Trimethylation profile of histones H3 lysine 4 and 9 in late preantral and early antral caprine follicles grown in vivo versus in vitro in the presence of anethole.

This study assessed the histones methylation profile (H3K4me3 and H3K9me3) in late preantral (PA) and early antral (EA) caprine follicles grown in vivo and in vitro, and the anethole effect during in vitro culture of PA follicles. Uncultured in vivo-grown follicles (PA, n = 64; EA, n = 73) were used as controls to assess the methylation profile and genes' expression related to apoptosis cascade (BAX, proapoptotic; BCL2, antiapoptotic), steroidogenesis (CYP17, CYP19A1), and demethylation (KDM1AX1, KDM1AX2, KDM3A). The isolated PA follicles (n = 174) were cultured in vitro for 6 days in α-MEM+ in either absence (control) or presence of anethole. After culture, EA follicles were evaluated for methylation, mRNA abundance, and morphometry. Follicle diameter increased after culture, regardless of treatment. The methylation profile and the mRNA abundance were similar between in vivo-grown PA and EA follicles. Anethole treatment led to higher H3K4me3 fluorescence intensity in EA follicles. The mRNA abundances of BAX, CYP17, and CYP19A1 were higher, and BCL2 and KDM3A were lower in in vitro-grown EA follicles than in vivo-grown follicles. In conclusion, in vitro follicle culture affected H3K4me3 fluorescence intensity, mRNA abundance of apoptotic genes, and steroidogenic and demethylase enzymes compared with in vivo-grown follicles.

Comprehensive proteomic profiling of early antral follicles from sheep.

The present study evaluates the proteome of early antral follicles from Ovis aries. Fifty follicles were collected from ovaries of adult ewes and extracted proteins were trypsin-digested, desalted and analyzed by LC-MS/MS. Genes were screened for potential modulation by miRNAs and protein data, subjected to functional enrichment analysis. Label-free mass spectrometry allowed the identification of 2503 follicle proteins, confirming vimentin, actin, lamin, heat shock proteins and histones as the most abundant ones. In silico analyses indicated that miRNAs modulate the expression of genes coding proteins of the sheep follicles involved in cell cycle, cell differentiation, aging, apoptosis, cell death, adipocyte differentiation, cell division. The most important biological processes associated with the follicle proteins were innate immune response, translation, adaptive immune response and protein folding, while molecular functions linked to the proteome of sheep antral follicles related to metal ion binding, ATP binding, oxygen binding, RNA binding and GTP binding, among others. Upload of 2503 Uniport accession codes through DAVID platform matched 1274 genes, associated with translation, metabolic process, proteolysis involved in cellular protein catabolic process, zona pellucida receptor complex and others. KEEG pathways analysis indicated genes correlated with ovine follicular development, with major pathways listed as carbon metabolism, biosynthesis of amino acids, glutathione metabolism, oxidative phosphorylation, fatty acid degradation and oocyte meiosis. This represents a comprehensive atlas of proteins expressed in sheep early antral follicles and will contribute to future identification of biomarkers for follicular development and oocyte maturation.

How in vitro maturation changes the proteome of ovine cumulus-oocyte complexes?

The present study evaluated the effects of in vitro maturation (IVM) on the proteome of cumulus-oocyte complexes (COCs) from ewes. Extracted COC proteins were analyzed by LC-MS/MS. Differences in protein abundances (p < 0.05) and functional enrichments in immature versus in vitro-matured COCs were evaluated using bioinformatics tools. There were 2550 proteins identified in the COCs, with 89 and 87 proteins exclusive to immature and mature COCs, respectively. IVM caused downregulation of 84 and upregulation of 34 proteins. Major upregulated proteins in mature COCs were dopey_N domain-containing protein, structural maintenance of chromosomes protein, ubiquitin-like modifier-activating enzyme 2. Main downregulated proteins in mature COCs were immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain 2, alpha-2-macroglobulin. Proteins exclusive to mature COCs and upregulated after IVM related to immune response, complement cascade, vesicle-mediated transport, cell cycle, and extracellular matrix organization. Proteins of immature COCs and downregulated after IVM were linked to metabolic processes, immune response, and complement cascade. KEGG pathways and miRNA-regulated genes attributed to downregulated and mature COC proteins related to complement and coagulation cascades, metabolism, humoral response, and B cell-mediated immunity. Thus, IVM influenced the ovine COC proteome. This knowledge supports the future development of efficient IVM protocols for Ovis aries.

