TNF-α promoter hypomethylation is frequent in oncopediatric patients who recovered from mucositis.

The aim of this study was to investigate the DNA methylation profile in genes encoding catalase (CAT) and superoxide dismutase (SOD3) enzymes, which are involved in oxidative stress mechanisms, and in genes encoding pro-inflammatory cytokines interleukin-6 (IL6) and tumor necrosis factor-alpha (TNF-α) in the oral mucosa of oncopediatric patients treated with methotrexate (MTX®). This was a cross-sectional observational study and the population comprised healthy dental patients (n = 21) and those with hematological malignancies (n = 64) aged between 5 and 19 years. Oral conditions were evaluated using the Oral Assessment Guide and participants were divided into 4 groups: 1- healthy individuals; 2- oncopediatric patients without mucositis; 3- oncopediatric patients with mucositis; 4- oncopediatric patients who had recovered from mucositis. Methylation of DNA from oral mucosal cells was evaluated using the Methylation-Specific PCR technique (MSP). For CAT, the partially methylated profile was the most frequent and for SOD3 and IL6, the hypermethylated profile was the most frequent, with no differences between groups. For TNF-α, the hypomethylated profile was more frequent in the group of patients who had recovered from mucositis. It was concluded that the methylation profiles of CAT, SOD3, and IL6 are common profiles for oral cells of children and adolescents and have no association with oral mucositis or exposure to chemotherapy with MTX®. Hypomethylation of TNF-α is associated with oral mucosal recovery in oncopediatric patients who developed oral mucositis during chemotherapy.

DNMT3B (rs2424913) polymorphism is associated with systemic lupus erythematosus alone and with co-existing periodontitis in a Brazilian population.

The association between Periodontitis and Systemic Lupus Erythematosus (SLE) has been primarily based on their similar pathophysiology and both are associated with genetic polymorphisms. OBJECTIVES: To investigate an association between the methylation-related gene polymorphisms DNMT3B (rs2424913) and MTHFR (rs1801133) to Systemic Lupus Erythematosus (SLE) and Periodontitis. METHODOLOGY: In total, 196 individuals of all genders aged 24 to 60 years old were allocated into four groups based on their systemic and periodontal status, namely: Healthy control (n=60), periodontitis (n=51), SLE (n=47), and SLE + periodontitis (n=38). Individuals with SLE were stratified according to disease activity (SLEDAI) in inactive or active. We performed polymorphism analysis using PCR-RFLP with genomic DNA from mouthwash. We analyzed data using Fisher's Exact, Chi-square test, and regression models. RESULTS: Periodontal status were similar in subjects with periodontitis alone and combined with SLE. SLE patients with periodontitis had a longer SLE diagnosis than SLE only (p=0.001). For DNMT3 B polymorphism, the periodontitis, SLE, and Inactive SLE + periodontitis groups showed a higher frequency of T allele and TT genotypes compared to healthy controls (p<0.05). Regression analyses showed that the TT genotype is a strong risk factor for periodontitis (OR=4.53; CI95%=1.13-18.05) and also for SLE without periodontitis (OR=11.57; CI95%=3.12-42.84) and SLE with periodontitis (OR=5.27; CI95%=1.25-22.11) when compared to control. CONCLUSION: SLE patients with periodontitis had a longer length of SLE diagnosis. The DNMT3B (rs2424913) polymorphism was associated with periodontitis and SLE alone or combined with periodontitis. Our study contributes to understanding the genetic mechanisms involved in periodontitis and SLE susceptibility.

Genetic polymorphisms of genes involved in oxidative stress and inflammatory management in oncopediatric patients with chemo-induced oral mucositis.

