidyl Methacrylate/pharmacology , Porphyromonas gingivalis/physiology , Methacrylates/pharmacology , Reference Values , Time Factors , Enzyme-Linked Immunosorbent Assay , Leukocytes, Mononuclear/metabolism , Cell Survival/drug effects , Reproducibility of Results , Analysis of Variance , Cytokines/analysis , Cytokines/drug effects , Apoptosis/drug effects , Statistics, Nonparametric , Necrosis
J. bras. nefrol ; 40(4): 333-338, Out.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-984593


ABSTRACT Introduction: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). Objective: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). Methods: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. Results: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. Conclusion: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.

RESUMO Introdução: A doença de Fabry (DF) é um distúrbio causado por mutações no gene que codifica a enzima lisossômica α-galactosidase A (α-GAL). A redução da atividade de α-GAL leva ao acúmulo progressivo de globotriaosilceramida (Gb3), também conhecida como CD77. O recente relato de aumento da expressão de CD77 em células sanguíneas de pacientes com DF indicou que essa molécula pode ser utilizada como um potencial marcador para o monitoramento da terapia de reposição enzimática (TRE). Objetivo: O objetivo deste estudo foi avaliar os níveis de CD77 ao longo da TRE em pacientes com DF (mutação V269M). Métodos: Foram avaliadas as flutuações na expressão de CD77 nas membranas das CMSP (células mononucleares do sangue periférico) em pacientes com DF submetidos à TRE e correlacionados com aqueles observados em diferentes tipos de células. Resultados: Uma maior expressão de CD77 foi encontrada em fagócitos de pacientes em comparação aos controles no início do estudo. Curiosamente, a variabilidade nos níveis de CD77 é maior em pacientes no início do estudo (340 - 1619 MIF) e após 12 meses de TRE (240 - 530 MIF) em comparação com o grupo controle (131 - 331 MFI). Além disso, analisando os níveis de CD77 em fagócitos de pacientes ao longo da TRE, encontramos uma diminuição constante nos níveis de CD77. Conclusão: O aumento nos níveis de CD77 nos fagócitos de portadores de Fabry, juntamente com a diminuição nos níveis de CD77 ao longo da TRE, sugerem que medir os níveis de CD77 nos fagócitos é uma ferramenta promissora para monitorar a resposta à TRE na DF.

Humans , Male , Female , Adult , Young Adult , Trihexosylceramides/biosynthesis , Leukocytes, Mononuclear/metabolism , Fabry Disease/drug therapy , Fabry Disease/blood , alpha-Galactosidase/therapeutic use , Enzyme Replacement Therapy , Trihexosylceramides/analysis , Leukocytes, Mononuclear/chemistry
Braz. j. med. biol. res ; 51(8): e7334, 2018. graf
Article in English | LILACS | ID: biblio-951739


Pregnancy-induced hypertension (PIH) causes significant maternal and fetal morbidity and mortality. A decreased number of regulatory T (Treg) cells is associated with the pathogenesis of PIH. The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical to normal pregnancy (NP) by promoting Treg cell development. However, the relationship between PD-1/PD-L1 and Treg differentiation in PIH has not been fully elucidated. In this study, venous blood was obtained from 20 NP and 58 PIH patients. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood. The levels of Treg-related cytokines (TGF-β, IL-10, and IL-35) in serum and PBMCs were measured by ELISA. The percentage of Treg cells in PBMCs was assessed by flow cytometry. The mRNA levels of Treg-specific transcription factor Foxp3 in PBMCs, and PD-1 and PD-L1 in Treg cells were detected by qRT-PCR. The protein levels of PD-1 and PD-L1 in Treg cells were evaluated by western blot. The serum levels of TGF-β, IL-10, IL-35, and Foxp3 mRNA expression and CD4+CD25+ Treg cell percentage in PBMCs were decreased in PIH. Furthermore, a significant increase of PD-1 in Treg cells was found in PIH compared with NP. In addition, PD-L1 Fc, an activator of PD-1/PD-L1 pathway, increased Treg cell percentage, enhanced Foxp3 mRNA expression, and elevated levels of TGF-β, IL-10, and IL-35 in PBMCs. However, anti-PD-L1 mAb exerted a reverse effect. These findings revealed that PD-L1 Fc had a favorable effect on Treg cell differentiation, indicating a potential therapeutic value of PD-1/PD-L1 pathway for PIH treatment.

