Karyotype of cryopreserved bone marrow cells

The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05). Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Disciplina de Hematologia e HemoterapiaUNIFESP, EPM, Disciplina de Hematologia e HemoterapiaSciEL

Methylene tetrahydrofolate reductase (MTHFR) and vascular endothelial growth factor (VEGF) polymorphisms in Brazilian patients with Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC)

OBJECTIVE: The folate pathway is involved in hepatic carcinogenesis and angiogenesis. Polymorphisms in genes related to such processes, including methylene tetrahydrofolate reductase (MTHFR) and vascular endothelial growth factor (VEGF)] may play an important role in the development of hepatocellular carcinoma (HCC). The objective of this study was to evaluate&nbsp;MTHFR&nbsp;and&nbsp;VEGF&nbsp;polymorphisms in Brazilian patients with hepatitis C virus (HCV)-related HCC. METHODS: A total of 119 patients diagnosed with confirmed HCC and HCV were included in the study. SNP genotyping assays were performed using real-time PCR. VEGFA (rs2010963, rs3025039, and rs833061) and MTHFRC677T (rs1801133, rs1801131) polymorphisms were evaluated. RESULTS: The C alleles of&nbsp;MTHFR&nbsp;(rs1801131) and&nbsp;VEGF&nbsp;(rs2010963) were associated with protection against the development of multinodular HCC, while the T allele of&nbsp;MTHFR&nbsp;(rs1801133) was associated with a higher risk of multinodular presentation [p=0.04 OR 1.835 CI (1.022-3.297)]. Multivariate analysis revealed that the GG/GC genotypes of&nbsp;VEGF&nbsp;rs2010963 were independently associated with multinodular tumors at diagnosis (p=0.013; OR 4.78 CI (1.38-16.67)]. CONCLUSION: Our results suggest that these polymorphisms may increase the risk of rapid tumor progression in patients with HCV infection. This subgroup of patients with HCC and who present polymorphism is more likely to be diagnosed with multinodular disease and not be amenable to receiving curative treatments. These data must be validated in larger cohorts, and the screening intervals can be customized based on genetic history

Association of a variant in the regulatory region of NADPH oxidase 4 gene and metabolic syndrome in patients with chronic hepatitis C

Abstract\ud \ud Background\ud Given the important contribution of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system to the generation of reactive oxygen species induced by hepatitis C virus (HCV), we investigated two single nucleotide polymorphisms (SNPs) in the putative regulatory region of the genes encoding NADPH oxidase 4 catalytic subunit (NOX4) and its regulatory subunit p22phox (CYBA) and their relation with metabolic and histological variables in patients with HCV.\ud \ud \ud Methods\ud One hundred seventy eight naïve HCV patients (49.3% male; 65% HCV genotype 1) with positive HCV RNA were genotyped using specific primers and fluorescent-labeled probes for SNPs rs3017887 in NOX4 and −675 T → A in CYBA.\ud \ud \ud Results\ud No association was found between the genotype frequencies of NOX4 and CYBA SNPs and inflammation scores or fibrosis stages in the overall population. The presence of the CA + AA genotypes of the NOX4 SNP was nominally associated with a lower alanine aminotransferase (ALT) concentration in the male population (CA + AA = 72.23 ± 6.34 U/L versus CC = 100.22 ± 9.85; mean ± SEM; P = 0.05). The TT genotype of the CYBA SNP was also nominally associated with a lower ALT concentration in the male population (TT = 84.01 ± 6.77 U/L versus TA + AA = 109.67 ± 18.37 U/L; mean ± SEM; P = 0.047). The minor A-allele of the NOX4 SNP was inversely associated with the frequency of metabolic syndrome (MS) in the male population (odds ratio (OR): 0.15; 95% confidence interval (CI): 0.03 to 0.79; P = 0.025).\ud \ud \ud Conclusions\ud The results suggest that the evaluated NOX4 and CYBA SNPs are not direct genetic determinants of fibrosis in HCV patients, but nevertheless NOX4 rs3017887 SNP could indirectly influence fibrosis susceptibility due to its inverse association with MS in male patients

