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

Hepatocellular carcinoma (HCC) in patients with Non-Alcoholic Fatty Liver Disease (NAFLD): screening, treatment and survival analysis in a Brazilian series

Objective: The aim of the present study was to evaluate the clinical features, Hepatocellular Carcinoma (HCC) screening, treatment modalities, and Overall Survival (OS) in a series of Non-Alcoholic Fatty Liver Disease-Related Hepatocellular Carcinoma (NAFLD-HCC) Brazilian patients. Methods: This was a cross-sectional study at the Instituto do Cancer do Estado de São Paulo, at the Faculdade de Medicina da Universidade de São Paulo with the approval of the local research ethics committee. NAFLD patients with HCC diagnosed, from May&nbsp;2010 to May&nbsp;2019, were included. Results: A total of&nbsp;131&nbsp;patients were included. Risk factors for NAFLD were present in&nbsp;94.7% of the patients. Only&nbsp;29% of patients were in the HCC screening program before diagnosis. HCC treatment was performed in&nbsp;84.7% of patients. Cumulative survival at the end of the first year was&nbsp;72%, second-year&nbsp;52%, and fifth-year&nbsp;32%. HCC screening before diagnosis was not significantly associated with higher cumulative survival. The independent factors associated with shorter general survival were BCLC C-D, p &lt; 0.001, and the size of the largest nodule&nbsp;&gt; 42&nbsp;mm, p&nbsp;=&nbsp;0.039. Conclusions: Although the efficacy of screening in our population regarding overall survival was hampered due to the sample size (29%&nbsp;had screening), BCLC stages&nbsp;C‒D and the size of the largest nodule larger than&nbsp;42&nbsp;mm were identified as independent factors of worse prognosis

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

Uso de celulas da medula ossea congeladas para estudo de cariotipo

O estudo citogenetico habitual utiliza-se de celulas frescas para a cultura, o que limita o numero de experimentos a serem realizados. Este fato tem despertado o interesse de alguns pesquisadores em avaliar o uso de celulas congeladas para analise de cariotipo. Muito pouco tem sido modificado nas tecnicas de congelamento e descongelamento desde sua descricao, apenas o aprimoramento das mesmas tornando-as menos complexas e mais utilizaveis. O presente trabalho analisa os efeitos de metodo simplificado de criopreservacao no estudo citogenetico de celulas de medula ossea, utilizando agentes crioprotetores de baixo e alto peso molecular, sem velocidade de congelamento controlada. Avalia, tambem, o uso de GM-CSF como fator estimulante de crescimento e sua interferencia na qualidade das metafases. Para avaliar o efeito proliferativo do GM-CSF, utilizou-se o indice mitotico nas culturas de celulas a fresco e congeladas. O teste de viabilidade celular, azul de Trypan, foi realizado em todas as amostras para determinar o efeito do congelamento sobre as celulas. A concentracao media de celulas congeladas foi de 25,0 x 106 celulas / mL. As celulas foram criopreservadas em Normosol R contendo ohydroxyethylstarcho (HES) 12%, albumina humana 8% e imetilsulfoxido (DMSO) 10%. As celulas de medula ossea foram adicionados volumes iguais de solucao crioprotetora e a concentracao final foi HES 6%, Albumina Humana 4% e DMSO 5%. Foram estudadas 25 amostras de 24 individuos, sendo 8 com LMC, 6 com LMA, 3 com SMD, 2 com LLA-T, 1 com linfoma linfoblastico leucemizado, 1 doador de medula ossea para transplante, 1 com anemia megaloblastica, 1 com LMA em remissao ha 2 anos e 1 com LLA em remissao ha 4 anos. As celulas de medula ossea foram separadas em dois grupos: um para cultura a fresco e o outro para congelamento. Foram feitas duas culturas: uma simples e outra com GM-CSF antes e apos o congelamento. Por volta de 3 meses, as celulas congeladas foram descongeladas rapidamente por imersao em banho Maria a 37&#61616; C sem agitacao e diluidas com tres volumes de meio RPMI 1640. O material foi ressuspenso e dividido em duas culturas, uma com GM-CSF e outra sem. O teste de viabilidade celular pre e pos congelamento mostrou valores medios de 98,8% e 78,5% respectivamente. O teste t pareado, foi estatisticamente significante (p = 0,000), confirmando que a criopreservacao induziu um decrescimo na viabilidade celular e consequentemente no indice mitotico. Obteve-se sucesso em 76% dos casos de cultura de celulas a fresco e em 52% das congeladas, embora os aspectos morfologicos foram semelhantes em ambas. Em relacao ao indice mitotico, nao houve diferenca estatisticamente significante pelo teste t entre as culturas com e sem GM-CSF (p = 0,084). O uso de GM-CSF nao melhorou a quantidade e qualidade das metafases. Os dados mostram que os estudos em celulas congeladas nao devem substituir os de celulas a fresco, salvo nos casos de auto transplante e em pesquisasBV UNIFESP: Teses e dissertaçõe