Humans , Female , Pregnancy , Leukocytes, Mononuclear/chemistry , Interleukins/metabolism , Interleukin-10/metabolism , Apoptosis , Hypertension, Pregnancy-Induced/metabolism , B7-H1 Antigen/metabolism , Enzyme-Linked Immunosorbent Assay , Leukocytes, Mononuclear/metabolism , Case-Control Studies , Blotting, Western , Transforming Growth Factor beta/metabolism , T-Lymphocytes, Regulatory/metabolism , Real-Time Polymerase Chain Reaction
Braz. j. med. biol. res ; 50(10): e6139, 2017. tab, graf
Article in English | LILACS | ID: biblio-888929


Augmenter of liver regeneration (ALR) is a thermostable cytokine that was originally identified to promote the growth of hepatocytes. This study was conducted to explore the expression and function of ALR in multiple myeloma (MM), a common hematologic malignancy. Real-time PCR and western blot analysis were performed to detect the expression of ALR in U266 human MM cells and healthy peripheral blood mononuclear cells (PBMCs). U266 MM cells were exposed to 20 or 40 μg/mL of recombinant ALR and tested for cell proliferation. Small interfering RNA-mediated silencing of ALR was done to investigate the role of ALR in cell proliferation, apoptosis, and cytokine production. Compared to PBMCs, U266 MM cells exhibited significantly higher levels of ALR at both the mRNA and protein levels. The addition of recombinant ALR protein significantly promoted the proliferation of U266 cells. In contrast, knockdown of ALR led to a significant decline in the viability and proliferation of U266 cells. Annexin-V/PI staining analysis demonstrated that ALR downregulation increased apoptosis in U266 MM cells, compared to control cells (20.1±1.1 vs 9.1±0.3%, P<0.05). Moreover, ALR depletion reduced the Bcl-2 mRNA level by 40% and raised the Bax mRNA level by 2-fold. Additionally, conditioned medium from ALR-depleted U266 cells had significantly lower concentrations of interleukin-6 than control cells (P<0.05). Taken together, ALR contributed to the proliferation and survival of U266 MM cells, and targeting ALR may have therapeutic potential in the treatment of MM.

Humans , Apoptosis/drug effects , Cell Proliferation/drug effects , Multiple Myeloma/metabolism , Proteins/pharmacology , Blotting, Western , Cell Line, Tumor , Cytokines/biosynthesis , Down-Regulation , Flow Cytometry , Leukocytes, Mononuclear/metabolism , Multiple Myeloma/immunology , Proteins/physiology , Real-Time Polymerase Chain Reaction , Recombinant Proteins/pharmacology , RNA, Small Interfering/metabolism
Braz. j. med. biol. res ; 50(8): e5163, 2017. graf
Article in English | LILACS | ID: biblio-888986


Pseudobrickellia brasiliensis (Asteraceae) is a plant commonly known as arnica-do-campo and belongs to the native flora of the Brazilian Cerrado. The alcoholic extract of the plant has been used as an anti-inflammatory agent in folk medicine, but the biological mechanism of action has not been elucidated. The present study evaluated the composition of P. brasiliensis aqueous extract and its effects on pro-inflammatory cytokine production and lymphocyte proliferation. The extracts were prepared by sequential maceration of P. brasiliensis leaves in ethanol, ethyl acetate, and water. Extract cytotoxicity was evaluated by trypan blue exclusion assay, and apoptosis and necrosis were measured by staining with annexin V-FITC and propidium iodide. The ethanolic (ETA) and acetate (ACE) extracts showed cytotoxic effects. The aqueous extract (AQU) was not cytotoxic. Peripheral blood mononuclear cells stimulated with phorbol myristate acetate and ionomycin and treated with AQU (100 μg/mL) showed reduced interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression. AQU also inhibited lymphocyte proliferative response after nonspecific stimulation with phytohemagglutinin. The aqueous extract was analyzed by liquid chromatography coupled with photodiode array detection and mass spectrometry. Quinic acid and its derivatives 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid, as well as the flavonoids luteolin and luteolin dihexoside, were detected. All these compounds are known to exhibit anti-inflammatory activity. Taken together, these findings demonstrate that P. brasiliensis aqueous extract can inhibit the pro-inflammatory cytokine production and proliferative response of lymphocytes. These effects may be related to the presence of chemical substances with anti-inflammatory actions previously reported in scientific literature.

