Evaluation of enemas containing sucralfate in tissue content of muc-2 protein in experimental model of diversion colitis / Avaliação de enemas com sucralfato no conteúdo tecidual da proteina muc-2 em colite de exclusão experimental

ABSTRACT Background: The effects of topical application of sucralfate (SCF) on the tissue content of MUC-2 protein have not yet been evaluated in experimental models of diversion colitis. Aim: To measure the tissue content of MUC-2 protein in the colonic mucosa diverted from fecal stream submitted to the SCF intervention. Methods: Thirty-six rats underwent derivation of intestinal transit through proximal colostomy and distal mucous fistula. The animals were divided into three groups which were submitted application of enemas with saline, SCF 1 g/kg/day and SCF 2 g/kg/day. Each group was divided into two subgroups, according to euthanasia was done after two or four weeks. The colitis diagnosis was established by histopathological study and the inflammatory intensity was evaluated by previously validated scale. The MUC-2 protein was identified by immunohistochemistry and the tissue content was measured computerized morphometry). Results: The application of enemas with SCF in the concentration of 2 g/kg/day reduced inflammatory score of the segments that were diverted from fecal stream. The content of MUC-2 in diverted colon of the animals submitted to the intervention with SCF, independently of intervention period and the used concentration, was significantly greater than animals submitted to the application of enemas containing saline (p< 0.01). The content of MUC-2 after the intervention with SCF in the concentration of 2 g/kg/day was significantly higher when compared to the animals submitted to the application containing SCF at concentration of 1.0 g/kg/day (p<0.01). The tissue content of MUC-2 reached the highest values after intervention with SCF in the concentration of 2 g/kg/day for four weeks (p<0.01). Conclusion: The preventive application of enemas containing SCF reduces the inflammatory score and avoids the reduction of tissue content of MUC-2, suggesting that the substance is a valid therapeutic strategy to preserve the mucus layer that covers the intestinal epithelium.

RESUMO Racional: Os efeitos da aplicação tópica de sucralfato (SCF) no conteúdo tecidual da proteína mucina-2 (MUC-2) ainda não foram avaliados em modelos experimentais de colite de exclusão. Objetivo: Mensurar o conteúdo tecidual da proteína MUC-2 na mucosa cólica sem trânsito intestinal submetida à intervenção com SCF. Método : Trinta e seis ratos foram submetidos à derivação intestinal por colostomia proximal terminal e fístula mucosa distal. Foram divididos em três grupos segundo recebessem clisteres contendo solução fisiológica (SF), SCF 1 g/kg/dia e SCF 2 g/kg/dia. Cada grupo foi dividido em dois subgrupos, segundo a eutanásia ser realizada após duas ou quatro semanas. O diagnóstico de colite foi estabelecido por estudo histopatológico e a intensidade inflamatória foi avaliada por escala validada. A expressão tecidual da MUC-2 foi identificada por imunoistoquímica e seu conteúdo mensurado por morfometria computadorizada. Resultados: A aplicação de clisteres com SCF na concentração de 2 g/kg/dia reduziu a intensidade inflamatória no cólon sem trânsito fecal. O conteúdo tecidual de MUC-2 no cólon sem trânsito dos animais submetidos à intervenção com SCF, independente do tempo de intervenção e da concentração utilizada, foi maior quando comparado aos animais tratados com SF (p<0,01). O conteúdo de MUC-2 após a intervenção com SCF na concentração de 2 g/kg/dia foi maior quando comparado aos animais submetidos à intervenção com concentração menor (p<0,01). O conteúdo de MUC-2 foi maior após intervenção com SCF na concentração de 2 g/kg/dia por quatro semanas (p<0,01). Conclusão: A aplicação preventiva de clisteres com SCF reduz o grau de inflamação e preserva o conteúdo tecidual de MUC-2, em segmentos desprovidos de trânsito intestinal, mostrando-se uma estratégia terapêutica válida para preservar a camada de muco que recobre o epitélio intestinal.

