Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet.

In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.

A summary of reptile and anuran amphibian species from Brazilian sandy coastal plains: 31 years of sampling efforts of the "Laboratório de Ecologia de Vertebrados, Universidade do Estado do Rio de Janeiro" / Um resumo das espécies de répteis e anfíbios anuros das planícies costeiras arenosas brasileiras: 31 anos de esforços de amostragem do "Laboratório de Ecologia de Vertebrados da Universidade do Estado do Rio de Janeiro"

Abstract Although currently there is already a set of studies regarding ecological aspects of some particular reptile and amphibian species living in Brazilian sandy coastal plains (including the so-called "restinga" and "campo nativo" habitats), there is comparatively few information on the species composition usually associated to these environments. During 31 years (1988-2019) of herpetological studies carried out in sandy coastal plains environments by our research team of the Laboratory of Vertebrate Ecology (Department of Ecology, Universidade do Estado do Rio de Janeiro, in Rio de Janeiro Brazil) we have surveyed reptile and amphibian communities and performed different studies with similar methods in 70 sites from 10 different states along the Brazilian coast. Our surveys resulted in records of 87 species of reptile (five turtles, two crocodylians, six amphisbaenians, 36 lizards and 39 snakes) from 24 families, and 77 species of anuran amphibians from nine families. We have studied multiple natural history topics for anurans and reptiles which resulted in the publication of some specific ecological studies, especially regarding some species, encompassing population and community ecology, foraging and feeding habits, species activity, thermoregulation, reproduction, use of microhabitats, and parasitism by ecto and endoparasites. Our results along these three decades have also contributed for the description of four new lizard species (Ameivula nativo, Glaucomastix littoralis, G. abaetensis and G. itabaianensis). Our studies constitute an important contribution to the knowledge of the ecology of anuran amphibians and reptiles in these ecosystems, as well as to the conservation of sandy coastal plains environment. The checklist presented in this study, based on our records of sandy coastal plains herpetofauna, provides for many localities along the Brazilian coast, the needed knowledge on species occurrence, including the presence of endemic and/or endangered species, which can be of value for many conservation actions.

Resumo Embora atualmente exista um conjunto de estudos sobre aspectos ecológicos de algumas espécies de répteis e de anfíbios que ocorrem nas planícies costeiras arenosas brasileiras (incluindo os chamados habitats de "restinga" e de "campo nativo"), há relativamente poucas informações sobre a composição de espécies geralmente associada a esses ambientes. Durante 31 anos (1988-2019) de estudos herpetológicos realizados em restingas por nossa equipe de pesquisa do Laboratório de Ecologia de Vertebrados (Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil) nós estudamos comunidades de répteis e de anfíbios e realizamos diferentes estudos com métodos semelhantes em 70 localidades de dez diferentes Estados ao longo da costa brasileira. Nossas pesquisas resultaram em registros de 87 espécies de répteis (cinco tartarugas, dois crocodilianos, seis anfisbênios, 36 lagartos e 39 serpentes) de 24 famílias, e 77 espécies de anfíbios anuros de nove famílias. Estudamos vários tópicos de história natural sobre anuros e répteis, que resultaram na publicação de alguns estudos ecológicos específicos, especialmente em relação a algumas espécies, abrangendo ecologia populacional e de comunidades, forrageamento e dieta, horário de atividade de espécies, termorregulação, reprodução, uso do microhabitat e parasitismo por ecto e endoparasitas. Nossos resultados ao longo dessas três décadas também contribuíram para a descrição de quatro novas espécies de lagartos (Ameivula nativo, Glaucomastix littoralis, G. abaetensis e G. itabaianensis). Nossos estudos constituem uma importante contribuição para o conhecimento da ecologia de répteis e de anfíbios anuros nesses ecossistemas, bem como para a conservação dos ecossistemas de restinga. A lista de espécies apresentada neste estudo, com base em nossos registros de herpetofauna das planícies costeiras arenosas, fornece para muitas localidades ao longo da costa brasileira o conhecimento necessário sobre a ocorrência de espécies, incluindo a presença de espécies endêmicas e/ ou ameaçadas de extinção, que podem ser úteis para muitas ações de conservação.

A summary of reptile and anuran amphibian species from Brazilian sandy coastal plains: 31 years of sampling efforts of the "Laboratório de Ecologia de Vertebrados, Universidade do Estado do Rio de Janeiro".