MiR-612, miR-637, and miR-874 can Regulate VEGFA Expression in Hepatocellular Carcinoma Cell Lines.

MicroRNAs (miRNAs) are short non-coding RNA molecules acting as important posttranscriptional gene and protein expression regulators in cancer. The study goal was to examine VEGFA (vascular endothelial growth factor A) expression in hepatocellular carcinoma (HCC) cell lines upon transfection miR-612, miR-637, or miR-874. Methods: MiR-612 mimics, miR-637 mimics, or miR-874 inhibitors were transfected using Lipofectamine RNAiMax in both HCC cell lines, HepG2 and HuH-7. Real-time PCR, Western blotting, and ELISA methods were used to evaluate VEGFA regulation by the miRNAs. Results: Gene and protein expression levels of VEGFA were down-expressed in both cell lines, HepG2 and HuH-7, transfected with miR-612 or miR-637. Transfection with miR-874 inhibitor showed an increase in VEGFA gene expression in HepG2 and HuH-7 cell lines; however, no regulation was observed on VEGFA protein expression by miR-874 inhibition. Correlation analysis between miRNAs and VEGFA protein expression showed that miR-637 and miR-874 expression present inversely correlated to VEGFA protein expression. Conclusions: VEGFA was down-regulated in response to hsa-miR-612 or hsa-miR-637 overexpression; however, the modulation of VEGFA by miR-874 was observed only at the gene expression and thus, needs further investigation.

Intra-abdominal hypertension, fluid balance, and adverse outcomes after orthotopic liver transplantation.

BACKGROUND: Intra-abdominal hypertension (IAH) is frequently encountered in critically ill surgical patients. We aimed to evaluate the incidence of IAH after orthotopic liver transplant (OLT) and its impact on organ function, hospital length-of-stay (LOS), and death. METHODS: This prospective, observational, cohort study evaluated consecutive adult patients admitted in the ICU after undergoing OLT. Intra-abdominal pressure (IAP) was measured every 4-6 h for 3 days. Worsening IAP was defined as a gradual increase in IAP over a period of time. Daily fluid balance was the daily sum of all intakes minus the output. RESULTS: IAH was observed in 48% of the patients within the first 3 days after ICU admission, while ACS was diagnosed in 15%. Patients with IAH had a higher positive fluid balance at day 1 (1764 mL [812-2733 mL] vs. 1301 mL [241-1904 mL], p = 0.025). Worsening IAH was associated with fewer days free of organ dysfunction. IAH within 72 h after ICU admission was independently associated with a composite outcome of death or a longer ICU LOS (odds ratio 2.9; CI 95% 1.02-8.25, p = 0.043). CONCLUSION: After OLT, nearly half of the patients presented IAH, that was associated with unfavorable outcomes.

Impact of Brazilian expanded criteria for liver transplantation in patients with hepatocellular carcinoma: a multicenter study.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the main indications for orthotopic liver transplantation (OLT). In Brazil, selection criteria for HCC is an expanded version of the Milan Criteria (MC), the so-called "Brazilian Milan Criteria" (BMC). Our aims were to evaluate post-OLT outcomes in patients with HCC and analyze the BMC performance. MATERIALS AND METHODS: We conducted a multicenter, retrospective cohort study, analyzing medical records of 1,059 liver transplant recipients with HCC. Tumor was staged according to MC and BMC and correlated with overall survival (OS) and disease-free survival (DFS). We compared the ability of MC and BMC to predict OS and DFS using Delta C-statistic. RESULTS: Post-OLT OS were 63% in five years and HCC recurrence was observed in 8% of patients. At diagnosis, 85% of patients were within MC. Patients within MC at diagnosis and in the explant showed a higher OS and DFS than patients outside MC and within BMC and patients outside both criteria (p < 0.001). Patients outside MC in the explant had an increased risk of tumor recurrence (HR: 3.78; p < 0.001) and poor survival (HR:1.77; p = 0.003). The BMC presented a lower performance than MC in properly classifying patients regarding recurrence risk. CONCLUSIONS: In a large Brazilian cohort of HCC patients submitted to liver transplantation, we observed satisfactory overall survival and recurrence rates. However, patients transplanted within the Brazilian expanded criteria had lower OS and DFS when compared to patients within MC, which may generate future discussions regarding the criteria currently used.

