Protocol to generate human liver spheroids to study liver fibrosis induced by metabolic stress.

Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic "livers in a dish." We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies.

Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

The Origin and Fate of Liver Myofibroblasts.

Liver fibrosis of different etiologies is a serious health problem worldwide. There is no effective therapy available for liver fibrosis except the removal of the underlying cause of injury or liver transplantation. Development of liver fibrosis is caused by fibrogenic myofibroblasts that are not present in the normal liver, but rather activate from liver resident mesenchymal cells in response to chronic toxic or cholestatic injury. Many studies indicate that liver fibrosis is reversible when the causative agent is removed. Regression of liver fibrosis is associated with the disappearance of activated myofibroblasts and resorption of the fibrous scar. In this review, we discuss the results of genetic tracing and cell fate mapping of hepatic stellate cells and portal fibroblasts, their specific characteristics, and potential phenotypes. We summarize research progress in the understanding of the molecular mechanisms underlying the development and reversibility of liver fibrosis, including activation, apoptosis, and inactivation of myofibroblasts.

Isolation of primary human liver cells from normal and nonalcoholic steatohepatitis livers.

Here, we present a protocol for isolating human hepatocytes and neural progenitor cells from normal and nonalcoholic steatohepatitis livers. We describe steps for perfusion for scaled-up liver cell isolation and optimization of chemical digestion to achieve maximal yield and cell viability. We then detail a liver cell cryopreservation and potential applications, such as the use of human liver cells as a tool to link experimental and translational research.

Evaluation of the Genetic Screening Processor for the Performance of Newborn Screening Tests

Abstract The collection of dried blood spots (DBSs) on filter paper has been a powerful tool in newborn screening (NBS) programs and in other fields. However, filter paper has been associated with some level of imprecision due to the filter paper matrix effect. In order to minimize measurement variations, these interferences should be evaluated by NBS assays. The aim of this study is to evaluate the performance of genetic screening processor (GSP) equipment in comparison with a widely used AutoDELFIA and to discuss the limitations and advantages of this new technology in NBS. We evaluated the performance of 3 NBS assays in DBS using GSP in comparison with AutoDELFIA. To determine the inaccuracy and the intra-assay precision, a comparative study and a replication experiment were performed. In the comparative study, human thyroid-stimulating hormone (hTSH) assay showed the highest correlation coefficient, followed by 17α-OH-progesterone and immunoreactive trypsinogen (IRT) assays. The results of the present study suggest that the GSP equipment and kits are suitable for implementation and have acceptable performance for NBS routine. Genetic screening processor assay tends to underestimate hTSH and IRT concentrations in the clinically relevant range when compared to AutoDELFIA assays. More studies are necessary to reevaluate cutoff values. Furthermore, the equipment has advantages when compared with AutoDELFIA, such as methodology with more specificity, reduction in the processing time, and randomized routine. This helps promoting faster dynamic technical processes and faster report generation.

Decreased Expression of GPER1 Gene and Protein in Goiter.

Goiter is more common in women, suggesting that estrogen could be involved in its physiopathology. The presence of classical estrogen receptors (ERα and ERß) has been described in thyroid tissue, suggesting a direct effect of estrogen on the gland. A nonclassic estrogen receptor, the G-protein-coupled estrogen receptor (GPER1), has been described recently in several tissues. However, in goiter, the presence of this receptor has not been studied yet. We investigated GPER1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. In normal thyroid (n = 16) and goiter (n = 19), GPER1 gene was expressed in all samples, while GPER1 protein was expressed in all samples of normal thyroid (n = 15) but in only 72% of goiter samples (n = 13). When comparing GPER1 gene and protein levels in both conditions, gene expression and protein levels were higher in normal thyroid than in goiter, suggesting a role of this receptor in this condition. Further studies are needed to elucidate the role of GPER1 in normal thyroid and goiter.

Validation of reference genes for normalization gene expression in reverse transcription quantitative PCR in human normal thyroid and goiter tissue.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been recognized as the most accurate method for quantifying mRNA transcripts, but normalization of samples is a prerequisite for correct data interpretation. So, this study aimed to evaluate the most stable reference gene for RT-qPCR in human normal thyroid and goiter tissues. Beta-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); succinate dehydrogenase, subunit A, flavoprotein (Fp) (SDHA); hypoxanthine phosphoribosyltransferase I (HPRTI); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ); and beta-2-microglobulin (B2M) were evaluated in 14 thyroid tissue samples (7 normal and 7 goiter tissues) by RT-qPCR. The mean Cq and the maximum fold change (MFC) and NormFinder software were used to assess the stability of the genes. As a result, ACTB gene was more stable than GAPDH, SDHA, HPRTI, YWHAZ, and B2M. In conclusion, ACTB could be used to normalize RT-qPCR data in normal thyroid and goiter tissues.

