Regulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver

Mu hiding resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetra-acetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. Conclusion: InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane. (HEPATOLOGY 2010;52:327-337)

SLC2A2 gene expression in kidney of diabetic rats is regulated by HNF-1 alpha and HNF-3 beta

We hypothesize that, in kidney of diabetic rats, hepatocyte nuclear factors (HNF-1 alpha. and HNF-3 beta) play a critical role in the overexpression of solute carrier 2A2 (SLC2A2) gene. Diabetic rats submitted or not to rapid (up to 12 h) and short-term (1, 4 and 6 days) insulin treatment were investigated. Twofold increase in GLUT2 mRNA was observed in diabetic, accompanied by significant increases in HNF-1 alpha and HNF-3 beta expression and binding activity. Additional 2-fold increase in GLUT2 mRNA and HNF-3 beta expression/activity was observed in 12-h insulin-treated rats. Six-day insulin treatment decreased GLUT2 mRNA and HNF-1 alpha expression and activity to levels of non-diabetic rats, whereas HNF-3 beta decreased to levels of non-insulin-treated diabetic rats. Our results provide evidence for a link between the overexpression of SLC2A2 gene and the transcriptional activity of HNF-1 alpha and HNF-3 beta in kidney of diabetic rats. Furthermore, recovery of SLC2A2 gene after 6-day insulin treatment also involves HNF-1 alpha and HNF-3 beta activity. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

O envolvimento do marcador inflamatório Heme Oxigenêse-1 na carciinogênese experimental de esôfago

Sabe-se que o refluxo crônico pode induzir lesão mucosa, estimular a proliferação de células e promover tumorigênese no esôfago distal. Ainda não é sabido por que apenas uma parcela dos pacientes com refluxo esofágico progrediram para uma metaplasia intestinal (Esôfago de Barrett) e adenocarcinoma. O estresse oxidativo parece exercer um papel importante nessa progressão . Assim sendo, examinamos o padrão de expressão da enzima Heme Oxigenase-1 (HO-1), enzima indutora do estresse oxidativo, em peças de esôfago obtidos de um estudo experimental com ratos que avaliou o papel do refluxo gástrico e duodenoesofágico na carcinogênese esofágica. Métodos: Uma amostra de três (3) peças de esôfago de cada grupo de ratos submetidos a tratamentos diferentes tiveram a expressão da enzima Heme Oxigenase-1 avaliada através de imunohistoquímica. Os ratos foram divididos nos seguintes grupos: (1) cardioplastia para induzir refluxo predominantemente gástrico, (2) anastomose esofagoduodenal para induzir refluxo duodenal, (3) sem tratamento, (4) cardioplastia+dietilnitrosamina (DEN), (5) anastomose esofagoduodenal +DEN, (6) DEN. Resultados: Não houve desenvolvimento de câncer ou metaplasia intestinal nos animais que não foram expostos ao refluxo de conteúdo duodenal. A expressão de HO-1 foi observada apenas em ratos submetidos à anastomose esofagoduodenal (Grupos 2 e 5) e a análise da média de intensidade de fluorescência demonstrou uma diferença significante de expressão de HO-1 (4,8 e 4,6 vezes respectivamente) comparando-se ao controle (Grupo 3) (p<0,05). O alvo principal para expressão da HO-1 foram as células inflamatórias dentro do tumor ou em áreas subepiteleiais. Os ratos expostos ao refluxo gástrico não desenvolveram tumores ou expressaram a HO-1. Conclusões: A esofagite de refluxo induzida por refluxo esofágico com conteúdo duodenal provocou estresse oxidativo considerável e pode desempenhar um papel importante na carcinogênese esofágica. O refluxo gástrico não foi suficiente para induzir estresse oxidativo neste modelo experimental.