OBJECTIVE: Oral mucositis (OM) is a painful inflammatory oral condition that affects children who undergo chemotherapy. Oxidative stress is a known OM mediator and pro-inflammatory cytokines contribute to the amplification of the immune response. To investigate the possible associations of rs4880 (superoxide dismutase 2, SOD2 47 C/T), rs7943316 (catalase, CAT -21 A/T), rs1800629 (tumor necrosis factor α, TNF- α -308 G/A), and rs1800795 (interleukin 6, IL-6 -174 G/C) polymorphisms with chemo-induced OM occurrence and severity in oncopediatric patients. METHODOLOGY: We conducted a single-center, observational cross-sectional study with sample collection of oral epithelial cells from 95 children and adolescents with hematological cancers who underwent chemotherapy, followed by genomic DNA extraction. Single-nucleotide polymorphisms (SNPs) were assessed with PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Demographic data and information concerning OM occurrence were obtained from dental charts of the multidisciplinary oral care team. Information on OM severity was obtained from appropriately-filled Oral Assessment Guide records. Descriptive and inferential statistics were conducted with Student's T test, chi-squared test, and Fisher's exact test, with p≤0.05. RESULTS: The mean age was 10 years-old and most patients were male individuals (57.89%). Female sex was considered a protective factor for OM occurrence (OR=4.83; CI=[1.14; 16.57]). The AA genotype for CAT was the most frequent amongst individuals with severe OM (p=0.04). The GA genotype for TNF- α was the most frequent amongst individuals without severe OM (p=0.03). For SOD2 and IL-6 , the most frequent genotypes were CT and GG respectively for all groups (p>0.05). CONCLUSION: The AA genotype for CAT -21 A/T was a tendency among the group with severe OM. Data on TNF- α -308 G/A were inconclusive. No associations were detected for SOD2 47 C/T and IL-6 -174 G/C polymorphisms in oncopediatric patients with chemo-induced oral mucositis.

DNMT3B (rs2424913) polymorphism is associated with systemic lupus erythematosus alone and with co-existing periodontitis in a Brazilian population

Abstract The association between Periodontitis and Systemic Lupus Erythematosus (SLE) has been primarily based on their similar pathophysiology and both are associated with genetic polymorphisms. Objectives: To investigate an association between the methylation-related gene polymorphisms DNMT3B (rs2424913) and MTHFR (rs1801133) to Systemic Lupus Erythematosus (SLE) and Periodontitis. Methodology: In total, 196 individuals of all genders aged 24 to 60 years old were allocated into four groups based on their systemic and periodontal status, namely: Healthy control (n=60), periodontitis (n=51), SLE (n=47), and SLE + periodontitis (n=38). Individuals with SLE were stratified according to disease activity (SLEDAI) in inactive or active. We performed polymorphism analysis using PCR-RFLP with genomic DNA from mouthwash. We analyzed data using Fisher's Exact, Chi-square test, and regression models. Results: Periodontal status were similar in subjects with periodontitis alone and combined with SLE. SLE patients with periodontitis had a longer SLE diagnosis than SLE only (p=0.001). For DNMT3 B polymorphism, the periodontitis, SLE, and Inactive SLE + periodontitis groups showed a higher frequency of T allele and TT genotypes compared to healthy controls (p<0.05). Regression analyses showed that the TT genotype is a strong risk factor for periodontitis (OR=4.53; CI95%=1.13-18.05) and also for SLE without periodontitis (OR=11.57; CI95%=3.12-42.84) and SLE with periodontitis (OR=5.27; CI95%=1.25-22.11) when compared to control. Conclusion: SLE patients with periodontitis had a longer length of SLE diagnosis. The DNMT3B (rs2424913) polymorphism was associated with periodontitis and SLE alone or combined with periodontitis. Our study contributes to understanding the genetic mechanisms involved in periodontitis and SLE susceptibility.

Genetic polymorphisms of genes involved in oxidative stress and inflammatory management in oncopediatric patients with chemo-induced oral mucositis