Determinação da acurácia do método qualitativo da medida da atividade da glicose-6-fosfato desidrogenase / Determination of the accuracy of the measurement method for dehydrogenase activity

A deficiência de glicose-6-fosfato desidrogenase (G6PD) é um problema de saúde pública que afeta aproximadamente 400 milhões de pessoas no mundo. No mercado, existem vários métodos que medem a atividade da G6PD. Os objetivos deste estudo foram determinar a acurácia do método de Brewer frente a um padrão de referência e estimar a prevalência de deficiência de G6PD na amostra. Foi realizado um estudo transversal de grupo de pacientes internados no HCPA com icterícia a esclarecer, no período de junho de 2004 a maio de 2005. Amostras foram processadas pelo método de Brewer e pelo método de Normalização da Hemoglobina, o qual foi usado como padrão ouro. Foi analisado para atividade da G6PD um total de 173 pacientes. A idade variou de 1 dia a 82 anos, sendo que 66 por cento da amostra possuía até 15 dias de vida. A atividade média e o desvio padrão da G6PD na amostra analisada foi de 17.67± 5,66 U/gHb. A freqüência estimada, pelo padrão ouro, da deficiência de G6PD, foi de 13 (7,7 por cento) pacientes com deficiência parcial ou total, e pelo método de Brewer foi de 14 (8,67 por cento). A sensibilidade do método de Brewer comparada com o método quantitativo da Normalização da Hemoglobina foi de 92,8 por cento e a especificidade foi de 98,7 por cento. A deficiência de G6PD é prevalente em nosso meio. Testes de baixo custo, tais como o teste de Brewer, podem ser utilizados como testes de triagem desta deficiência, principalmente no monitoramento de recém-nascidos que estão sob o risco de desenvolver icterícia neonatal.

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is a public health problem which affects about 400 millions of people all over the world. Some methods that measure the activity of G6PD have already been developed. Thus, the aim of this study was to evaluate the accuracy of the Brewer's method compared with a standard reference and estimate the prevalence of G6PD deficiency in the sample. A cross-sectional study of a group of patients in HCPA presenting with jaundice was carried out from June 2004 to May 2005. Samples were processed by the metahaemoglobin reduction test (Brewer's method) and by the method of Haemoglobin Normalization, which was used as the standard reference. A total of 173 patients were analyzed for G6PD activity. The ages varied from one day to 82 years old with 66 percent of the sample being less than 16 days old. The mean activity and standard deviation of G6PD for the analyzed sample was 17.67 ± 5.66 U/gHb. The estimated frequencies of G6PD deficiency for the standard reference and Brewer's method were 13 (7.7 percent) subjects (total or partial deficiency) and 14 (8.67 percent), respectively. When the Brewer's method was compared with the quantitative method of Hemoglobin Normalization, it showed a sensitivity of 92.8 percent and specificity of 98.7 percent. As G6PD deficiency predominates in our society, low cost tests, such as the Brewer's test can be used for screening this deficiency, mainly to monitor newborn babies who are at risk of developing jaundice.

Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in patients from the southern Brazilian city of Porto Alegre, RS

Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) deficiency is one of the most common human enzymopathies throughout the world. Although most affected individuals are asymptomatic, there is a risk of neonatal jaundice and acute hemolytic anemia which can be triggered by infection, some pharmaceuticals and, in older individuals, eating fava beans. We characterized the molecular basis of G6PDH deficiency in a sample of 348 adults from Porto Alegre (population about 1.5 million), the capital of the southernmost Brazilian state of Rio Grande do Sul. Genomic DNA was extracted from peripheral blood leukocytes. We studied the three G6PDH mutations that appear to be the most frequent in Southern Brazil, the G202A and A376G A minus (A-) variants and the C563T Mediterranean (Med) variant. From July 2004 to October 2005, 348 patients (162 Females plus 186 males, age range 0 to 82 years) from Porto Alegre were referred to our laboratory for G6PDH analysis, 36 (9.7 percent) of which showed deficient G6PDH activity. These 36 patients and 34 randomly-selected non-deficient control individuals were submitted to molecular analysis which revealed a predominance of G6PDH A- allele among the deficient patients. The prevalence of the G6PDH A- variant agrees with its distribution among the ethnic groups that colonized RS, especially those of African, Portuguese, Spanish, and Italian origin.

Prevalence of G6PD deficiency in newborns in the south of Brazil.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked disorder which causes neonatal jaundice in most cases, and in association with intake of drugs or certain foods (for example fava) can cause haemolytic crises. The aim of this study was to determine the prevalence of G6PD deficiency in Rio Grande do Sul (RS), the southernmost state of Brazil. We tested 2799 newborn blood samples. A commercial kit was used for the quantitative measurement of G6PD activity. Of the 2799 samples, 39 (1.4%) exhibited total deficiency, 178 (6.4%) exhibited intermediate deficiency and 2582 (92.2%) were normal. We found no correlation between G6PD deficiency and ethnic origin, but a high prevalence of patients with partial deficiency could be associated with the type of colonization of RS. The combined prevalence for both types of deficiency (complete and partial) was 7.9% among the newborn population. This finding is important as both types of deficiency must receive same kind of preventive care.