Câncer do colo uterino: rastreamento, marcador biológico e polimorfismo genético

Existem fortes evidências de que os programas de rastreamento baseados em citologia resultaram em diminuição significativa da incidência e mortalidade por câncer do colo do útero, no entanto, um excesso substancial de tratamento de lesões intraepiteliais de baixo grau que dificilmente progrediriam para carcinoma cervical resulta da baixa especificidade do tradicional rastreio citológico. A detecção precoce das lesões através do rastreamento citológico e a avaliação do grau histológico em espécimes cervicais são fundamentais, entretanto não permitem identificar quais pacientes terão maior probabilidade de progressão para lesões de alto grau e carcinoma invasivo. A busca de potenciais marcadores de prognóstico; objetivando o entendimento da progressão das lesões intraepiteliais é de suma importância. Acredita-se que fatores imunoregulatórios, imunogenéticos e proteínas do ciclo celular estejam intimamente envolvidos no processo de carcinogênese. Considerando o exposto, a proposta do projeto foi inicialmente avaliar a eficácia da citologia oncológica no rastreamento do câncer cervical, foi investigado ainda o polimorfismo do gene do fator de transcrição FOXP3 e a expressão da proteína do ciclo celular P63 (P63) associados respectivamente a diagnóstico e prognóstico das lesões cervicais. Em um primeiro momento foi realizado estudo transversal que envolveu 3194 mulheres. As participantes foram submetidas à citologia e biópsia de colo dirigida por colposcopia e os resultados foram comparados para verificar-se a acurácia do teste de Papanicolaou na detecção de lesões intraepiteliais e câncer cervical. Posteriormente, realizou-se estudo comparativo do tipo observacional estratificado em três grupos: Grupo 1: 16 casos com diagnóstico histopatológico de metaplasia/cervicite, considerados normais, Grupo 2: 11 casos com lesão de baixo grau (LSIL) e Grupo 3: 15 casos com lesão de alto grau (HSIL) ou carcinoma epidermoide de colo. Um total de 42 participantes respondeu a um questionário epidemiológico padronizado sobre as características demográficas, hábitos pregressos, história reprodutiva e de comportamento sexual. Após exame colposcópico, foram coletados fragmentos de espécimes cervicais para a pesquisa da expressão proteica da P63 por imunohistoquímica. Amostras de sangue periférico foram coletadas para extração do DNA e detecção do polimorfismo do gene FOXP3. No primeiro estudo em que se avaliou a acurácia do teste de Papanicolaou, encontrou-se sensibilidade de 0,83, valor preditivo positivo (VPP) de 0,77 e especificidade de 0,23 no rastreamento das lesões cervicais e câncer de colo. viii Melhores resultados foram observados quando se avaliou a acurácia diagnóstica para lesões de alto grau e carcinoma com VPP de 0,99 e especificidade de 0,84.No estudo subsequente onde se comparou a expressão da proteína P63 observou-se maior número de núcleos marcados no grupo com lesões intraepiteliais de alto grau e câncer quando comparado ao grupo com biópsias negativas (p=0,0004). No último estudo pesquisou-se a associação do polimorfismo do gene FOXP3 com lesões intraepiteliais cervicais sendo evidenciada maior prevalência do genótipo heterozigoto, CT, no grupo com lesões de colo na histopatologia (p=0,027). Mulheres com lesões intraepiteliais de baixo ou alto grau e câncer de colo de útero apresentam maior expressão da proteína P63 e maior prevalência de genótipo heterozigoto do gene FOXP3 em comparação com as sem lesões cervicais. A associação da pesquisa da expressão da proteína e do polimorfismo do gene pode tornar os exames utilizados atualmente para a avaliação diagnóstica e prognóstica das lesões de colo uterino mais efetivos em detectar quais as mulheres com maior risco para progressão para câncer

Avaliação histológica da próstata de cães adultos sexualmente intactos

Avaliou-se histologicamente a próstata de 30 cães adultos e idosos sexualmente intactos que apresentavam ou não sintomatologia clínica de doença prostática, e verificou-se a incidência de possíveis alterações da glândula. Dentre as alterações encontradas, a hiperplasia prostática benigna constituiu o diagnóstico mais comum, 85,6% (n=24), seguida por prostatite crônica, 64,3% (n=18), displasia do epitélio glandular, 42,8% (n=12), atrofia do epitélio glandular, 39,3% (n=11), infiltrado inflamatório focal, 25% (n=7), dilatação glandular focal, 21,4% (n=6), prostatite aguda, 7,1% (n=2), metaplasia escamosa, 3,6%, (n=1), metástase de neoplasia sistêmica, 3,6% (n=1) e abscesso prostático, 3,6% (n=1). Como em muitos casos os cães são assintomáticos, ressalta-se a importância da realização rotineira de exames clínicos específicos, como o toque retal e a ultrassonografia, para o diagnóstico precoce e o tratamento das afecções prostáticas.