Although currently there is already a set of studies regarding ecological aspects of some particular reptile and amphibian species living in Brazilian sandy coastal plains (including the so-called "restinga" and "campo nativo" habitats), there is comparatively few information on the species composition usually associated to these environments. During 31 years (1988-2019) of herpetological studies carried out in sandy coastal plains environments by our research team of the Laboratory of Vertebrate Ecology (Department of Ecology, Universidade do Estado do Rio de Janeiro, in Rio de Janeiro Brazil) we have surveyed reptile and amphibian communities and performed different studies with similar methods in 70 sites from 10 different states along the Brazilian coast. Our surveys resulted in records of 87 species of reptile (five turtles, two crocodylians, six amphisbaenians, 36 lizards and 39 snakes) from 24 families, and 77 species of anuran amphibians from nine families. We have studied multiple natural history topics for anurans and reptiles which resulted in the publication of some specific ecological studies, especially regarding some species, encompassing population and community ecology, foraging and feeding habits, species activity, thermoregulation, reproduction, use of microhabitats, and parasitism by ecto and endoparasites. Our results along these three decades have also contributed for the description of four new lizard species (Ameivula nativo, Glaucomastix littoralis, G. abaetensis and G. itabaianensis). Our studies constitute an important contribution to the knowledge of the ecology of anuran amphibians and reptiles in these ecosystems, as well as to the conservation of sandy coastal plains environment. The checklist presented in this study, based on our records of sandy coastal plains herpetofauna, provides for many localities along the Brazilian coast, the needed knowledge on species occurrence, including the presence of endemic and/or endangered species, which can be of value for many conservation actions.

A multiple endpoint approach reveals potential in vitro anticancer properties of thymoquinone in human renal carcinoma cells.

Thymoquinone (TQ) is a monoterpene isolated from the oil of Nigella sativa seeds. The aim of this work was to evaluate the cytotoxic effects induced by TQ and its impact on the migration and invasion potential of 786-O human renal cancer cells. These cells were exposed to TQ (1-100 µM) for 24 and 48 h and cell viability assessed using the Crystal Violet and MTS assays. TQ treatment clearly decreased cell viability in a concentration- and time-dependent manner. TQ exposure moderately increased intracellular ROS levels and co-incubation with reduced glutathione markedly increased cell viability. Moreover, the effect of TQ in the cell cycle distribution was evaluated using flow cytometry, and an increase in the sub-G1 population was observed, especially at 30 µM, along with an increase in the % of apoptotic cells. TQ did not show genotoxic effects at a non-cytotoxic concentration (1.0 µM). At this concentration level, TQ significantly decreased the collective migration of 786-O cells, whereas it had no effect in chemotactic migration. TQ also decreased the invasiveness potential of 786-O cells, as evaluated by the transwell invasion assay. Overall, these results suggest that TQ presents an anticancer potential in the context of renal cancer, warranting further investigation.

Role of aquaporin-7 in ghrelin- and GLP-1-induced improvement of pancreatic ß-cell function after sleeve gastrectomy in obese rats.

BACKGROUND/OBJECTIVES: Glycerol is a key metabolite for lipid accumulation in insulin-sensitive tissues as well as for pancreatic insulin secretion. We examined the role of aquaporin-7 (AQP7), the main glycerol channel in ß-cells, and AQP12, an aquaporin related to pancreatic damage, in the improvement of pancreatic function and steatosis after sleeve gastrectomy in diet-induced obese rats. SUBJECTS/METHODS: Male Wistar obese rats (n=125) were subjected to surgical (sham operation and sleeve gastrectomy) or dietary (pair-fed to the amount of food eaten by sleeve-gastrectomized animals) interventions. The tissue distribution and expression of AQPs in the rat pancreas were analyzed by real-time PCR, western blotting and immunohistochemistry. The effect of ghrelin isoforms and glucagon-like peptide 1 (GLP-1) on insulin secretion, triacylglycerol (TG) accumulation and AQP expression was determined in vitro in RIN-m5F ß-cells. RESULTS: Sleeve gastrectomy reduced pancreatic ß-cell apoptosis, steatosis and insulin secretion. Lower ghrelin and higher GLP-1 concentrations were also found after bariatric surgery. Acylated and desacyl ghrelin increased TG content, whereas GLP-1 increased insulin release in RIN-m5F ß-cells. Sleeve gastrectomy was associated with an upregulation of AQP7 together with a normalization of the increased AQP12 levels in the rat pancreas. Interestingly, ghrelin and GLP-1 repressed AQP7 and AQP12 expression in RIN-m5F ß-cells. AQP7 protein was negatively correlated with intracellular lipid accumulation in acylated ghrelin-treated cells and with insulin release in GLP-1-stimulated ß-cells. CONCLUSIONS: AQP7 upregulation in ß-cells after sleeve gastrectomy contributes, in part, to the improvement of pancreatic steatosis and insulin secretion by increasing intracellular glycerol used for insulin release triggered by GLP-1 rather than for ghrelin-induced TG biosynthesis.