Abstract Oral mucositis (OM) is a painful inflammatory oral condition that affects children who undergo chemotherapy. Oxidative stress is a known OM mediator and pro-inflammatory cytokines contribute to the amplification of the immune response. Objective: To investigate the possible associations of rs4880 (superoxide dismutase 2, SOD2 47 C/T), rs7943316 (catalase, CAT −21 A/T), rs1800629 (tumor necrosis factor α, TNF- α −308 G/A), and rs1800795 (interleukin 6, IL-6 −174 G/C) polymorphisms with chemo-induced OM occurrence and severity in oncopediatric patients. Methodology: We conducted a single-center, observational cross-sectional study with sample collection of oral epithelial cells from 95 children and adolescents with hematological cancers who underwent chemotherapy, followed by genomic DNA extraction. Single-nucleotide polymorphisms (SNPs) were assessed with PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Demographic data and information concerning OM occurrence were obtained from dental charts of the multidisciplinary oral care team. Information on OM severity was obtained from appropriately-filled Oral Assessment Guide records. Descriptive and inferential statistics were conducted with Student's T test, chi-squared test, and Fisher's exact test, with p≤0.05. Results: The mean age was 10 years-old and most patients were male individuals (57.89%). Female sex was considered a protective factor for OM occurrence (OR=4.83; CI=[1.14; 16.57]). The AA genotype for CAT was the most frequent amongst individuals with severe OM (p=0.04). The GA genotype for TNF- α was the most frequent amongst individuals without severe OM (p=0.03). For SOD2 and IL-6 , the most frequent genotypes were CT and GG respectively for all groups (p>0.05). Conclusion: The AA genotype for CAT −21 A/T was a tendency among the group with severe OM. Data on TNF- α −308 G/A were inconclusive. No associations were detected for SOD2 47 C/T and IL-6 −174 G/C polymorphisms in oncopediatric patients with chemo-induced oral mucositis.

ABCG2 polymorphism, age and leukocyte count may contribute to oral mucositis in oncopediatric patients.

The study investigated the relationship between genetic polymorphisms and the development of oral mucositis in pediatric patients undergoing chemotherapy involving methotrexate. A longitudinal study was conducted with 64 patients, and oral mucositis was evaluated by the modified Oral Assessment Guide, which aims to diagnose and classify oral mucositis. Epithelial cells were obtained by mouthwash and DNA was extracted. The polymorphisms MTHFR (rs1801133), DNMT3B (rs2424913), ABCC2 (rs717620), ABCG2 (rs2231137) and ABCG2 (rs2231142) were analyzed by PCR-RFLP method. Demographic, hematological and biochemical data were collected from medical records. Statistical analysis was performed using the SPSS software adopting a p-value of 0.05. Male sex predominated (56.2%), and the mean age was 10.8 years (± 4.9). Oral mucositis affected 65.6% of the patients, of which 61.9% developed the severe form of the disease. For the ABCG2 gene (rs2231142), the rare A allele and CA genotype were more frequent in individuals with mucositis (p= 0.02; RR = 0.60; CI = 0.387 - 0.813). The severity of the disease was mainly observed in younger patients (median = 9 years; p=0.02). Patients with severe oral mucositis presented lower leukocytes count (median = 2.150 mm3) compared to patients with the mild/moderate form (median = 4.200 mm3; p=0.03). Female patients and each 10,000-platelet increase were protective factors against the onset of oral mucositis (p=0.02). It is concluded that rs2231142 polymorphism increases the likelihood of oral mucositis and younger patients and patients with low leukocytes counts are more likely to develop severe form.

ABCG2 polymorphism, age and leukocyte count may contribute to oral mucositis in oncopediatric patients

Abstract The study investigated the relationship between genetic polymorphisms and the development of oral mucositis in pediatric patients undergoing chemotherapy involving methotrexate. A longitudinal study was conducted with 64 patients, and oral mucositis was evaluated by the modified Oral Assessment Guide, which aims to diagnose and classify oral mucositis. Epithelial cells were obtained by mouthwash and DNA was extracted. The polymorphisms MTHFR (rs1801133), DNMT3B (rs2424913), ABCC2 (rs717620), ABCG2 (rs2231137) and ABCG2 (rs2231142) were analyzed by PCR-RFLP method. Demographic, hematological and biochemical data were collected from medical records. Statistical analysis was performed using the SPSS software adopting a p-value of 0.05. Male sex predominated (56.2%), and the mean age was 10.8 years (± 4.9). Oral mucositis affected 65.6% of the patients, of which 61.9% developed the severe form of the disease. For the ABCG2 gene (rs2231142), the rare A allele and CA genotype were more frequent in individuals with mucositis (p= 0.02; RR = 0.60; CI = 0.387 - 0.813). The severity of the disease was mainly observed in younger patients (median = 9 years; p=0.02). Patients with severe oral mucositis presented lower leukocytes count (median = 2.150 mm3) compared to patients with the mild/moderate form (median = 4.200 mm3; p=0.03). Female patients and each 10,000-platelet increase were protective factors against the onset of oral mucositis (p=0.02). It is concluded that rs2231142 polymorphism increases the likelihood of oral mucositis and younger patients and patients with low leukocytes counts are more likely to develop severe form.