Humans , Anti-Inflammatory Agents/pharmacology , Asteraceae/chemistry , Cell Proliferation/drug effects , Interferon-gamma/drug effects , Lymphocytes/drug effects , Plant Extracts/pharmacology , Tumor Necrosis Factor-alpha/drug effects , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Interferon-gamma/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Plant Extracts/chemistry , Time Factors , Tumor Necrosis Factor-alpha/metabolism
Braz. j. med. biol. res ; 49(3): e4853, Mar. 2016. tab, graf
Article in English | LILACS | ID: lil-771933


The objective of this study was to examine the relationship between the expression of B cell activating factor (BAFF) and BAFF receptor in patients with disease activity of systemic lupus erythematosus (SLE). Real-time RT-PCR was used to examine BAFF mRNA expression in peripheral blood monocytes of active and stable SLE patients and healthy controls. The percentage of BAFF receptor 3 (BR3) on B lymphocytes was measured by flow cytometry. Soluble BAFF levels in serum were assayed by ELISA. Microalbumin levels were assayed by an automatic immune analysis machine. BAFF mRNA and soluble BAFF levels were highest in the active SLE group, followed by the stable SLE group, and controls (P<0.01). The percentage of BR3 on B lymphocytes was downregulated in the active SLE group compared with the stable SLE group and controls (P<0.01). BAFF mRNA levels and soluble BAFF levels were higher in patients who were positive for proteinuria than in those who were negative (P<0.01). The percentage of BR3 on B lymphocytes was lower in patients who were positive for proteinuria than in those who were negative (P<0.01). The BAFF/BR3 axis may be over-activated in SLE patients. BAFF and BR3 levels may be useful parameters for evaluating treatment.

Humans , Male , Female , Adolescent , Adult , Middle Aged , Young Adult , B-Cell Activating Factor/metabolism , B-Cell Activation Factor Receptor/metabolism , Lupus Erythematosus, Systemic/metabolism , Albuminuria/urine , B-Cell Activating Factor/analysis , B-Cell Activating Factor/genetics , B-Cell Activation Factor Receptor/analysis , B-Cell Activation Factor Receptor/genetics , B-Lymphocytes/metabolism , Biomarkers/metabolism , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic/immunology , RNA, Messenger/genetics , RNA, Messenger/metabolism
Arch. argent. pediatr ; 113(5): 404-410, oct. 2015. tab
Article in Spanish | LILACS | ID: lil-757061


Introducción. Los trastornos crónicos (TC) en etapas tempranas de la vida pueden influir en diferentes dimensiones de la calidad de vida relacionada con la salud (CVRS) de los niños/as. Objetivo. Comparar la CVRS de niños/as con TC confirmados, con TC declarados y sin TC. Población y método. Estudio transversal en el marco de una investigación mayor realizada en escuelas de Córdoba y Bahía Blanca, y en los hospitales Italiano y Garrahan de Buenos Aires, Argentina, en 2012. La presencia de TC fue establecida por diagnóstico médico en hospitales o por declaración de sus cuidadores en escolares. Los niños/as de 8 a 12 años respondieron el cuestionario KIDSCREEN-52 sobre CVRS, una escala de desarrollo puberal y una escala de recursos económicos familiares. Los cuidadores indicaron el nivel educativo materno. Se estimó la asociación entre TC y CVRS ajustada por sexo, edad, desarrollo puberal, nivel educativo materno y nivel socioeconómico. Resultados. Participaron 670 duplas niños/as-cuidadores, 13,3% (n= 89) con TC confirmados (muestras de hospitales), 14,5% (n= 97) escolares con trastornos declarados y el resto eran escolares sanos. La edad promedio fue 10,2 años (desvío estándar= 1,01); 54,8% fueron niñas. Tener TC confirmados se asoció a una mayor frecuencia de bajo bienestar físico (OR 2,61; IC 95%: 1,43-4,76), mientras que la presencia de TC declarados se asoció con bajas puntuaciones en bienestar psicológico (OR 1,96; IC 95%: 1,063,63), autopercepción (OR 2,22; IC 95%: 1,283,87) y relación con los padres (OR 2,04; IC 95%: 1,21-3,44). Conclusiones. Los niños/as con TC confirmados mostraron con mayor frecuencia malestar físico y los que tenían TC declarados manifestaron malestar en áreas psicosociales, en comparación con los niños/as sin trastorno.