Effect of interferon-alfa on the IGF system gene expression in patients with Hepatitis C

A presente investigação teve por objetivo estudar o papel do eixo GH-IGF-IGFBP no tecido hepático e nos linfócitos T e B de pacientes portadores do vírus da hepatite C, em terapia com INF-alfa e ribavirina nos períodos pré e pós-tratamento. Dados da literatura têm evidenciado o envolvimento do IGF-I na modulação da resposta imune, bem como sua atuação como fator de crescimento para células imunológicas, além de apresentar valor preditivo na avaliação da reserva hepática desses pacientes. O número final de pacientes abordados perfez o total de 80. Destes, 39 iniciaram tratamento conforme protocolo estabelecido pela equipe clínica. Quatro pacientes foram excluídos durante o curso do tratamento, sendo 2 por óbito e 2 por transtorno depressivo grave com ideação suicida. Dos 35 pacientes que concluíram o tratamento, 18 apresentaram resposta virológica ao final do tratamento. Destes, 15 pacientes (43%) obtiveram resposta virológica sustentada (RVS). As médias das U.A. (unidades arbitrárias) das enzimas AST e ALT foram estatisticamente diferentes entre os períodos pré e pós-tratamento, tanto para os pacientes que apresentaram RVS quanto para aqueles que não apresentaram tal resposta. No grupo de pacientes com RVS, observou-se que a média dos níveis basais de AST foi menor quando comparada à do grupo de pacientes sem RVS. A média das concentrações plasmáticas basais de IGF-I livre no grupo de pacientes com RVS foi estatisticamente maior quando comparada à do grupo de pacientes não respondedores. As médias das concentrações de IGFBP3 circulante no período pós-tratamento foram estatisticamente diferentes entre os grupos com e sem RVS. Um aumento do conteúdo de mRNA do gene do IGF-IR foi observado em tecido hepático de todos os pacientes com HCC quando comparado com o tecido hepático normal. Este resultado foi confirmado por análise imuno-histoquímica para o IGF-IR. Nos pacientes que apresentaram RVS a magnitude de expressão de mRNA do IGF-IR em amostras de tecido hepático após o tratamento foram estatisticamente menores em relação ao basal. Tal fato não foi observado no grupo de pacientes não respondedores. Não houve diferença estatisticamente significativa entre a média do conteúdo de mRNA do IGF-I em tecido hepático dos grupos de pacientes com e sem RVS, pré ou pós-tratamento. A média do conteúdo de mRNA do IGF-I em linfócitos T no grupo de pacientes com RVS foi estatisticamente maior em relação à do grupo de pacientes não respondedores quando se consideram os períodos pré e pós-tratamento conjuntamente. Não se observou diferença estatisticamente significativa entre as médias do conteúdo de mRNA do IGF-IR nos grupos com e sem RVS, pré ou pós-tratamento. Não houve diferença estatisticamente significativa entre as médias do conteúdo de mRNA de IGF-I e IGF-IR em linfócitos B de pacientes portadores do VHC, com e sem RVS e nos períodos pré e pós-tratamento. A diminuição de expressão de mRNA do IGF-IR em tecido hepático, observada no grupo de pacientes com RVS, sugere uma melhora da doença hepática. A hipótese de que um efeito do INF-? sobre os componentes do sistema IGF possa contribuir para este achado não pode ser descartada, porém, é provável que este efeito não seja tão importante, pois não se observou diferença nos níveis de expressão do IGF-IR hepático no grupo de pacientes não respondedores. Embora a supra-regulação (\"up-regulation\") do IGF-IR possa participar da regeneração hepática, é preciso elucidar se o aumento da expressão do IGF-IR na HCC resulta da ativação direta do gene pelo VHC ou se é uma conseqüência da agressão ao parênquima. As concentrações plasmáticas de IGF-I livre >1,35 ng/mL puderam ser consideradas preditivas da resposta ao tratamento da hepatite C com uma razão de probabilidade (\"odds ratio\") de 17,33±1,02 (Limite de confiança: 2,26-127,34)The current investigation aimed to study the role of the GH-IGF-IGFBP axis in liver tissue and in T and B lymphocytes in patients with hepatitis C virus (HCV) before and after therapeutic regimen based on interferon alfa-2a or alfa-2b (3 million U SC 3x/wk) and ribavirin (1000-1200 mg qid). It has been shown that IGF-I plays an important role in the modulation of the immune response, besides its role as a growth factor for the immunologic cells. It also presents a predictive value in the evaluation of the hepatic reserve of these patients. Among 80 patients enrolled for this investigation, 39 began treatment with interferon-? and ribavirin, according to an established protocol. Two patients were excluded during treatment due to severe depressive disorders accompanied by suicidal thoughts and 2 patients died. Of the 35 patients who concluded the treatment, 18 eventually presented virological response. Of these, 15 (43%) maintained sustained virological response (SVR). The levels of AST and ALT enzymes in both pre and post-treatment periods were statistically different for both patients with SVR and those who did not present such response. In the group with SVR, aminotransferases basal levels were statistically lower when compared to the group of patients without SVR. In the group of non-responsive patients, the average of the scores of parenchyma activity was statistically lower in post-treatment period when compared to pre-treatment period. Furthermore, comparing post-treatment periods in both groups with and without SVR, the average of the scores of parenchyma activity was statistically lower in the group without SVR when compared to the group with SVR. Mean plasma concentrations of free IGF-I before treatment in patients who eventually achieved SVR was statistically higher in comparison to the group of non-responsive patients. Mean plasma concentrations of IGFBP3 were statistically higher in the group with RVS when compared to the group of patients without SVR. An increase of IGF-IR mRNA content was observed in hepatic tissue from all patients with CHC in comparison to normal liver. These results were confirmed by immunohistochemical analysis for the IGF-IR. IGF-IR mRNA content in liver tissue samples from patients who achieved SVR after treatment was statistically lower than that observed before treatment There was not statistical difference between IGF-I mRNA content in hepatic tissues from both groups of patients with and without SVR, in pre and post-treatment periods. IGF-I mRNA expression in T lymphocytes from patients with SVR was statistically higher in comparison to the non-responsive group of patients, considering both pre and post-treatment periods altogether. No statistical difference was observed in IGF-IR mRNA expression in both groups of patients with and without SVR, in pre and post-treatment periods. The statistical analysis did not disclose any statistically significant differences in IGF-I or IGF-IR mRNA expressions in B lymphocytes from patients with or without SVR, in pre and post-treatment periods. A decrease in hepatic IGF-IR mRNA content observed in patients who achieved SVR after therapy, suggested an improvement in hepatic damage. The hypothesis that the INF-? affects components of the IGF system contributing to these findings could not be discarded. However, it is unlike that these effects would play relevant role because any differences were observed in the hepatic IGF-IR mRNA expression in non-responsive patients. It remains to be elucidated whether IGF-IR up-regulation would be involved in hepatocyte regeneration or CHC would result from direct activation of IGF-IR gene by HCV and/or as a consequence of chronic aggression to hepatic parenchyma. Plasma concentration of free IGF-I >1.35 ng/ml was considered to be a predictive response to the treatment of hepatitis C with a probability ratio (odds ratio) of 17.33±1.02 (confidence interval: 2.26 -127.34

