Navegando por Palavras-chave "Flow Cytometry"
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- ItemAcesso aberto (Open Access)Aldefluor protocol to sort keratinocytes stem cells from skin(Acta Cirurgica Brasileira, 2017) Ribeiro Noronha, Samuel Marcos [UNIFESP]; Gragnani, Alfredo [UNIFESP]; Calado Pereira, Thiago Antonio [UNIFESP]; Alves Correa, Silvana Aparecida [UNIFESP]; Bonucci, Jessica [UNIFESP]; Ferreira, Lydia Masako [UNIFESP]Purpose: To investigate the use Aldefluor r and N, N-Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor r aliquots were prepared and maintained at temperature between 2 to 8 degrees C, or stored at -20 degrees C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor r 1.5 mu l and DEAB 15 mu l for 1 x 10(6) cells, incubated at 37 degrees C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. Conclusion: Aldefluor r has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.
- ItemAcesso aberto (Open Access)Evaluation of lymphocyte levels in a random sample of 218 elderly individuals from São Paulo city(Associação Brasileira de Hematologia e Hemoterapia e Terapia Celular, 2011-10-01) Teixeira, Daniela [UNIFESP]; Longo-Maugéri, Ieda Maria [UNIFESP]; Santos, Jair Licio Ferreira; Duarte, Yeda Aparecida Oliveira; Lebrão, Maria Lucia; Bueno, Valquiria [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)BACKGROUND: Age-associated changes in the immune system cause decreased protection after vaccination and increased rates of infections and tumor development. METHODS: Lymphocyte percentages were compared by gender and age to establish differences between subtypes. Three mL blood samples were obtained from 218 randomly selected individuals (60-101 years old) who live in São Paulo city. Blood was lysed with Tris phosphate buffer and stained for 30 minutes with monoclonal antibodies (CD3PerCP, CD4FITC, CD8Pe, CD19Pe) for analysis by flow cytometry. Statistical analysis was by ANOVA. RESULTS: The percentage of CD4+ T cells (p-value = 0.005) and the CD4/CD8 ratio (p-value = 0.010) were lower in men, whereas the percentage of CD8+ T cells was lower (p-value = 0.002) in women; the percentage of B cells (CD19+ ) was similar between groups. Individuals grouped by gender and age range and compared showed a drop in CD4+ cells in 75 to 79-year-old men (female: 46.1% ± 8.1% and male: 38.8% ± 10.5%; p-value = 0.023). Also, the 80 to 84-year-old group of men had a higher percentage of CD8+ (female: 20.8% ± 8.2%, and male: 27.2% ± 8.2%; p-value = 0.032). Low percentages of B cells were detected in men in the 75 to 79-year-old (p-value = 0.003), 85 to 89-year-old (p-value = 0.020) and older than 90 year old (p-value = 0.002) age ranges. CONCLUSION: Elderly men present with more changes in lymphocyte subsets compared to elderly women. These findings could demonstrate impairment in the immune response since the lower CD4+ in men would provide less help to B cells (also lower in men) in terms of antibody production. In addition, the increase in CD8+ cells in this group could represent chronic inflammation observed during the aging process.
- ItemAcesso aberto (Open Access)Evaluation of renal function and immune system cells in elderly individuals from São Paulo City(Faculdade de Medicina / USP, 2013-01-01) Teixeira, Daniela [UNIFESP]; Longo-Maugéri, Ieda Maria [UNIFESP]; Duarte, Yeda Aparecida Oliveira; Lebrão, Maria Lucia; Bueno, Valquiria [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); University of São Paulo Epidemiology DepartmentOBJECTIVES: Both renal function and immune system function decline with age. Although controversial, a significant number of studies have shown that the decline in kidney function is associated with the worsening of the immune system. These findings are reinforced by the increased susceptibility to infections and deficient immunization coverage after vaccination both in patients with chronic renal disease and in elderly individuals. Our objective was to evaluate a non-institutionalized elderly population from São Paulo City and correlate the estimated glomerular filtration rate with the percentage of lymphocytes in circulation. METHODS: A random population of 237 individuals (107 men and 130 women), ranging in age from 60 to 101 years, who were enrolled in the Health, Well-Being and Aging Study was evaluated for renal function (Modification on Diet in Renal Disease formula) and lymphocyte percentage (flow cytometry). RESULTS: Aging was associated with a decrease in the estimated glomerular filtration rate in both male and female individuals. We did not identify a significant correlation between the estimated glomerular filtration rate and either the percentage of CD4, CD8, and B cells or CD4/CD8 ratio. The median percentage of CD8+ T cells was significantly lower in individuals with an estimated glomerular filtration rate >60 mL/min/1.73 m². CONCLUSIONS: In this study, no statistical correlation was found between the estimated glomerular filtration rate and either the lymphocyte phenotype (CD4+,CD8+, and CD19+ cells) or the CD4/CD8 ratio in blood.