Introduction. Chronic conditions (CCs) in the early stages of life may have an impact on various dimensions of health-related quality of life (HRQoL) in children. Objective. To compare HRQoL in children with confirmed CCs, reported CCs, and without CC. Population and Method. Cross-sectional study conducted in 2012 in the context of a larger research study carried out at schools in Córdoba and Bahía Blanca, and at Hospital Italiano of Buenos Aires and Hospital Prof. Dr. Juan P. Garrahan at Buenos Aires. The presence of a chronic condition was established by medical diagnosis at the hospital or as reported by schoolchildren's caregivers. Eight-to-twelve year-old children completed the KIDSCREEN-52 questionnaire on HRQoL, a pubertal development scale, and a family financial resource scale. The association between CCs and HRQoL adjusted by sex, age, pubertal development, maternal education level, and socioeconomic level was estimated. Results. Six hundred and seventy children/ caregiver dyads participated; 13.3% (n= 89) had confirmed CCs, 14.5% (n= 97) were schoolchildren with reported CCs, and the rest corresponded to healthy schoolchildren. Their average age was 10.2 years old (standard deviation= 1.01); 54.8% were girls. Having a confirmed CC was associated with a higher frequency of low physical wellbeing (odds ratio --OR--: 2.61; 95% confidence interval --95% CI--:1.43-4.76), while the presence of a reported CC was associated with a low score in psychological well-being (OR: 1.96; 95% CI: 1.06-3.63), self-perception (OR: 2.22; 95% CI: 1.28-3.87), and parent relations (OR: 2.04; 95% CI: 1.21-3.44). Conclusions. Children with confirmed CCs showed a higher frequency of physical discomfort, and those with reported CCs showed discomfort in psychosocial areas compared to children without CCs.

Humans , Cytokines/genetics , Cytokines/metabolism , Proteome , Transcriptome , Cluster Analysis , Computational Biology , Gene Expression Profiling , Leukocytes, Mononuclear/metabolism , Proteomics
Arch. argent. pediatr ; 113(5): 411-418, oct. 2015. graf, tab
Article in Spanish | LILACS | ID: lil-757062


Introducción. Existen evidencias de la asociación de determinantes sociales con la salud infantil. Objetivo. Identificar características sociodemográficas asociadas a desigualdades en la salud infantil y evaluar el efecto acumulado sobre la salud de factores de riesgo basados en estas características. Población y métodos. Evaluamos niños de 4-13 años, de Bariloche, entre junio de 2008 y mayo de 2009. Características sociodemográficas consideradas: nivel socioeconómico, educación materna, embarazo adolescente, cobertura médica, inseguridad y hábitos familiares. Valoramos la percepción parental de la salud física y socioemocional, el estado nutricional y la salud bucal en relación con dichas características y con la acumulación de factores de riesgo. Utilizamos encuesta, antropometría y examen bucal. Resultados. Participaron 180 escolares. El nivel educativo materno se asoció con la salud física, socioemocional y bucal del niño. El porcentaje de niños con piezas faltantes o caries fue 77% entre aquellos cuyas madres, como máximo, habían completado el primario, comparado con 13% entre aquellos cuyas madres habían completado estudios terciarios/universitarios. La posibilidad de percepción de salud física y socioemocional no óptima aumentó con cada factor de riesgo 1,8 y 1,4 veces, respectivamente, y la posibilidad de caries o piezas faltantes se duplicó con cada factor de riesgo adicional. El 27,3% de los escolares presentó sobrepeso y el 8,7%, obesidad, y no se encontró asociación con características sociodemográficas. Conclusiones. El bajo nivel socioeconómico familiar y educativo materno se asoció con una mayor prevalencia de resultados de salud desfavorables. Múltiples factores de riesgo tienen un efecto acumulado sobre la percepción parental de la salud física y socioemocional y la salud bucal.