Effect of interferon-alfa on the IGF system gene expression in patients with Hepatitis C

A presente investigação teve por objetivo estudar o papel do eixo GH-IGF-IGFBP no tecido hepático e nos linfócitos T e B de pacientes portadores do vírus da hepatite C, em terapia com INF-alfa e ribavirina nos períodos pré e pós-tratamento. Dados da literatura têm evidenciado o envolvimento do IGF-I na modulação da resposta imune, bem como sua atuação como fator de crescimento para células imunológicas, além de apresentar valor preditivo na avaliação da reserva hepática desses pacientes. O número final de pacientes abordados perfez o total de 80. Destes, 39 iniciaram tratamento conforme protocolo estabelecido pela equipe clínica. Quatro pacientes foram excluídos durante o curso do tratamento, sendo 2 por óbito e 2 por transtorno depressivo grave com ideação suicida. Dos 35 pacientes que concluíram o tratamento, 18 apresentaram resposta virológica ao final do tratamento. Destes, 15 pacientes (43%) obtiveram resposta virológica sustentada (RVS). As médias das U.A. (unidades arbitrárias) das enzimas AST e ALT foram estatisticamente diferentes entre os períodos pré e pós-tratamento, tanto para os pacientes que apresentaram RVS quanto para aqueles que não apresentaram tal resposta. No grupo de pacientes com RVS, observou-se que a média dos níveis basais de AST foi menor quando comparada à do grupo de pacientes sem RVS. A média das concentrações plasmáticas basais de IGF-I livre no grupo de pacientes com RVS foi estatisticamente maior quando comparada à do grupo de pacientes não respondedores. As médias das concentrações de IGFBP3 circulante no período pós-tratamento foram estatisticamente diferentes entre os grupos com e sem RVS. Um aumento do conteúdo de mRNA do gene do IGF-IR foi observado em tecido hepático de todos os pacientes com HCC quando comparado com o tecido hepático normal. Este resultado foi confirmado por análise imuno-histoquímica para o IGF-IR. Nos pacientes que apresentaram RVS a magnitude de expressão de mRNA do IGF-IR em amostras de tecido hepático após o tratamento foram estatisticamente menores em relação ao basal. Tal fato não foi observado no grupo de pacientes não respondedores. Não houve diferença estatisticamente significativa entre a média do conteúdo de mRNA do IGF-I em tecido hepático dos grupos de pacientes com e sem RVS, pré ou pós-tratamento. A média do conteúdo de mRNA do IGF-I em linfócitos T no grupo de pacientes com RVS foi estatisticamente maior em relação à do grupo de pacientes não respondedores quando se consideram os períodos pré e pós-tratamento conjuntamente. Não se observou diferença estatisticamente significativa entre as médias do conteúdo de mRNA do IGF-IR nos grupos com e sem RVS, pré ou pós-tratamento. Não houve diferença estatisticamente significativa entre as médias do conteúdo de mRNA de IGF-I e IGF-IR em linfócitos B de pacientes portadores do VHC, com e sem RVS e nos períodos pré e pós-tratamento. A diminuição de expressão de mRNA do IGF-IR em tecido hepático, observada no grupo de pacientes com RVS, sugere uma melhora da doença hepática. A hipótese de que um efeito do INF-? sobre os componentes do sistema IGF possa contribuir para este achado não pode ser descartada, porém, é provável que este efeito não seja tão importante, pois não se observou diferença nos níveis de expressão do IGF-IR hepático no grupo de pacientes não respondedores. Embora a supra-regulação (\"up-regulation\") do IGF-IR possa participar da regeneração hepática, é preciso elucidar se o aumento da expressão do IGF-IR na HCC resulta da ativação direta do gene pelo VHC ou se é uma conseqüência da agressão ao parênquima. As concentrações plasmáticas de IGF-I livre >1,35 ng/mL puderam ser consideradas preditivas da resposta ao tratamento da hepatite C com uma razão de probabilidade (\"odds ratio\") de 17,33±1,02 (Limite de confiança: 2,26-127,34)The current