Immune modulation by the hepatitis C virus core protein.

Hepatitis C virus (HCV) infection is currently the most important cause of chronic viral hepatitis in the world and one of the most frequent indications for liver transplantation. HCV uses different strategies to evade the innate and adaptive immune response, and this evasion plays a key role in determining viral persistence. Several HCV viral proteins have been described as immune modulators. In this review, we will focus on the effect of HCV nucleocapsid core protein in the function of immune cells and its correlation with the findings observed in HCV chronically infected patients. Effects on immune cell function related to both extracellular and intracellular HCV core localization will be considered. This review provides an updated perspective on the mechanisms involved in HCV evasion related to one single HCV protein, which could become a key tool in the development of new antiviral strategies able to control and/or eradicate HCV infection.

The role of epigenetic modifiers in extended cultures of functional hepatocyte-like cells derived from human neonatal mesenchymal stem cells.

The development of predictive in vitro stem cell-derived hepatic models for toxicological drug screening is an increasingly important topic. Herein, umbilical cord tissue-derived mesenchymal stem cells (hnMSCs) underwent hepatic differentiation using an optimized three-step core protocol of 24 days that mimicked liver embryogenesis with further exposure to epigenetic markers, namely the histone deacetylase inhibitor trichostatin A (TSA), the cytidine analogue 5-azacytidine (5-AZA) and dimethyl sulfoxide (DMSO). FGF-2 and FGF-4 were also tested to improve endoderm commitment and foregut induction during Step 1 of the differentiation protocol, being HHEX expression increased with FGF-2 (4 ng/mL). DMSO (1%, v/v) when added at day 10 enhanced cell morphology, glycogen storage ability, enzymatic activity and induction capacity. Moreover, the stability of the hepatic phenotype under the optimized differentiation conditions was examined up to day 34. Our findings showed that hepatocyte-like cells (HLCs) acquired the ability to metabolize glucose, produce albumin and detoxify ammonia. Global transcriptional analysis of the HLCs showed a partial hepatic differentiation degree. Global analysis of gene expression in the different cells revealed shared expression of gene groups between HLCs and human primary hepatocytes (hpHeps) that were not observed between HepG2 and hpHeps. In addition, bioinformatics analysis of gene expression data placed HLCs between the HepG2 cell line and hpHeps and distant from hnMSCs. The enhanced hepatic differentiation observed was supported by the presence of the hepatic drug transporters OATP-C and MRP-2 and gene expression of the hepatic markers CK18, TAT, AFP, ALB, HNF4A and CEBPA; and by their ability to display stable UGT-, EROD-, ECOD-, CYP1A1-, CYP2C9- and CYP3A4-dependent activities at levels either comparable with or even higher than those observed in primary hepatocytes and HepG2 cells. Overall, an improvement of the hepatocyte-like phenotype was achieved for an extended culture time suggesting a role of the epigenetic modifiers in hepatic differentiation and maturation and presenting hnMSC-HLCs as an advantageous alternative for drug discovery and in vitro toxicology testing.