Introduction. There is evidence of an association between social determinants and child health. Objective. To identify sociodemographic characteristics related to child health inequalities and to analize the cumulative effect on health of risk factors based on these characteristics. Population and Methods. We evaluated 4-13 year-old children in Bariloche between June 2008 and May 2009. The following sociodemographic characteristics were taken into account: socioeconomic level, maternal education, adolescent pregnancy, medical coverage, unsafeness, and family habits. We assessed parental perception of physical, and social and emotional health, nutritional status and oral health in relation to these characteristics and the accumulation of risk factors. We used survey, anthropometry and oral examination. Results. One hundred and eighty students participated. The level of maternal education was associated with the child's physical, social and emotional, and oral health. The percentage of children with missing teeth or cavities reached 77% among those whose mothers had, at most, completed primary school, compared to 13% among those whose mothers had completed tertiary school or university. The possibility of perceiving a non-optimal physical, and social and emotional health increased 1.8 and 1.4 times with each risk factor, respectively, and the possibility of having missing teeth or cavities was twice as much with each additional risk factor. Overweight and obesity was observed in 27.3% and 8.7% of students, respectively, and no relationship was found with sociodemographic characteristics. Conclusions. A low family socioeconomic level and a low maternal education level were associated with a higher prevalence of unfavorable health outcomes. Multiple risk factors have an cumulative effect on parental perception of physical, social and emotional, and oral health.

Humans , Cell Transformation, Viral/genetics , Gene Expression Profiling , Transcriptome , Antigens, Surface/genetics , Antigens, Surface/metabolism , Cell Line, Transformed , Cell Proliferation , Cells, Cultured , Flow Cytometry , Gene Expression , Genes, Viral , Genotype , /genetics , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Transcription, Genetic , Virus Latency
Mem. Inst. Oswaldo Cruz ; 110(6): 781-785, Sept. 2015. tab, graf
Article in English | LILACS | ID: lil-763092


Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.

Adult , Female , Humans , Male , Middle Aged , Young Adult , Endemic Diseases , Genetic Predisposition to Disease , /genetics , /metabolism , Paracoccidioidomycosis/immunology , Polymorphism, Genetic/immunology , Enzyme-Linked Immunosorbent Assay , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Microsatellite Repeats , Paracoccidioidomycosis/epidemiology , Promoter Regions, Genetic/genetics , Statistics, Nonparametric
Rev. latinoam. enferm. (Online) ; 23(4): 642-650, July-Aug. 2015. tab
Article in English | LILACS, BDENF | ID: lil-761700


AbstractObjectives: develop and validate the content of a tool about nursing care production.Method: the data were collected between 2011 and 2013, based on focus groups, the application of semistructured questionnaires (prototype test) and the Delphi technique. The focus groups were used to produce the instrument items and held at three hospitals in the interior of the State of São Paulo, involving 20 nurses. A panel of 10 experts evaluated the instrument.Results: after two phases of the Delphi technique, the tool consisted of eight items. The content validity index of the scale corresponded to ≥0.9 and the content validity of the items ranged between 0.8 and 1.0, indicating the maintenance of the structure and content. The assertion on the applicability in daily nursing practice showed a content validity index of the scale equal to 0.8.Conclusion: this study permitted the development and content validation of scale on nursing care production, equipping the nurses in their management practice.

ResumoObjetivos:desenvolver e validar o conteúdo de um instrumento sobre produção do cuidado de enfermagem.Método:a coleta de dados ocorreu entre 2011 e 2013, a partir de grupos focais, aplicação de questionários semiestruturados (teste do protótipo) e técnica Delphi. Os grupos focais foram utilizados para geração de itens do instrumento e realizados em três hospitais do interior do Estado de São Paulo, com a participação de 20 enfermeiros. A apreciação do instrumento foi conduzida por um painel de 10 especialistas.Resultados:após duas fases da técnica Delphi, o instrumento passou a ser constituído por oito itens. O índice de validade do conteúdo da escala foi de ≥0,9 e a validade dos conteúdos dos itens apresentou variação de 0,8 a 1,0, indicando a manutenção da estrutura e do conteúdo. A afirmativa referente à aplicabilidade na prática diária do enfermeiro apresentou índice de validade do conteúdo da escala de 0,8.Conclusão:este estudo possibilitou desenvolver e validar o conteúdo de uma escala sobre produção do cuidado de enfermagem, instrumentalizando os enfermeiros em sua prática gerencial.