Resumo O presente estudo investigou a relação entre cinco polimorfismos genéticos e o desenvolvimento de mucosite oral em pacientes pediátricos recebendo quimioterapia com metrotexato. O estudo longitudinal foi conduzido com 64 pacientes e a mucosite oral avaliada pelo Oral Assessment Guide modificado, que tem como objetivo diagnosticar e classificar a mucosite oral. Células epiteliais bucais foram obtidas por bochecho e o DNA foi extraído. Os polimorfismos MTHFR (rs1801133), DNMT3B (rs2424913), ABCC2 (rs717620), ABCG2 (rs2231137) e ABCG2 (rs2231142), foram analisados pela técnica de PCR-RFLP. Dados demográficos, hematológicos e bioquímicos foram coletados a partir de registros médicos. Análise estatística foi realizada utilizando o software SPSS adotando um valor de p=0,05. Observou-se que, o sexo masculino foi predominante (56,2%), e a idade média foi de 10,8 anos (± 4.9). A mucosite oral acometeu 65,6% dos pacientes, dos quais, 61,9% desenvolveram a forma grave da doença. Para o gene ABCG2 (rs2231142), o alelo raro A e o genótipo CA foram mais frequentes em indivíduos com mucosite (p= 0.02; RR = 0.60; CI = 0.387 - 0.813). A gravidade da doença foi observada principalmente em pacientes mais jovens (mediana = 9 anos; p=0.02). Além disso, os pacientes com mucosite oral grave apresentaram menor contagem de leucócitos (mediana = 2150 mm3) em comparação aos pacientes com a forma leve/moderada (mediana = 4200 mm3; p=0.03). Pacientes do sexo feminino e aumento a cada 10.000 plaquetas foram fatores de proteção contra o aparecimento de mucosite oral (p=0.02). Concluiu-se que a presença do polimorfismo rs2231142 aumenta o risco de o paciente desenvolver a mucosite oral, bem como pacientes mais jovens e menor contagem de leucócitos contribui com a severidade.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis.

BACKGROUND: Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. OBJECTIVE: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and -149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. METHODOLOGY: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. RESULTS: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. CONCLUSION: The polymorphism -149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

Food Intervention with Folate Reduces TNF-α and Interleukin Levels in Overweight and Obese Women with the MTHFR C677T Polymorphism: A Randomized Trial.

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism associated with body fat accumulation could possibly trigger an inflammatory process by elevating homocysteine levels and increasing cytokine production, causing several diseases. This study aimed to evaluate the effects of food intervention, and not folate supplements, on the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in overweight and obese women with the MTHFR C677T polymorphism. A randomized, double-blind eight-week clinical trial of 48 overweight and obese women was conducted. Participants were randomly assigned into two groups. They received 300 g of vegetables daily for eight weeks containing different doses of folate: 95 µg/day for Group 1 and 191 µg/day for Group 2. MTHFR C677T polymorphism genotyping was assessed by digestion with HinfI enzyme and on 12% polyacrylamide gels. Anthropometric measurements, 24-h dietary recall, and biochemical analysis (blood folic acid, vitamin B12, homocysteine (Hcy), TNF-α, IL-1ß, and IL-6) were determined at the beginning and end of the study. Group 2 had a significant increase in folate intake (p < 0.001) and plasma folic acid (p < 0.05) for individuals with the cytosine-cytosine (CC), cytosine-thymine (CT), and thymine-thymine (TT) genotypes. However, only individuals with the TT genotype presented reduced levels of Hcy, TNF-α, IL-6, and IL-1ß (p < 0.001). Group 1 showed significant differences in folate consumption (p < 0.001) and folic acid levels (p < 0.05) for individuals with the CT and TT genotypes. Food intervention with folate from vegetables increased folic acid levels and reduced interleukins, TNF-α, and Hcy levels, mainly for individuals with the TT genotype.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis

Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

Methylation profile of the ADRB3 gene and its association with lipid profile and nutritional status in adults

BACKGROUND: Defects in DNA methylation have been shown to be associated with metabolic diseases such as obesity, dyslipidemia, and hypercholesterolemia. To analyze the methylation profile of the ADRB3 gene and correlate it with lipid profile, lipid intake, and oxidative stress based on malondialdehyde (MDA) and total antioxidant capacity (TAC), homocysteine and folic acid levels, nutritional status, lifestyle, and socioeconomic variables in an adult population. A cross-sectional epidemiological study representative of the East and West regions of the municipality of João Pessoa, Paraíba state, Brazil, enrolled 265 adults of both genders. Demographic, lifestyle, and socioeconomic questionnaires and a 24-h recall questionnaire were applied by trained interviewers' home. Nutritional and biochemical evaluation (DNA methylation, lipid profile, MDA, TAC, homocysteine and folic acid levels) was performed. RESULTS: DNA hypermethylation of the ADRB3 gene, analyzed in leukocytes, was present in 50% of subjects and was associated with a higher risk of being overweight (OR 3.28; p = 0.008) or obese (OR 3.06; p = 0.017), a higher waist-hip ratio in males (OR 1.17; p = 0.000), greater intake of trans fats (OR 1.94; p = 0.032), higher LDL (OR 2.64; p = 0.003) and triglycerides (OR 1.81; p = 0.031), and higher folic acid levels (OR 1.85; p = 0.022). CONCLUSIONS: These results suggest that epigenetic changes in the ADRB3 gene locus may explain the development of obesity and non-communicable diseases associated with trans-fat intake, altered lipid profile, and elevated folic acid. Because of its persistence, DNA methylation may have an impact in adults, in association with the development of non-communicable diseases. This study is the first population-based study of the ADRB3 gene, and the data further support evaluation of ADRB3 DNA methylation as an effective biomarker.

Global and gene-specific DNA methylation and hydroxymethylation in human skin exposed and not exposed to sun radiation

Abstract: Background: epigenomes can be influenced by environmental factors leading to the development of diseases. Objective: To investigate the influence of sun exposure on global DNA methylation and hydroxymethylation status and at specific sites of the miR-9-1, miR-9-3 and MTHFR genes in skin samples of subjects with no history of skin diseases. Methods: Skin samples were obtained by punch on sun-exposed and sun-protected arm areas from 24 corpses of 16-89 years of age. Genomic DNA was extracted from skin samples that were ranked according to Fitzpatrick's criteria as light, moderate, and dark brown. Global DNA methylation and hydroxymethylation and DNA methylation analyses at specific sites were performed using ELISA and MSP, respectively. Results: No significant differences in global DNA methylation and hydroxymethylation levels were found among the skin areas, skin types, or age. However, gender-related differences were detected, where women showed higher methylation levels. Global DNA methylation levels were higher than hydroxymethylation levels, and the levels of these DNA modifications correlated in skin tissue. For specific sites, no differences among the areas were detected. Additional analyses showed no differences in the methylation status when age, gender, and skin type were considered; however, the methylation status of the miR-9-1 gene seems to be gender related. Study limitations: there was no separation of dermis and epidermis and low sample size. Conclusion: sun exposure does not induce changes in the DNA methylation and hydroxymethylation status or in miR-9-1, miR-9-3 and MTHFR genes for the studied skin types.

Global and gene-specific DNA methylation and hydroxymethylation in human skin exposed and not exposed to sun radiation.