In vitro long-term culture of isolated ovine preantral follicles: Influence of ethanol on steroid production, oocyte meiotic resumption, and metabolomic profile.

Ethanol is used routinely to dilute cell culture media supplements with little or no water solubility. This study evaluates the effect of low concentration of ethanol on the follicular development, oocyte maturation, hormone production, gene expression, and metabolomics profile of spent culture medium after long-term culture of isolated ovine preantral follicles. For this, follicles were cultured for 18 days in α-Minimum Essential Medium+ alone (control treatment) or supplemented with 100 ng/mL recombinant bovine FSH (rbFSH treatment) or with 0.2%-v/v ethanol (ethanol treatment). Ethanol treatment increased the percentage of degenerated follicles and oocytes significantly, however, it showed the highest estradiol secretion. Also, the rate of meiosis resumption was higher in ethanol treatment than Control treatment. Ethanol treatment decreased the mRNA levels of B-cell lymphoma 2 (BCL2), BCL2 associated X, Aquaporin 3, Connexin 43, Inhibin Subunit Beta A, kit ligand, Heat Shock Protein (HSP A1A) significantly when compared to the Control treatment. However, mRNA levels of cytochrome P450 family 19, and FSH receptors were significantly higher in ethanol treatment than in the Control treatment. The levels of some metabolites, which are likely amino acids, lipids, an analog of Cyclic guanosine monophosphate, and a derivative of phosphoinositol phosphate metabolism, had higher relative concentrations in ethanol and rbFSH treatments than the Control treatment. In conclusion, ethanol addition augmented the follicular and oocyte degeneration rates but increased the estradiol production and the meiotic resumption. Furthermore, the follicular metabolomic profile was similar between ethanol and rbFSH treatments being both treatments; however, different from the Control treatment.

Differential expression of angiogenesis-related miRNAs and VEGFA in cirrhosis and hepatocellular carcinoma.

INTRODUCTION: Liver cirrhosis (LC) is a heterogeneous liver disease, the last stage of liver fibrosis, and the major risk factor for hepatocellular carcinoma (HCC). Our study aimed to evaluate the expression of microRNAs and the endothelial vascular growth factor (VEGFA) gene in LC and HCC. MATERIAL AND METHODS: The sample group consisted of 46 tissue samples: 21 of LC, 15 of HCC, and 10 of non-tumoural and non-cirrhotic liver tissue (control group). MiRNAs were chosen based on a mirDIP prediction database as regulators of the VEGFA gene. Gene expression of VEGF and miRNAs was quantified by real-time quantitative polymerase chain reaction. VEGFA protein expression was evaluated by ELISA. RESULTS: VEGFA gene expression was significantly overexpressed in LC compared to the control group (p < 0.0001). Hsa-miR-206 (p = 0.0313) and hsa-miR-637 (p = 0.0156) were down-expressed in LC. In HCC, hsa-miR-15b (p = 0.0010), hsa-miR-125b (p = 0.0010), hsa-miR-423-3p (p = 0.0010), hsa-miR-424 (p = 0.0313), hsa-miR-494 (p < 0.0001), hsa-miR-497 (p < 0.0001), hsa-miR-612 (p = 0.0078), hsa-miR-637 (p < 0.0001), and hsa-miR-1255b (p = 0.0156) presented down-expression. CONCLUSIONS: Overexpression of VEGFA in LC suggests impairment of angiogenesis in this tissue. The differential expression of microRNAs in LC and HCC observed in our study can lead to the evaluation of possible biomarkers for these diseases.

Hepatocellular carcinoma recurrence after liver transplantation in a Brazilian multicenter study: clinical profile and prognostic factors of survival.