ResumenObjetivos:desarrollar y validar el contenido de un instrumento sobre producción del cuidado de enfermería.Método:los datos fueron recolectados entre 2011 y 2013, a partir de grupos focales, aplicación de cuestionarios semiestructurados (prueba del prototipo) y técnica Delphi. Los grupos focales fueron utilizados para generar ítems del instrumento y organizados en tres hospitales del interior del Estado de São Paulo, con la participación de 20 enfermeros. La apreciación del instrumento fue conducida por un panel de 10 especialistas.Resultados:tras dos fases de la técnica Delphi, el instrumento pasó a ser constituido por ocho ítems. El índice de validez de contenido de la escala fue ≥0,9 y la validez de los contenidos mostró variación de 0,8 a 1,0, indicando la manutención de la estructura y del contenido. La afirmativa respecto a la aplicabilidad en la práctica diaria del enfermero mostró índice de validez del contenido de la escala de 0,8.Conclusión:este estudio permitió desarrollar y validar el contenido de una escala sobre producción del cuidado de enfermería, instrumentalizando los enfermeros en su práctica gerencial.

Humans , Male , Female , Adult , Middle Aged , Aged , /blood , Gene Expression Regulation , Graft Rejection/blood , Kidney Transplantation , Leukocytes, Mononuclear/metabolism , Age Factors , /immunology , Graft Rejection/immunology , Graft Rejection/pathology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/pathology , Postoperative Period , Time Factors
Clinics ; 70(2): 120-125, 2/2015. tab, graf
Article in English | LILACS | ID: lil-741425


OBJECTIVES: To explore the microendoscopic discectomy technique and inclusion criteria for the treatment of recurrent lumbar disc herniation and to supply feasible criteria and technical notes to avoid complications and to increase the therapeutic effect. METHODS: A consecutive series of 25 patients who underwent posterior microendoscopic discectomy for recurrent lumbar disc herniation were included. The inclusion criteria were as follows: no severe pain in the lumbar region, no lumbar instability observed by flexion-extension radiography and no intervertebral discitis or endplate damage observed by magnetic resonance imaging. All patients were diagnosed by clinical manifestations and imaging examinations. RESULTS: Follow-up visits were carried out in all cases. Complications, such as nerve injuries, were not observed. The follow-up outcomes were graded using the MacNab criteria. A grade of excellent was given to 12 patients, good to 12 patients and fair to 1 patient. A grade of excellent or good occurred in 96% of cases. One patient relapsed 3 months after surgery and then underwent lumbar interbody fusion and inner fixation. The numerical rating scale of preoperative leg pain was 7.4± 1.5, whereas it decreased to 2.1±0.8 at 7 days after surgery. The preoperative Oswestry disability index of lumbar function was 57.5±10.0, whereas it was 26.0±8.5 at 7 days after surgery. CONCLUSION: In these cases, microendoscopic discectomy was able to achieve satisfactory clinical results. Furthermore, it has advantages over other methods because of its smaller incision, reduced bleeding and more efficient recovery. .

Humans , Centrifugation/methods , Leukocytes, Mononuclear/metabolism , Transfection/methods , Cell Survival/physiology , Leukocytes, Mononuclear/cytology , RNA Interference/physiology , RNA, Small Interfering/genetics
Gut and Liver ; : 370-380, 2015.
Article in English | WPRIM | ID: wpr-203889


BACKGROUND/AIMS: This study investigated the expression of T cell immunoglobulin- and mucin-domain-containing molecule 3 (TIM-3), human beta-defensin (HBD)-2, forkhead box protein 3 (FOXP3), and the frequency of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) in children with Crohn's disease (CD) during infliximab therapy. METHODS: We enrolled 20 CD patients who received infliximab treatment for 1 year. Peripheral blood and colonic mucosal specimens were collected from all CD patients and from healthy control individuals. RESULTS: A significant difference in TIM-3 mRNA expression was evident in peripheral blood mononuclear cells and colonic mucosa between CD patients before infliximab therapy and the healthy controls (p<0.001 and p=0.005, respectively). A significant difference in HBD-2 mRNA expression was found in colonic mucosa between CD patients before infliximab therapy and the healthy controls (p=0.013). In the active phase of CD, at baseline, the median percentage of T cells that were CD25+ FOXP3+ was 1.5% (range, 0.32% to 3.49%), which increased after inflixmab treatment for 1 year to 2.2% (range, 0.54% to 5.02%) (p=0.008). CONCLUSIONS: Our study suggests that both the adaptive and innate immune systems are closely linked to each other in CD pathogenesis. And the results of our study indicate that it could be a useful therapeutic tool, where restoration of TIM-3, HBD-2 and the function of Tregs may repair the dysfunctional immunoregulation in CD.