BACKGROUND: epigenomes can be influenced by environmental factors leading to the development of diseases. OBJECTIVE: To investigate the influence of sun exposure on global DNA methylation and hydroxymethylation status and at specific sites of the miR-9-1, miR-9-3 and MTHFR genes in skin samples of subjects with no history of skin diseases. METHODS: Skin samples were obtained by punch on sun-exposed and sun-protected arm areas from 24 corpses of 16-89 years of age. Genomic DNA was extracted from skin samples that were ranked according to Fitzpatrick's criteria as light, moderate, and dark brown. Global DNA methylation and hydroxymethylation and DNA methylation analyses at specific sites were performed using ELISA and MSP, respectively. RESULTS: No significant differences in global DNA methylation and hydroxymethylation levels were found among the skin areas, skin types, or age. However, gender-related differences were detected, where women showed higher methylation levels. Global DNA methylation levels were higher than hydroxymethylation levels, and the levels of these DNA modifications correlated in skin tissue. For specific sites, no differences among the areas were detected. Additional analyses showed no differences in the methylation status when age, gender, and skin type were considered; however, the methylation status of the miR-9-1 gene seems to be gender related. STUDY LIMITATIONS: there was no separation of dermis and epidermis and low sample size. CONCLUSION: sun exposure does not induce changes in the DNA methylation and hydroxymethylation status or in miR-9-1, miR-9-3 and MTHFR genes for the studied skin types.

Age-related Changes in DNA Methylation Status of hTERT Gene Promoter of Oral Epithelial Cells

The purpose of this study was to investigate the effect of aging on the DNA methylation status of two genes involved in tumorigenesis (telomerase gene hTERT and DNA repair gene- MLH1) and one in metabolism (methylenetetrahydrofolate reductase gene- MTHFR) in oral epithelial cells. DNA methylation analysis was performed by Methylation Sensitive Restriction Enzymes (MSRE) of healthy oral epithelial cells of child (6-10 years, n=21), young (20-25 years, n=19) and elderly (over 60 years, n=25). The results for the hTERT gene showed significant variation in the methylation frequency at CpG dinucleotides among the groups (p=0.0001), with the methylated condition more frequently in children and young people. In relation to MLH1 and MTHFR, no differences were observed among the groups and the unmethylated condition were present in most individuals (p>0.05). Thus, it was concluded that aging of oral epithelial cells was associated with hypomethylation of the hTERT gene promoter and this could be a promising marker for screening a set of age-related alterations.

Light microscope observation of circulating human lymphocytes cultured in vitro

The purpose of this work was to study the isolation and a light microscopy technique for cultured lymphocytes. Blood samples were obtained by venipuncture with an anticoagulant added and centrifuged in a Percoll density gradient to separate the leukocytes. Lymphocytes were placed in 25 cm ³ tissue culture flasks at 37ºC. After culturing, they were fixed and stained with the methods used for blood smears. Results showed that not all fixing solutions and stains were an equally good choice for cultured lymphocytes.

Os linfócitos são células importantes do sistema imune e têm sido largamente utilizados em estudos morfológicos. Entretanto, a literatura sobre técnicas de preparação dessas células é escassa e antiga, especialmente para linfócitos cultivados in vitro. Portanto, o objetivo desse estudo foi relatar com detalhes as técnicas de isolamento e microscopia de luz de linfócitos mantidos em cultura. Amostras de sangue foram obtidas por punção venosa e centrifugadas em gradiente de densidade de Percoll, para separar os leucócitos. Os linfócitos foram mantidos em frascos de cultura de 25 cm³ a 37ºC. Após a cultura, as células foram fixadas e coradas de acordo com a metodologia utilizada para esfregaços sanguíneos. Nossos resultados mostraram que nem todos os fixadores e corantes utilizados para esfregaços sanguíneos são uma boa escolha para linfócitos cultivados in vitro.

Cell death induced by tamoxifen in human blood lymphocytes cultivated in vitro / Morte celular induzida pelo tamoxifeno em linfócitos humanos cultivados in vitro

Many chemotherapeutic agents with a potential against solid tumors or leukemia can cause lymphopenia. Tamoxifen (TAM) is a synthetic non-steroidal anti-estrogen drug employed in female breast cancer treatment. The present study investigated the capacity of TAM to induce cell death in human lymphocytes cultivated in vitro. Lymphocytes were obtained from young (25-30 years; n = 3) and elderly women (58-77 years; n = 3) and cultivated for 24 or 48h, with or without TAM (20 µM). After the culture, cell viability, immunocytochemical response and ultrastructure were evaluated. TAM affected lymphocytes in a time- dependent manner, and cells obtained from elderly women were the most sensitive to TAM. Immunocytochemical analysis evidenced higher frequency of apoptosis in treated cells, and the ultrastructural study revealed autophagic vacuoles, differing from the controls. In summary, the treated lymphocytes were affected by TAM, leading to cell death by apoptosis and autophagy.