Hepatic concentration of heat shock protein 70 kD (Hsp70) in broilers subjected to different thermal treatments

1. The relationship between repeated thermal treatments and hepatic synthesis of Hsp 70 was studied in broiler chickens.2. Sixty broilers were submitted to 5 different treatments (12 birds each) from day 1 to day 42. Four groups were kept in a thermoneutral environment and subjected to 0, 1, 2 and 3 heat stress episodes at 35 degrees C for 4 h per week (TN-0, TN-1, TN-2 and TN-3, respectively). The last group (HT-35) was reared at a room temperature of 35 degrees C.3. From 39 to 42 old, the birds experienced acute heat stress at 41 degrees C. Resistance to heat stress was evaluated by the time taken for rectal temperature to increase by 3 degrees C above the pre-treatment value. Livers were collected (before and after heat stress) and Hsp70 was determined using Western Blot analysis with monoclonal anti-Hsp70 antibody.4. Resistance to heat stress and concentration of Hsp70 were higher in those birds subjected to more heat stress episodes during the experimental period (TN-3) and HT-35. A positive correlation was observed between Hsp70 concentration and the time taken for a 3 degrees C increase in rectal temperature (r=0.42; P<0.01).5. Exposing birds to episodes of heat stress (35 degrees C) during rearing may improve their resistance to acute heat stress, but the previous thermal history did not seem to influence the hepatocyte Hsp70 content after exposure to more severe heat stress (41 degrees C).

Avaliação clínica da infecção experimental de bezerros com Salmonella Dublin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Controle da neoplasia maligna do colo de útero: a resolutividade na atenção básica

A quantitative, descriptive, cross-sectional and retrospective study, using technical procedures of document consultation from secondary sources and health household survey with application form for face to face inter views, with the assent nº.039/2011 from the Ethics Committee of the Federal University of Rio Grande do Norte. The aim of this study was to analyze the cervix cancer control in the area47 of the Health Family Centre Nova Natal II. The cancer cervix is the second most common cancer among women worldwide. In Brazil screening for early detection and treatment of disease has been poorly done and follow-up to reduce mortality has not been executed. From a total of1170women belonging to area 47, who under went screening by the Pap test in the period from 2005 to 2010,was elected a sample of 38 women with positive cervical changes, over 18 years old. The calculation of frequency analysis of socio demographic and clinical and epidemiological selected variables with the results of cervical changes, using the X2 test and taking as significance level of p<0.05 was not statistically significant. The predominant age range was 25 to 64 years (68.9%), most no white women (60.5%), predominantly with primary education (57.9%), most married (68.4%) and housewives (68.4%) with early age of sexual activity (86.8%), the minority smokers (13.2%), with a sexual partner (36.8%). At the time of interview, 42.1% of the women voiced complaint of discharge, while only 2.6% reported bleeding. In relation to the occurrence of STDs (including HPV), 10.5% of women reported being a carrier. The use of oral contraceptives was 32.3% of women, from 2 to 4 years (44.4%). The result of the last screening test performed, showed prevalence of immature squamous metaplasia (55.3%), followed by intraepithelial low- grade lesion (including the cytopathologic HPV effect and cervical intra epithelial neoplasia grade I) (31.6%); intraepithelial high-grade lesion (including cervical intraepithelial neoplasia grade II and III) (7.9%), atypical squamous non neoplastic cells (5.3%). There was no squamous cell carcinoma and adenocarcinoma. Most women received information about the action that should be done after the last screening test result (55.3%), but how to perform follow, most women did not report having done so (55.3%). The follow-up group of women studied, with varying degrees of cervical abnormalities, should only be completed with the discharge by cure, established inconsecutive negative cytology, a goal that is not being achieved in the area 47 of the Health Family Centre of Nova Natal II