investigation aimed to study the role of the GH-IGF-IGFBP axis in liver tissue and in T and B lymphocytes in patients with hepatitis C virus (HCV) before and after therapeutic regimen based on interferon alfa-2a or alfa-2b (3 million U SC 3x/wk) and ribavirin (1000-1200 mg qid). It has been shown that IGF-I plays an important role in the modulation of the immune response, besides its role as a growth factor for the immunologic cells. It also presents a predictive value in the evaluation of the hepatic reserve of these patients. Among 80 patients enrolled for this investigation, 39 began treatment with interferon-? and ribavirin, according to an established protocol. Two patients were excluded during treatment due to severe depressive disorders accompanied by suicidal thoughts and 2 patients died. Of the 35 patients who concluded the treatment, 18 eventually presented virological response. Of these, 15 (43%) maintained sustained virological response (SVR). The levels of AST and ALT enzymes in both pre and post-treatment periods were statistically different for both patients with SVR and those who did not present such response. In the group with SVR, aminotransferases basal levels were statistically lower when compared to the group of patients without SVR. In the group of non-responsive patients, the average of the scores of parenchyma activity was statistically lower in post-treatment period when compared to pre-treatment period. Furthermore, comparing post-treatment periods in both groups with and without SVR, the average of the scores of parenchyma activity was statistically lower in the group without SVR when compared to the group with SVR. Mean plasma concentrations of free IGF-I before treatment in patients who eventually achieved SVR was statistically higher in comparison to the group of non-responsive patients. Mean plasma concentrations of IGFBP3 were statistically higher in the group with RVS when compared to the group of patients without SVR. An increase of IGF-IR mRNA content was observed in hepatic tissue from all patients with CHC in comparison to normal liver. These results were confirmed by immunohistochemical analysis for the IGF-IR. IGF-IR mRNA content in liver tissue samples from patients who achieved SVR after treatment was statistically lower than that observed before treatment There was not statistical difference between IGF-I mRNA content in hepatic tissues from both groups of patients with and without SVR, in pre and post-treatment periods. IGF-I mRNA expression in T lymphocytes from patients with SVR was statistically higher in comparison to the non-responsive group of patients, considering both pre and post-treatment periods altogether. No statistical difference was observed in IGF-IR mRNA expression in both groups of patients with and without SVR, in pre and post-treatment periods. The statistical analysis did not disclose any statistically significant differences in IGF-I or IGF-IR mRNA expressions in B lymphocytes from patients with or without SVR, in pre and post-treatment periods. A decrease in hepatic IGF-IR mRNA content observed in patients who achieved SVR after therapy, suggested an improvement in hepatic damage. The hypothesis that the INF-? affects components of the IGF system contributing to these findings could not be discarded. However, it is unlike that these effects would play relevant role because any differences were observed in the hepatic IGF-IR mRNA expression in non-responsive patients. It remains to be elucidated whether IGF-IR up-regulation would be involved in hepatocyte regeneration or CHC would result from direct activation of IGF-IR gene by HCV and/or as a consequence of chronic aggression to hepatic parenchyma. Plasma concentration of free IGF-I >1.35 ng/ml was considered to be a predictive response to the treatment of hepatitis C with a probability ratio (odds ratio) of 17.33±1.02 (confidence interval: 2.26 -127.34