Muitos agentes quimioterápicos com potencial contra tumores sólidos ou leucemias podem causar linfopenia. O Tamoxifeno (TAM) é um agente antiestrógeno não-esteroidal empregado no tratamento de câncer de mama feminino. O presente trabalho investigou a capacidade do TAM em induzir morte celular em linfócitos humanos cultivados in vitro. Os linfócitos foram obtidos de mulheres jovens (25-30 anos; n = 3) e idosas (58-77 anos; n = 3) e cultivados por 24 ou 48h, com ou sem TAM (20 µM). Após a cultura, foram analisadas a viabilidade celular, a resposta imunocitoquímica e a ultraestrutura. Os resultados indicam que o Tamoxifeno induziu morte celular em linfócitos de ambos os grupos, entretanto, as células das mulheres idosas apresentaram-se mais sensíveis ao tratamento. A análise imunocitoquímica mostrou maior frequência de apoptose nas células tratadas e o estudo ultraestrutural revelou vacúolos autofágicos nos linfócitos expostos ao Tamoxifeno. Em conclusão, nosso estudo revelou que o TAM induziu morte celular por apoptose e autofagia.

Alterações no Epigenoma e o Hábito de Fumar / Epigenome Changes and the Smoking Habit

Cigarro apresentam capacidade de alterar o genoma dascélulas e levar ao desenvolvimento tumoral. Recentemente,a capacidade do cigarro em alterar o epigenoma também temsido investigada. Epigenoma se refere a informações dogenoma que não estão relacionadas com a sequência debases do DNA. Objetivo: O presente estudo teve comoobjetivo realizar uma revisão sobre o hábito de fumar e suaassociação com o padrão alterado de metilação de DNA emamostras tumorais. Método: Foi realizada revisão integrativada literatura através de busca eletrônica na base de dadosPubMed. O período temporal considerado no estudo foi de2000 a 2010. Os artigos foram selecionados considerandosea acessibilidade e excluindo-se revisões e pesquisascom linhagens celulares e animais. Resultados: Os artigosrevelaram que o cigarro pode alterar o epigenoma mesmo deórgãos não diretamente expostos como a boca e os pulmões,sugerindo que o cigarro tem um efeito generalizado,provavelmente alterando de alguma forma todo o sistema doindivíduo. Ainda, outros artigos relataram alterações no padrãode metilação do DNA em tecidos saudáveis de fumantes.Conclusão: O câncer associado ao hábito de fumar pode terum mecanismo epigenético envolvido inclusive em órgãosnão diretamente expostos. A presença de alteraçõesepigenéticas em tecidos saudáveis pode servir de alertapara o clínico responsável pelo paciente, uma vez que já foisugerido que alterações epigenéticas podem atuar comomarcadores para diversos tipos de câncer, sendo assimuma ferramenta para diagnóstico e direcionamento dotratamento...

Environmental factors such as cigarette agentsare capable of altering the genome of the cells and lead totumor development. Recently, the ability of the cigarette tocause alterations in the epigenome has also been investigated.Epigenome refers to information of the genome that is notrelated to the sequence of DNA bases. Objective: The aim ofthis study was to conduct a literature review about smokingand its association with the altered pattern of DNA methylationin tumor samples. Method: The research considered articlespublished from 2000 to 2010 in Pubmed database. Articleswere selected by accessibility, excluding reviews andresearch on cell lines and animals. Results: The selectedarticles revealed that smoking can alter the epigenome evenin organs not directly exposed as the mouth and lung,suggesting that cigarette smoking has a widespread effect,probably changing in some way the whole system of theindividual. Some articles have reported changes in the patternof DNA methylation in healthy tissues of smokers. Conclusion:Smoking-associated cancers may have an epigeneticmechanism involved even in organs not directly exposed.The presence of epigenetic changes in healthy tissue canserve as a warning to the clinician responsible for the patient,since it has been suggested to be as strong biomarker formany types of cancers and then it is a tool for diagnosis andtreatment course...