A summary of reptile and anuran amphibian species from Brazilian sandy coastal plains: 31 years of sampling efforts of the Laboratório de Ecologia de Vertebrados, Universidade do Estado do Rio de Janeiro

Abstract Although currently there is already a set of studies regarding ecological aspects of some particular reptile and amphibian species living in Brazilian sandy coastal plains (including the so-called restinga and campo nativo habitats), there is comparatively few information on the species composition usually associated to these environments. During 31 years (1988-2019) of herpetological studies carried out in sandy coastal plains environments by our research team of the Laboratory of Vertebrate Ecology (Department of Ecology, Universidade do Estado do Rio de Janeiro, in Rio de Janeiro Brazil) we have surveyed reptile and amphibian communities and performed different studies with similar methods in 70 sites from 10 different states along the Brazilian coast. Our surveys resulted in records of 87 species of reptile (five turtles, two crocodylians, six amphisbaenians, 36 lizards and 39 snakes) from 24 families, and 77 species of anuran amphibians from nine families. We have studied multiple natural history topics for anurans and reptiles which resulted in the publication of some specific ecological studies, especially regarding some species, encompassing population and community ecology, foraging and feeding habits, species activity, thermoregulation, reproduction, use of microhabitats, and parasitism by ecto and endoparasites. Our results along these three decades have also contributed for the description of four new lizard species (Ameivula nativo, Glaucomastix littoralis, G. abaetensis and G. itabaianensis). Our studies constitute an important contribution to the knowledge of the ecology of anuran amphibians and reptiles in these ecosystems, as well as to the conservation of sandy coastal plains environment. The checklist presented in this study, based on our records of sandy coastal plains herpetofauna, provides for many localities along the Brazilian coast, the needed knowledge on species occurrence, including the presence of endemic and/or endangered species, which can be of value for many conservation actions.

Resumo Embora atualmente exista um conjunto de estudos sobre aspectos ecológicos de algumas espécies de répteis e de anfíbios que ocorrem nas planícies costeiras arenosas brasileiras (incluindo os chamados habitats de restinga e de campo nativo), há relativamente poucas informações sobre a composição de espécies geralmente associada a esses ambientes. Durante 31 anos (1988-2019) de estudos herpetológicos realizados em restingas por nossa equipe de pesquisa do Laboratório de Ecologia de Vertebrados (Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil) nós estudamos comunidades de répteis e de anfíbios e realizamos diferentes estudos com métodos semelhantes em 70 localidades de dez diferentes Estados ao longo da costa brasileira. Nossas pesquisas resultaram em registros de 87 espécies de répteis (cinco tartarugas, dois crocodilianos, seis anfisbênios, 36 lagartos e 39 serpentes) de 24 famílias, e 77 espécies de anfíbios anuros de nove famílias. Estudamos vários tópicos de história natural sobre anuros e répteis, que resultaram na publicação de alguns estudos ecológicos específicos, especialmente em relação a algumas espécies, abrangendo ecologia populacional e de comunidades, forrageamento e dieta, horário de atividade de espécies, termorregulação, reprodução, uso do microhabitat e parasitismo por ecto e endoparasitas. Nossos resultados ao longo dessas três décadas também contribuíram para a descrição de quatro novas espécies de lagartos (Ameivula nativo, Glaucomastix littoralis, G. abaetensis e G. itabaianensis). Nossos estudos constituem uma importante contribuição para o conhecimento da ecologia de répteis e de anfíbios anuros nesses ecossistemas, bem como para a conservação dos ecossistemas de restinga. A lista de espécies apresentada neste estudo, com base em nossos registros de herpetofauna das planícies costeiras arenosas, fornece para muitas localidades ao longo da costa brasileira o conhecimento necessário sobre a ocorrência de espécies, incluindo a presença de espécies endêmicas e/ ou ameaçadas de extinção, que podem ser úteis para muitas ações de conservação.

Sex differences in hepatic and intestinal contributions to nevirapine biotransformation in rats.

The understanding of the intestine contribution to drug biotransformation improved significantly in recent years. However, the sources of inter-individual variability in intestinal drug biotransformation, namely sex-differences, are still elusive. Nevirapine (NVP) is an orally taken anti-HIV drug associated with severe idiosyncratic reactions elicited by toxic metabolites, with women at increased risk. As such, NVP is a good model to assess sex-dimorphic metabolism. The aim of this study was to perform a comparative profiling of NVP biotransformation in rat intestine and liver and evaluate whether or not it is organ- and sex-dependent. Therefore, nevirapine-containing solutions were perfused through the intestine, in a specially designed chamber, or incubated with liver slices, from male and female Wistar rats. The levels of NVP and its Phase I metabolites were quantified by HPLC-UV. Liver incubation experiments yielded the metabolites 2-, 3-, 8-, and 12-OH-NVP, being 12-OH-NVP and 2-OH-NVP the major metabolites in males and females, respectively. Inter-sex differences in the metabolic profile were also detected in the intestine perfusion experiments. Herein, the metabolites 3- and 12-OH-NVP were only found in male rats, whereas 2-OH-NVP levels were higher in females, both in extraluminal (p<0.01) and intraluminal media. The metabolite 8-OH-NVP was not detected in the intraluminal media from either males or females. In this study, important inter-sex differences were detected in both organs, providing further clues to the sex-dimorphic profile of NVP toxicity. Moreover, an extra-hepatic contribution to NVP biotransformation was observed, strengthening the relevance of the intestinal contribution in the biotransformation of orally taken-drugs.