Expressão imuno-histoquímica das integrinas a2ß1, a3ß1, e a5ß1 em folículos pericoronários espessados e cistos dentígeros incipientes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Upper gastrointestinal histopathological findings in children and adolescents with nonulcer dyspepsia with helicobacter pylori infection

Objectives: The aim of the study was to investigate the histopathological lesions in the upper gastrointestinal mucosa associated with Helicobacter pylori infection in children with nonulcer dyspepsia.Methods: A cross-sectional case-control study was performed on 185 Brazilian children and adolescents (4-17 years, mean 9.5 +/- 2.7 years), 63.2% girls, submitted to upper gastrointestinal endoscopy. The histopathological lesions of the esophageal and gastric mucosa were analyzed in biopsy samples.Results: H pylori infection was identified in 96 children (51.8%). Moderate to severe chronic active gastritis was present in antrum (70.5%) and corpus (45.2%), with higher grading in antrum than in corpus (P<0.05). The topographic distribution of inflammation was pangastritis (61.9%), followed by antral (32.1%) and corpus (5.9%). H pylori density was higher in antrum than in corpus. Intestinal metaplasia was not found in the H pylori-infected group, nor was significant gastric atrophy. The scores for esophagitis were significantly higher (P<0.05) in the noninfected group (1.4 +/- 0.8) than in the H pylori-infected group (1.07 +/- 0.9), with significant negative correlation (r = 0.29; P<0.05) with the scores of gastric inflammation.Conclusions: The prevalence of H pylori infection was high among children with dyspepsia and associated with moderate/severe degrees of gastric inflammation. The high scores of esophagitis in the noninfected group point to 2 distinct groups of pathological conditions sharing similar clinical patterns.

Subchronic Toxicity Evaluation of a Treated Urban Sewage Sludge

Disposal of tons of sludge produced daily by sewage treatment plants in large cities is a serious problem. Because recycling and application in agriculture have been proposed, the Brazilian National Environmental Council (CONAMA, 2006) issued a legal norm that regulates the use of the sewage sludge (SS) in crops. Due to the complex chemical nature of such products, characterization by analytical methods for health and environmental risk assessment has severe limitations. To overcome such limitations, it is necessary to (1) assess the toxicological potential of SS and (2) identify possible adverse effects in vivo in order to provide critical information for future environmental regulations. The present study was conducted to determine the potential toxicity of SS obtained from a representative urban treatment plant located in the São Paulo State, Brazil. Male and female Wistar rats were fed ad libitum a pelleted diet containing varying amounts of SS. No relevant clinical, hematological, urinary, or gross organ morphological alterations were observed in both genders of rats orally exposed to SS at up to 3.8 g/kg/d for 90 d. Sewage slude produced increased incidence of centrilobular hepatocyte hyperplasia at the high dose and significantly increased aspartate aminotransferease (AST) activities at all doses in both genders. Although the present data indicate some liver involvement, these alterations were considered adaptative and not toxicologically relevant, as the responses were relatively mild, not dose dependent, and no other parameters were markedly affected. The present results may contribute to the establishment of protocols for potential usage in SS agricultural soil application.

Folic acid supplementation during early hepatocarcinogenesis: cellular and molecular effects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Agaricus blazei (Himematsutake) does not alter the development of rat diethylnitrosamine-initiated hepatic preneoplastic foci