- ItemSomente MetadadadosHeteromorfismos Q. caracterização de uma amostra de mongolóides brasileiros e comparação com uma amostra de negróides brasileiros(Universidade Federal de São Paulo (UNIFESP), 1991) Toledo, Silvia Regina Caminada de [UNIFESP]; Andrade, Joyce Anderson Duffles [UNIFESP]
- ItemAcesso aberto (Open Access)Keratinocyte growth factor, interleukins (1 beta, 6, 8, 10, 12), and tumor necrosis factor alpha in culture medium of dermal fibroblast of burned patients(Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia, 2014-01-01) Noronha, Samuel Marcos Ribeiro de [UNIFESP]; Corrêa, Silvana Aparecida Alves [UNIFESP]; Klepp, Anthony Gueratto; Ipolito, Michele Zampieri; Ferreira, Lydia Masako [UNIFESP]; Gragnani, Alfredo [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)PURPOSE:To evaluate the level of cytokines and keratinocyte growth factor (KGF) or Fibroblast Growth Factor 7 (FGF-7) in the culture medium of cultured human dermal fibroblasts from patients with large burn in comparison to small burn.METHODS:Fibroblasts of 10 patients (four large burns, four small burns and two controls) were initiated by the enzymatic method using collagenase. Cytokines and KGF in the supernatant of the culture medium was measured by, respectively, flow cytometry using Cytometric Bead Array Human Inflammation kit (CBA, BD Biosciences, USA) and the enzyme immunoassay method using the Quantikine (r) Human KGF. The experiments were performed in triplicate.RESULTS:The expression of IL-12 protein in patients with large burns showed a tendency to increase. IL- 6, IL- 10, and IL- 1beta were observed no difference. For IL - 8, TNF - alpha and KGF was observed a significant difference between the expression in large and small burned patient.CONCLUSION:That IL-8, TNF-alpha and KGF showed higher expression in cultured fibroblasts of large burned patients.
- ItemSomente MetadadadosSubpopulações de células B e T foliculares em pacientes com síndrome do PI3KCD ativado (APDS)(Universidade Federal de São Paulo (UNIFESP), 2021) Velasco, Helena Fleck [UNIFESP]; Pinto, Maria Isabel De Moraes [UNIFESP]; Universidade Federal de São PauloIntroduction: Inborn Immunity Errors are a heterogeneous group of genetic diseases that affect the functioning of the immune system. Activated PI3Kδ syndrome (Activated PI3K delta syndrome - APDS) is one of the new inborn erros of immunity described because of the evolution of molecular biology. APDS is caused by heterozygous gain-of-function mutations in genes encoding the enzyme complex phosphoinositide 3-kinase (PI3K). The main clinical manifestations are pulmonary infections, high susceptibility to virus infections, benign lymphoproliferation and predisposition to lymphoma. Patients with APDS have defects in humoral and cellular immunity and consequently do not respond adequately to vaccines. Objectives: To evaluate follicular B and T cell subpopulations in patients with APDS. To compare laboratory findings with the clinical manifestations of affected patients. To describe from a clinical and laboratory point of view a family with an unprecedented PI3K gain-of-function mutation. Methods: All twelve patients with APDS who were evaluated at the Allergy and Clinical Immunology clinic at UNIFESP were selected. Among these patients, seven belonged to a family that was also described separately for presenting a new genetic variant of APDS, p.529insHEK. Clinical data were collected from all patients and for laboratory evaluation, peripheral blood samples were collected the same patients and 12 controls matched by sex and age. Absolute counts of T, B and NK lymphocytes and percentage of B and follicular T lymphocytes subsets was performed by flow cytometry. Results: Most patients had recurrent respiratory tract infections, pneumonia and bronchiectasis. Some patients also had lymphoproliferation. Compared to controls, patients with APDS had significantly lower numbers of B cells, TCD3, TCD4, TCD8 and CD19. Lower numbers of B lymphocytes appear to be associated with a reduction in the number of memory B cells. Unlike the literature, the patients in the study did not show an increase in transitional B cells. The value of total follicular T cells diverged between the different variants, but all presented a reduced number of follicular T cells 17 (Tfh17). Patients with the new genetic variant of APDS, p.529insHEK presented varied clinical manifestations, however with a similar pattern in infections and in autoimmunity/lymphoproliferation, which confirms the importance of studies of families with rare diseases, especially for a better understanding of this group of disease. Conclusion: Patients with mutations in the PIK3CD gene have varied symptoms but with similar clinical features. The value of total follicular T cells diverged among the different genetic variants, but all presented a reduced number of follicular T cells 17 which may be related to defects in the production of antibodies found in APDS. Patients with the new genetic variant of APDS, p.529insHEK. presented varied clinical manifestations, however with a similar pattern in infections and in autoimmunity/lymphoproliferation, which confirms the importance of studies of families with rare diseases, especially for a better understanding of this group of disease.