BACKGROUND: Liver transplantation (LT) is the treatment of choice for patients with unresectable early hepatocellular carcinoma (HCC). Post-LT HCC recurrence rates range from 8 to 20% and still impact on overall survival (OS). The aim of our study was to evaluate the impact of HCC recurrence on post-LT survival and analyze prognostic factors among those patients with recurrence. PATIENTS AND METHODS: We carried out a national, multicenter, retrospective cohort study in Brazil. Medical records of 1119 LT recipients with HCC were collected. Data from patients with post-LT HCC recurrence were analyzed and correlated with post-relapse survival. RESULTS: OS of the 1119 patients included in the study was 63% over 5 years. Post-LT HCC recurrence occurred in 86 (8%) patients. The mean time to recurrence was 12 months. Sites of recurrence were extrahepatic in 55%, hepatic in 27%, and both hepatic and extrahepatic in 18%. Recurrence treatment was performed in 50 (64%) cases, mostly with sorafenib. Post-relapse survival rates were 34% at 1 year and 13% at 5 years. Univariable analysis identified α-fetoprotein more than 1000 ng/ml at relapse, recurrence treatment, extrahepatic location, and time to recurrence more than 2 years as prognostic factors. In multivariable analysis, recurrence treatment, extrahepatic location, and time to recurrence more than 2 years were independent predictors of better survival. CONCLUSION: In a large Brazilian cohort of LT recipients with HCC, post-LT HCC recurrence occurred in 8% and impacted significantly on the OS. Patients with early recurrence presented a worse prognosis. However, treatment of recurrence improved outcomes, highlighting the importance of early diagnosis.

Patients on the waiting list for liver transplantation: caregiver burden and stress.

Over the last few decades, informal caregivers of patients with chronic diseases have received more attention, and there is a growing volume of studies demonstrating high rates of burden, stress, and mental disorders in this group of individuals. The objective of this study was to evaluate the burden, stress, and psychosocial characteristics of informal caregivers of liver transplantation candidates. Participants were assessed by individual evaluations with the following instruments: a semistructured interview, the Caregiver Burden Scale, the Inventário de Sintomas de Stress para Adultos de Lipp, and the Beck Depression Inventory. The Mann-Whitney test was used for statistical analysis with a significance level of 0.05. The characteristics of the study group (n = 61) were similar to those of groups in other studies with respect to gender (82% were women), kinship (64% were spouses), and age (the mean age was 47.6 years). The main stressors identified by the participants were as follows: doubts about ways to react in a crisis or in emergency situations (42.6%), mood swings of the patient (29.5%), and care involving food and medications (27.9%). Approximately 25% of the caregivers reported that they felt unprepared to adequately perform their roles. Data analysis indicated a greater burden overall on caregivers when the patient's Model for End-Stage Liver Disease score was greater than or equal to 15 points (P = 0.041). Furthermore, caregivers of patients with alcoholic liver disease showed higher depression (P = 0.034) and overall burden scores (P = 0.031) versus caregivers of patients with liver disease due to other etiologies. In conclusion, the participants showed significantly high levels of burden, stress, and depression. Support measures and caregiver preparation should be implemented by health care providers.

Mannose-binding lectin gene polymorphisms are not associated with susceptibility to hepatitis C virus infection in the Brazilian Amazon region.

The present study compares the genotype frequencies between two population groups composed by 73 hepatitis C virus (HCV)-infected patients and 92 seronegative controls and investigates the role of allele variants as a possible factor in the susceptibility to HCV infection and the influence on disease progression. The identification of MBL*B and MBL*C alleles was performed by restriction fragment length polymorphism analysis of the 349-bp product using BanI and MboII restriction enzymes, respectively, and a polymerase chain reaction-sequence-specific polymorphism for discrimination of MBL*D. The analysis of allele and genotype frequencies between an HCV-infected group and seronegative controls did not indicate significant differences. The comparison of chronically infected subjects with and without liver cirrhosis was also not statistically significant. The odds ratio estimations were not significant, and the values obtained cannot suggest that the presence of allele variant MBL*B could have some influence in the risk of HCV infection progression to liver cirrhosis and that the presence of allele MBL*D could confer some protection against disease progression, but a larger sample size is necessary to confirm the present results.

Pseudo-aneurisma de artéria hepática comum direita em mulher jovem com Lupus Eritematoso Sistêmico / Pseudoaneurysm of the right common hepatic artery in a young woman with SystemicLupus Erythematosus

Aneurismas de artéria hepática comum são lesões vasculares raras e representam a segunda incidência deaneurismas de artérias viscerais. As principais etiologias são: aterosclerose (32%) e trauma (22%). Outrascausas menos comuns são vasculites, procedimentos diagnósticos e terapêuticos percutâneos e complicação anastomótica de transplante de fígado. O diagnóstico de aneurisma intra-hepático é difícil antes de suaruptura, o qual está associado a uma mortalidade superior a 20% dos casos. Este artigo relata a história de uma paciente do sexo feminino, de 26 anos, portadora de Lupus Eritematoso Sistêmico (LES) que apresentavahá 2 meses dor tipo cólica na parte superior do abdome com irradiação para dorso. O diagnóstico de pseudo-aneurisma de artéria hepática comum foi feito após investigação da dor abdominal com métodos de imagem.Para aneurisma intra-hepático, o tratamento de escolha é a embolização, e este procedimento tem aumentado a sobrevida desses pacientes. Em alguns casos de aneurismas proximais, torna-se necessária a ressecção do fígado ou mesmo de transplante hepático.