The modifying potential of crude extracts of the mushroom Agaricus blazei Murrill (Himematsutake) on the development and growth of glutathione S-transferase placental form (GST-P)-positive liver foci (liver preneoplastic lesion) was investigated in adult male Wistar rats. Six groups of animals were used. Groups 2 to 5 were given a single i.p. injection of 200 mg/kg b.w. of diethylnitrosamine (DEN) and groups 1 and 6 were treated with saline at the beginning of the experiment. After 2 weeks, animals of groups 3 to 6 were orally treated with three dose levels of aqueous extracts of the mushroom A. blazei (1.2, 5.6, 11.5, and 11.5 mg/ml of dry weight of solids) for 6 weeks. All animals were subjected to two-thirds partial hepatectomy at week 3 and sacrificed at week 8. Two hours before sacrifice, ten animals of each group were administered a single i.p injection of 100 mg/kg of bromodeoxyuridine (BrdU). Apoptotic bodies and BrdU-positive hepatocyte nuclei were quantified in liver sections stained for hematoxylin and eosin (H&E) (eosinophilic foci) and simultaneously stained for GST-P expression (GST-P-positive foci), respectively. The 6-week treatment with A. blazei did not alter the development (number and size) of GST-P-positive foci and did not affect the growth kinetics of liver normal parenchyma or foci in DEN-initiated animals. Our results indicate that the treatment with aqueous extracts of the mushroom A. blazei during the post-initiation stage of rat liver carcinogenesis does not exert any protective effect against the development of GST-P-positive foci induced by DEN. (Cancer Sci 2003; 94: 188-192).

Comparison of synthetic hydroxyapatite and porous polyethylene implants in eviscerated rabbit eyes

Purpose: Synthetic hydroxyapatite and porous polyethylene (Polipore) spheres were placed in rabbits' eviscerated cavities to evaluate tissue reaction and volume maintenance.Methods. Fifty-six Norfolk albino rabbits underwent unilateral evisceration and implantation of synthetic hydroxyapatite (H group, 28 animals) or porous polyethylene spheres (P group, 28 animals). Postoperative reactions, animal behavior, and socket conditions were monitored. Light microscopy and morphometric evaluation with statistical analysis of the exenterated orbits were performed at 7, 15, 30, 60, 90, 120, and 180 days. Scanning electron microscopy was appraised 7, 60, and 180 days after surgery.Results: Two animals from the H group and 1 from the P group had extrusion 7 days after surgery. Throughout the experimental period, the synthetic hydroxyapatite caused more inflammation than the porous polyethylene material. Ingrowth in the sphere occurred 7 to 15 days after the surgery in both groups, and the tissue reaction became denser at approximate to60 to 90 days, when bony metaplasia began in the H group. Volume maintenance was better in the P group and with a smaller pseudocapsule surrounding the implanted sphere than in the H group.Conclusions: Clinical findings demonstrated mild inflammation inside the sphere and in the pseudocapsule surrounding it and better cavity volume maintenance in the P group animals. The authors consider porous polyethylene a more suitable material than synthetic hydroxyapatite for use in anophthalmic cavity reconstruction.

Influence of aqueous extract of Agaricus blazei on rat liver toxicity induced by different doses of diethylnitrosamine

The modifying potential of prior administration of an aqueous extract of the mushroom Agaricus blazei Murrill (Agaricaceae) (Ab) on hepatotoxicity induced by different doses of diethylnitrosamine (DEN) in male Wistar rats was evaluated. During 2 weeks, animals of groups G3 (Ab+DEN50), G5 (Ab+DEN100), G7 (Ab+DEN200), and G8 (Ab-treated) were treated with the A. blazei through drinking water. After this period, groups G2 (DEN50), G3 (Ab+DEN50), G4 (DEN100) G5 (Ab+DEN100), G6 (DEN200), and G7 (Ab+DEN200) were given a single i.p. injection of 50, 100 and 200 mg/kg of DEN, respectively, while groups G1 (nontreated) and G8 (Ab-treated) were treated with 0.9% NaCl only. All animals were killed 48 h after DEN or NaCl treatments. The hepatocyte replication rate was estimated by the index of the proliferating cell nuclear antigen (PCNA) positive hepatocytes and the appearance of putative preneoplastic hepatocytes through expression of the enzyme glutathione S-transferase placental form (GSTP). After DEN-treatment, ALT levels, PCNA labeling index, and the number of GST-P positive hepatocytes were lower in rats that received A. blazei treatment and were exposed to 100 mg/kg of DEN. Our findings suggest that previous treatment with A. blazei exerts a hepatoprotective effect on both liver toxicity and hepatocarcinogenesis process induced by a moderately toxic dose of DEN. (C) 2002 Elsevier B.V. Ireland Ltd. All rights reserved.