- ItemSomente MetadadadosSubpopulações de monócitos no sangue periférico de pacientes com Doença de Behçet – há associação com manifestações clínicas e atividade de doença?(Universidade Federal de São Paulo (UNIFESP), 2021) Del Padre, Talita Cardoso Gazzito [UNIFESP]; Souza, Alexandre Wagner Silva De [UNIFESP]; Universidade Federal de São PauloIntroduction: Behçet's disease (BD) is a systemic inflammatory disease, classified as variable vessel vasculitis, characterized by recurrent episodes of mucocutaneous, ocular, joint, vascular, gastrointestinal, and neurological manifestations. Monocytes participate in the activation of neutrophils in BD; however, no study has assessed changes in the distribution of monocyte subpopulations in peripheral blood of BD patients. Objective: To evaluate the frequency and absolute number of classical, intermediate, and non-classical monocytes in the peripheral blood of BD patients and in healthy controls. In addition, associations between monocyte subpopulations in peripheral blood and disease activity, severity of clinical manifestations and therapy were analyzed. Methods: A cross-sectional study was carried out including 49 BD patients, 28.6% of them with active disease, and 39 healthy controls. Flow cytometry was performed to assess monocyte subpopulations in peripheral blood with the following markers: CD14, CD16 and CD66b. The monocyte subpopulations were subdivided into classical (CD14+CD16-), intermediate (CD14+CD16dim) and non-classical (CD14dimCD16high). CD66b was used to exclude neutrophils from the analysis. Results were expressed as median and interquartile range of the number of cells x 106/L and as a percentage of cells over the total number of monocytes. Results: A relationship was observed between BD and a lower absolute number of monocytes [349.16 (279.40-408.66) vs. 439.25 (312.81-532.58); p = 0.020] and classical monocytes [275.38 (175.50-322.07) vs. 380.80 (279.92-499.17); p <0.0001], in addition to a lower percentage of classical monocytes [84.7% (76.4-91.2) vs. 89.5% (86.0-94.0); p = 0.002] compared to the control group, respectively. On the other hand, BD was related to a higher number [30.46 (20.25-66.29) vs. 21.08 (13.48-25.64); p <0.0001] and percentage [9.2% (5.5, -16.9) vs. 4.9% (3.0-6.7); p < 0.0001] of intermediate monocytes in peripheral blood compared to the control group, respectively. There were no differences between BD and controls regarding non-classical monocytes in peripheral blood. In BD, there was no relationship between subpopulations of monocytes in peripheral blood and xii disease activity, severe manifestations of BD or glucocorticoid therapy, immunosuppressive or biological agents. However, the use of colchicine was been associated with a higher number of non-classical monocytes in peripheral blood [41.21 (11.47-59.04) vs. 15.19 (9.92-22.53); p = 0.035]. Conclusion: BD patients presented an altered distribution of monocyte subpopulations in peripheral blood as compared to healthy controls with a lower number of total monocytes, a lower number and percentage of classical monocytes and a higher number and percentage of intermediate monocytes. An increase in the number of non-classical monocytes was associated with the use of colchicine in BD.