Hepatic artery aneurysms are rare vascular lesions and represent a second incidence of aneurysms ofvisceral arteries. The main etiologies are atherosclerosis (32%) and trauma (22%). Other less common causesinclude vasculitis, diagnostic procedures, percutaneous therapies and as an anastomotic complication ofliver transplantation. Diagnosis of an intra-hepatic aneurysm before its rupture is difficult; ruptures areassociated to mortality in more that 20% of the cases. This work reports the case of a 26-year-old femalepatient with systemic lupus erythematosus who presented with a colic-type pain in the upper abdomen withirradiation to the back for two months. The diagnosis of hepatic artery pseudoaneurysm was made after animaging investigation of the abdominal pain. The first-line treatment for intra-hepatic aneurysms is embolization,which improves the patients’ survival rate. In some cases of proximal aneurysms, resection of the liver oreven liver transplantation is required.

As Organizações de Procura de Órgãos (OPOs) são efetivas? Análise de sete anos de atividade de uma OPO brasileira / Are the Organ Procurement Organizations (OPO) effective? A sevenyear

Introdução: A falta de órgãos humanos é o princial fator que limita o aumento do número dos transplantes (Tx) no Brasil. O governo do estado implantou as Organizações de Procura de Órgãos (OPOs) para aumentar a oferta de órgãos para Tx, entretanto ainda são escassos os relatos que demonstram a efetividade dessas OPOs. Objetivo: Esse trabalho teve o objetivo de analisar a evolução de uma OPO regional (OPO-SJRP) e avaliar seu desempenho em 7 anos (1997 a 2003). Casuística e método: Foram analisadas retrospectivamente as causas de não-efetivação das doações de MO / C e 1781 notificações de potenciais doadores (PD), sendo n= 1323 para córneas (C) e 458 para múltiplos órgãos (MO), que resultaram em 745 doadores de C e 166 de MO. Resultados: A análise mostrou redução de 25% nas notificações de PD. Entretanto, ocorreu aumento dos doadores efetivos de MO de 5 pmp/ano para 19 pmp/ano e nos de C de 36 pmp/ano para 63 pmp/ano. Na OPOSJRP as causas de não-efetivação das doações de MO foram principalmente a recusa familiar (RF) (20%) e a contra-indicação médica (CIM) (38%). Para as doações de C, a RF ocorreu em 35% e a CIM em 13%. A OPOSJRP apresenta taxas médias anuais de notificação para MO e de doação efetiva de 34 pmp e 12 pmp respectivamente, maiores que as demais OPOs do estado. Conclusão: A OPO-SJRP aumentou significativamente nos últimos 7 anos o número de doadores efetivos para transplantes de MO e de C e possui taxas de notificação e de doação pmp/ano similares ou maiores do que às das outras OPOs do Estado de S. Paulo

Linfoma de tecido linfático associado a mucosa MALT, primário de fígado, com infiltraçäo de vesícula biliar e medula óssea / Mucosa associated lymphatic tissue lymphoma, MALT, primarily from the liver, with infiltration of gall bladder and bone marrow

Linfomas hepáticos primários do fígado säo raros. Linfomas indolentes de expressäo B do tipo tecido linfático associado a mucosa säo ainda mais raros e inusitados, estando descritos na literatura somente quatro casos. Os autores apresentam neste relato um caso de paciente do sexo feminino, com 73 anos de idade com queixas dolorosas e sintomatologia de comprometimento biliar. O diagnóstico de linfoma do tecido linfático associado a mucosa (MALT) foi realizado acidentalmente pelo encontro de anormalidades hepáticas no momento do ato cirúrgico. O aspecto hepático era micronodular com a presença de lesäo ulcerada, que biopsiadas revelaram o diagnóstico. O estadiamento da moléstia comprovou infiltraçäo da medula óssea pelo linfoma, dado este infrequente, e a endoscopia digestiva alta foi normal, näo revelando comprometimento deste aparelho, dado comum e mais freqüente deste tipo de linfoma.