Adolescent , Case-Control Studies , Colon/immunology , Crohn Disease/drug therapy , Female , Forkhead Transcription Factors/metabolism , Gastrointestinal Agents/therapeutic use , Humans , Infliximab/therapeutic use , Intestinal Mucosa/immunology , Leukocytes, Mononuclear/metabolism , Male , Membrane Proteins/metabolism , T-Lymphocytes, Regulatory/immunology , beta-Defensins/metabolism
Mem. Inst. Oswaldo Cruz ; 109(8): 999-1004, 12/2014. tab, graf
Article in English | LILACS | ID: lil-732606


The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Adult , Humans , Middle Aged , /immunology , Epitopes/immunology , Interferon-gamma/metabolism , Latent Tuberculosis/diagnosis , Mycobacterium tuberculosis/immunology , Tuberculin Test , Algorithms , Antigens, Bacterial/analysis , Brazil , Bacterial Proteins/blood , Biomarkers/analysis , /metabolism , Chaperonins/blood , Enzyme-Linked Immunospot Assay , Epitope Mapping , Healthy Volunteers , HLA-DR Antigens/immunology , Latent Tuberculosis/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Phosphate-Binding Proteins/blood
Braz. j. med. biol. res ; 47(8): 662-669, 08/2014. tab, graf
Article in English | LILACS | ID: lil-716275


Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25+/highCD127Ø/lowFoxP3+, and effector T cells were defined as CD25+CD127+FoxP3Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4+TREG and CD28+TREG cells and an increased frequency of CD40L+TREG cells. There was a decrease in the TREG/effector-T ratio for GITR+, HLA-DR+, OX40+, and CD45RO+ cells, and an increased ratio of TREG/effector-T CD40L+ cells in patients with SLE. In addition, CD40L+TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.

Adult , Female , Humans , Male , Middle Aged , Antigens, Surface/metabolism , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic/immunology , T-Lymphocytes, Regulatory/immunology , Analysis of Variance , /analysis , /analysis , /analysis , /analysis , /analysis , Flow Cytometry , Forkhead Transcription Factors/analysis , Glucocorticoid-Induced TNFR-Related Protein/analysis , HLA-DR Antigens/analysis , /analysis , /analysis , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/physiopathology , Programmed Cell Death 1 Receptor/analysis , /analysis , Statistics, Nonparametric
Braz. j. med. biol. res ; 47(7): 540-547, 07/2014. tab, graf
Article in English | LILACS | ID: lil-712968


Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Animals , Female , Humans , Male , Mice , Gene Expression/physiology , Immunoglobulin Fragments/biosynthesis , Intercellular Adhesion Molecule-1/immunology , Protein Refolding , Protein Renaturation , Single-Chain Antibodies/biosynthesis , Antigen-Antibody Complex , Anti-Inflammatory Agents/pharmacology , Antibodies, Monoclonal/biosynthesis , Cell Adhesion , Chromatography , Dialysis , Enzyme-Linked Immunosorbent Assay , Ear Auricle/drug effects , Escherichia coli/genetics , Genetic Vectors , Immunoglobulin Fragments/pharmacology , Inclusion Bodies/metabolism , Intercellular Adhesion Molecule-1/drug effects , Leukocytes, Mononuclear/metabolism , Plasmids , Protein Engineering/methods , Single-Chain Antibodies/pharmacology , Xylenes/pharmacology
Braz. j. otorhinolaryngol. (Impr.) ; 80(3): 208-212, May-June/2014. graf
Article in English | LILACS | ID: lil-712989


INTRODUCTION: Sinonasal polyposis (NP) is a chronic inflammatory pathology of the nasal/paranasal cavities which affects from 1%-4% of the population. Although polyps seem to be a manifestation of chronic inflammation in both allergic and non-allergic subjects, the pathogenesis of nasal polyposis remains unknown. HLA-G molecules are a kind of no classic class I antigen with anti-inflammatory and tolerogenic properties. Little attention has been paid to the role of HLA-G chronic inflammatory disorders. OBJECTIVE: The aim of this study is to investigate the expression of HLA-G in the NP. MATERIALS AND METHODS: Prospective study involving samples of patients presenting with nasal polyposis that were subjected to the immunohistochemistry technique. After a skin prick test, all patients were divided into atopic and nonatopic groups and classified as asthmatic or non-asthmatic. RESULTS: Immunohistochemical staining demonstrated a higher expression of the HLA-G molecule in samples from nonatopic than in those from atopic patients, and was significantly lower in the non-asthmatic patients. CONCLUSION: These results indicate that HLA-G may play an important role in the pathology of nasal polyposis. Considering the anti-inflammatory properties of HLA-G, this study suggests that it could reduce susceptibility to atopy and asthma. .