Microbiota and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH)

Genetic predisposition, the intestinal microbiota (IM) and environmental factors, such as sedentary lifestyle and inadequate diet, should be considered as critical factors for the development of nonalcoholic fatty liver disease (NAFLD). Recently, some studies have demonstrated an association between dysbiosis and NAFLD; however, the exact mechanisms that lead to intestinal membrane damage, bacterial translocation and inflammation are not well elucidated. Due to the relevance of this theme, the IM and its metabolites have received special attention in recent years in an attempt to better understand the mechanisms related to the prevention, physiopathology, and treatment of NAFLD. In this paper, we provide a review of the human IM and its role in diet, obesity, and the development/progression of NAFLD/NASH, as well as the use of prebiotics and probiotics in the modulation of IM

Índices glicolipídicos no hipotireoidismo tratado associado à doença hepática gordurosa não-alcoólica

CONTEXT: Thyroid hormones may interfere with regulation of lipid and carbohydrate metabolism as well as with severity of nonalcoholic fatty liver disease (NAFLD), however results are still debated. OBJECTIVES: Retrospective evaluation of clinical and metabolic correlations between hypothyroidism and NAFLD was the target. METHODS: Clinical, biochemical and histological investigation of 103 NAFLD patients exhibiting drug-treated hypothyroidism was conducted. RESULTS: Steatosis was present in 32.0% of the population and nonalcoholic steatohepatitis in 68.0%. Females were the majority in both groups, with age of 50.0 ± 1.5 and 56.0 ± 1.1 years, respectively. Hypothyroidism was not rare (15.5%), and multivariate analysis confirmed positive correlation with this disease for insulin (r = 0.213, P = 0.03), glucose homeostasis index "HOMA" (r = 0.221, P = 0.02), aspartate aminotransferase (r = 0.234, P = 0.01) and triglycerides above 150 mg/dL (r = 0.233, P = 0.01). No association between hypothyroidism and steatohepatitis could be established. CONCLUSION: A link could be identified between hypothyroidism and markers of glucose and lipid homeostasis, but not with severity of NAFLD. The lack of correlation with liver biopsy requires further studies.CONTEXTO: Os hormônios tireoidianos podem interferir na regulação do metabolismo de lipídios e carboidratos e também na gravidade da doença hepática gordurosa não-alcoólica (DHGNA), porém os resultados ainda são debatidos. OBJETIVOS: Avaliar retrospectivamente correlações clínicas e metabólicas entre hipotireoidismo e DHGNA. MÉTODOS: Em 103 pacientes com DHGNA confirmada por biopsia e também hipotireoidismo recebendo tratamento, procedeu-se à investigação clínica, bioquímica e histológica. RESULTADOS: A esteatose foi observada em 32,0% e a esteatohepatite não-alcoólica em 68,0% da população. O sexo feminino foi mais frequente nas duas circunstâncias, com idade média de 50,0 ± 1,5 e 56,0 ± 1,1 anos, respectivamente. O hipotireoidismo não foi raro (15,5%), sendo que na análise multivariada insulina (r = 0,213, P = 0,03), índice de homeostase glicídica HOMA (r = 0,221, P = 0,02), aspartato aminotransferase (r = 0,234, P = 0,01) e triglicerídeos acima de 150 mg/dL (r = 0,233, P = 0,01) foram correlacionados positivamente com hipotireoidismo. A associação entre hipotireoidismo e esteatohepatite não pôde ser estabelecida neste estudo. CONCLUSÃO: O hipotireoidismo vinculou-se à piora de alguns marcadores do metabolismo glicolipídico, porém não a lesões histológicas mais avançadas. A falta de correlação com a biopsia do fígado requer maiores estudos