Blockade of proteinase-activated receptor-4 inhibits the eosinophil recruitment induced by eotaxin-1 in the pleural cavity of mice.

OBJECTIVE AND DESIGN: Although proteinase-activated receptor (PAR)-4 has been implicated in inflammation, its role in regulating eosinophil recruitment in response to chemoattractants has not yet been demonstrated. To investigate the contribution of proteinases and PAR-4 activation to eosinophil migration in response to eotaxin-1 or leukotriene B(4) (LTB(4)), the effects of aprotinin or PAR-4 antagonist trans-cinnamoyl-YPGKF-NH(2) (tcY-NH(2)) on eosinophil migration induced by these chemoattractants were investigated. METHODS: BALB/c mice were pretreated with aprotinin or tcY-NH(2) (30 µg/mouse) prior to intrapleural injection of LTB(4) or eotaxin-1 and the number of infiltrating eosinophils was determined 48 h later. RESULTS: Aprotinin (1 mg/kg) inhibited eosinophil recruitment induced by eotaxin-1 (p < 0.01), but not that induced by LTB(4). Moreover, tcY-NH(2) treatment inhibited eosinophil recruitment in response to eotaxin-1 (p < 0.01 by ANOVA/Tukey post-test). CONCLUSION: These data suggest that aprotinin-inhibited proteinases participate in eosinophil migration induced by eotaxin-1 and that PAR-4 activation plays an important role in regulating this migration.

Attribution as a function of agential distance in a causal chain.

The hypothesis that causality and blame will be differently affected by agential distance within a two-step causal chain was tested. A hypothetical medical accident was presented to 360 female college students in India, who gave causality, blame, or punishment judgments about either the proximal agent or the distal agent. The study had a 2 (proximal vs. distal agent) x 2 (high vs. low extenuation) x 2 (mild vs. severe outcome) x 3 (casuality, blame, or punishment judgments) fully crossed, between-subjects factorial design, with 15 participants per cell. In support of the basic hypothesis, more blame and punishment were assigned to the distal agent than to the proximal agent, whereas agential distance did not affect causal attribution. Extenuation was effective on causality and blame judgments only when the outcome was mild, and there was no effect of extenuation on punishment judgments, irrespective of outcome severity.

[Clinical and epidemiological study of blood donors seropositive for Trypanosoma cruzi]. / Estudio clínico y epidemiológico en donantes de sangre seropositivos por Trypanosoma cruzi.

BACKGROUND: The prevalence of positive antibodies for Chagas disease in a blood bank of a public hospital in Santiago is 1.2%. These positive individuals should be informed about their condition and further studied. AIM: To perform a serological confirmation, an epidemiological and clinical assessment of blood donors seropositive for Chagas disease in a blood bank of Santiago. SUBJECTS AND METHODS: One hundred eleven seropositive donors, detected between 1994 and 1996, were studied. Serological reactions were confirmed with ELISA and indirect immunofluorescence reactions. Those confirmed as positive were subjected to a clinical-epidemiological questionnaire, physical examination, EKG and barium enema or swallow according to symptoms. RESULTS: Seventy individuals were confirmed as positive for Chagas disease. Most of them lived at least once in an endemic zone for Chagas disease or in a sun-dried clay brick house. Forty-percent of individuals knew reduviid bugs and 14% had been bitten by these insects. Twenty six percent of these subjects had an abnormal EKG (61% with bradycardia). No digestive diseases attributable to Chagas disease were detected. CONCLUSIONS: The high prevalence of problems attributable to Chagas disease among seropositive blood donors underscores the need of an epidemiological follow up of these individuals.