INTRODUÇÃO: Polipose nasossinusal (PNS) é uma patologia inflamatória crônica das cavidades nasais/paranasais que afeta 1%-4% da população. Embora os pólipos pareçam ser uma manifestação de inflamação crônica em ambos os indivíduos alérgicos e não alérgicos, a patogênese da polipose nasal permanece desconhecida. Moléculas HLA-G são antígenos não clássicos da classe I com propriedades anti-inflamatórias e tolerogênicas. Pouca atenção tem sido dada ao papel do HLA-G em doenças inflamatórias crônicas. OBJETIVO: Investigar a expressão de HLA-G na PNS. MATERIAIS E MÉTODOS: Estudo prospectivo de pacientes com polipose nasal que foram submetidas à técnica de imuno-histoquímica. Após realizarem teste cutâneo, os pacientes foram divididos em grupos atópicos e não atópicos e classificados como asmáticos ou não asmáticos. RESULTADO: A coloração imuno-histoquímica mostrou uma maior expressão da molécula HLA-G em pacientes não atópicos do que naqueles atópicos e foi significativamente inferior nos pacientes não asmáticos. CONCLUSÃO: Os resultados indicam que o HLA-G pode ter um papel importante na patologia da polipose nasal. Considerando as propriedades anti-inflamatórias do HLA-G, este estudo sugere que ele poderia reduzir a susceptibilidade a atopia e asma. .

Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , HLA-G Antigens/biosynthesis , Histocompatibility Antigens Class I/biosynthesis , Nasal Polyps/immunology , Biomarkers/metabolism , Chronic Disease , Cohort Studies , HLA-G Antigens/immunology , Histocompatibility Antigens Class I/immunology , Immunohistochemistry , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Nasal Polyps/metabolism , Nasal Polyps/pathology , Prospective Studies
Braz. j. med. biol. res ; 47(4): 307-3015, 8/4/2014. graf
Article in English | LILACS | ID: lil-705765


Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Humans , Young Adult , /blood , Flow Cytometry/standards , Leukocytes, Mononuclear/metabolism , Trypan Blue , Cell Count , Cell Separation , Cell Survival , Cell Membrane/physiology , Fluorescence , Immunophenotyping , Indicators and Reagents/standards , Multiprotein Complexes/standards , Professional Competence , Propidium/standards , Staining and Labeling , Serum Albumin, Bovine/standards
Braz. j. med. biol. res ; 47(1): 42-49, 01/2014. tab, graf
Article in English | LILACS | ID: lil-697672


Membranous nephropathy (MN), characterized by the presence of diffuse thickening of the glomerular basement membrane and subepithelial in situ immune complex disposition, is the most common cause of idiopathic nephrotic syndrome in adults, with an incidence of 5-10 per million per year. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of human MN, but the specific biomarkers of MN have not been fully elucidated. As a result, our knowledge of the alterations in histone methylation in MN is unclear. We used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyze the variations in a methylated histone (H3K9me3) in peripheral blood mononuclear cells from 10 MN patients and 10 healthy subjects. There were 108 genes with significantly different expression in the MN patients compared with the normal controls. In MN patients, significantly increased activity was seen in 75 H3K9me3 genes, and decreased activity was seen in 33, compared with healthy subjects. Five positive genes, DiGeorge syndrome critical region gene 6 (DGCR6), sorting nexin 16 (SNX16), contactin 4 (CNTN4), baculoviral IAP repeat containing 3 (BIRC3), and baculoviral IAP repeat containing 2 (BIRC2), were selected and quantified. There were alterations of H3K9me3 in MN patients. These may be candidates to help explain pathogenesis in MN patients. Such novel findings show that H3K9me3 may be a potential biomarker or promising target for epigenetic-based MN therapies.

Adult , Female , Humans , Male , Glomerulonephritis, Membranous/genetics , Histones/genetics , Leukocytes, Mononuclear/metabolism , Lysine/genetics , Case-Control Studies , Chromatin Immunoprecipitation , Glomerulonephritis, Membranous/metabolism , Histones/metabolism , Lysine/metabolism , Methylation