Fatty Pancreas: Disease or Finding?

Despite a growing number of investigative studies on pancreatic fat deposition, there remains no clear indication regarding the clinical relevance of fat infiltration in the pancreas, also called fatty pancreas (FP). An individual’s body weight is correlated with their pancreatic weight. Moreover, lipid infiltration causes disorders that compromise not only morphology but also metabolic functions. Fat infiltration leads to insulin resistance, type II diabetes mellitus, and pancreatic cancer; however, knowledge about pancreatic fat content and aspects related to the clinical profile remains unclear in the literature. The present review describes the current knowledge of FP, including its pathophysiology and clinical implications, as well as lifestyle changes in FP

Non-pharmacological management options for MAFLD: a practical guide

Lifestyle changes should be the main basis for any treatment for metabolic dysfunction–associated fatty liver disease (MAFLD), aiming to increase energy expenditure, reduce energy intake and improve the quality of nutrients consumed. As it is a multifactorial disease, approaches such as physical exercise, a better dietary pattern, and possible pharmacological intervention are shown to be more efficient when used simultaneously to the detriment of their applications. The main treatment for MAFLD is a lifestyle change consisting of diet, activity, exercise, and weight loss. The variables for training prescription such as type of physical exercise (aerobic or strength training), the weekly frequency, and the intensity most indicated for the treatment of MAFLD remain uncertain, that is, the recommendations must be adapted to the clinical conditions comorbidities, and preferences of each subject in a way individual. This review addresses recent management options for MAFLD including diet, nutrients, gut microbiota, and physical exercise