The transcontinental variability of nonalcoholic fatty liver disease

Aim: To compare the phenotype of lean versus overweight (OW) and obese (OB) subjects with non-alcoholic fatty liver disease (NAFLD) across multiple continents.Methods: A retrospective study of histologically defined subjects from a single center each in France (Fr), Brazil (Br), India (In) and United States (US) was performed.Results: A total of 70 lean [body mass index (BMI) &lt; 25 kg/m2] subjects (Fr:Br:In:US: 16:19:22:13) with NAFLD were compared to 136 OW (BMI &gt; 25 kg/m2, BMI &lt; 29 kg/m2) (n = 28:33:52:23) and 224 OB subjects (BMI &gt; 29 kg/m2) (n = 81:11:22:103). Lean French subjects had the lowest incidence of type 2 diabetes while those from Brazil (P &lt; 0.01) had the highest. Lean subjects had similar low-density lipoprotein-cholesterol, but higher high-density lipoprotein-cholesterol compared to obese subjects in all regions. In both lean and obese subjects, there were both insulin-sensitive and insulin-resistant subjects. Lean French subjects were most insulin-sensitive while those from Brazil were mostly insulin-resistant. For each weight category, subjects from India were more insulin-sensitive than those from other regions. Disease activity increased from lean to overweight to obese in France but was similar across weight categories in other regions.Conclusion: The phenotype of NAFLD in lean subjects varies by region. Some obese subjects with NAFLD are insulin-sensitive. We hypothesize that genetics and region-specific disease modifiers account for these differences

A rodent model of NASH with cirrhosis, oval cell proliferation and hepatocellular carcinoma

Background/Aims: Hepatocellular carcinoma (HCC) is a well recognized complication of advanced NASH (non-alcoholic steatohepatitis). We sought to produce a rat model of NASH, cirrhosis and HCC. Methods: Adult Sprague-Dawley rats, weighing 250-300 g, were fed a choline-deficient, high trans-fat diet and exposed to DEN in drinking water. After 16 weeks, the animals underwent liver ultrasound (US), sacrifice and assessment by microscopy, immunohistochemistry and transmission electron microscopy (TEM). Results: US revealed steatosis and focal lesions in 6 of 7. All had steatohepatitis defined as inflammation, advanced fibrosis and ballooning with Mallory-Denk bodies (MDB) with frank cirrhosis in 6. Areas of more severe injury were associated with anti-CK19 positive ductular reaction. HCC, present in all, were macro-trabecullar or solid with polyhedral cells with foci of steatosis and ballooned cells. CK19 was positive in single or solid nests of oval cells and in neoplastic hepatocytes. TEM showed ballooning with small droplet fat, dilated endoplasmic reticulum and MDB in non-neoplastic hepatocytes and small droplet steatosis in some cancer cells. Conclusions: This model replicated many features of NASH including steatohepatitis with ballooning, fibrosis, cirrhosis and hepatocellular carcinoma. Oval cell proliferation was evident and the presence anti-CK 19 positivity in the cancer suggests oval cell origin of the malignancy. (C) 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.Alves Queiroz Family Fund for Research in Sao PauloThe University of Virginia Cancer Center, Commonwealth Cancer FoundationMr. Jack P Chamber