Tissue sulfomucin and sialomucin content in colon mucosa without intestinal transit subjected to intervention with Curcuma longa (curcumin)

Abstract Purpose: To measure the tissue sulfomucin and sialomucin content of the colon mucosa without fecal flow, subjected to intervention with curcumin, and the influence of the concentration used and the intervention time. Methods: Thirty-six rats were subjected to proximal right colostomy and distal mucous fistula. They were divided into two groups according to whether sacrifice was performed two or four weeks after the intervention. Each group was divided into three subgroups according to the enema applied daily: saline alone; curcumin at 50 mg/kg/day or curcumin at 200 mg/kg/day. Acid mucins were diagnosed using the Alcian blue technique. The mucin content was quantified by means of computer-assisted image analysis. The significance level of 5% was used throughout (p < 0.05). Results: There were dose-related increases in the quantities of sulfomucins in the animals subjected to interventions with curcumin, both after two weeks (p < 0.00001) and after four weeks (p < 0.00001). There were increases in sialomucin quantity that were concentration-related (p < 0.00001) and time-related (p < 0.00001). Conclusion: Curcumin enemas increase the quantity of acid mucins in the intestinal flow in the excluded colon, with dose and time dependency.

Claudin-3 and occludin content in the glands of colonic mucosa devoid from fecal stream submitted to topical intervention with oil extract of Curcuma longa

Abstract Purpose: To evaluate the inflammatory intensity and measure the tissue content of the proteins claudin-3 and occludin in the colonic mucosa without fecal stream submit to intervention with curcumin. Methods: Thirty-six rats were submitted to a proximal colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, curcumin at 50 mg/kg/day or 200 mg/kg/day. Colitis was diagnosed by histological analysis. Claudin-3 and occludin were determined by immunohistochemistry. The tissue content of claudin-3 and occludin were quantified by computer-assisted image analysis. Mann-Whitney, Student t and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). Results: Curcumin at both concentrations reduces the inflammation and preserves the tissue content of the proteins claudin-3 and occludin, which was related to the concentration used and to the time of the intervention. Conclusion: The application of enemas with curcumin reduces inflammation and preserves the tissue content of the proteins claudin-3 and occludin in the colonic mucosa devoid from the fecal stream.

Histochemical differences along the intestine of Corydoras paleatus (Siluriformes: Callichthyidae) / Diferencias histoquímicas a lo largo del intestino de Corydoras paleatus (Siluriformes: Callichthyidae)

AbstractThe Neotropical catfish Corydoras paleatus is a facultative air-breather and the caudal half of the intestine is involved in gas exchange. In South America, air-breathing fishes are found in tropical or sub-tropical freshwaters where the probability of hypoxia is high. The aim of this study was to characterize by traditional histochemical and lectinhistochemical methods the pattern of carbohydrate in the intestinal mucosa. Intestine samples were taken from 25 healthy adult specimens collected in Buenos Aires (Argentina). Samples were fixed by immersion in 10 % buffered formalin and routinely processed and embedded in paraffin wax. Subsequently, these sections were incubated in the biotinylated lectins battery. Labeled Streptavidin-Biotin (LSAB) system was used for detection, diaminobenzidine as chromogen and haematoxylin as a contrast. To locate and distinguish glycoconjugates (GCs) of the globet cells, we used the following histochemical methods: PAS; PAS*S; KOH/ PA*S; PA/Bh/KOH/PAS; KOH/PA*/Bh/PAS; Alcian Blue and Toluidine Blue at different pHs. Microscopically, the general structure of vertebrate intestine was observed and showed all the cell types characteristic of the intestinal epithelium. The cranial sector of catfish intestine is a site of digestion and absorption and its structure is similar to other fish groups. In contrast, enterocytes of the caudal portion are low cuboidal cells; and between these, globet cells and capillaries are observed, these latter may reach the mucosal lumen. Underlying the epithelium, observed a well-developed lamina propria-submucosa made of connective tissue; this layer was highly vascularized and did not exhibit glands. According to histochemistry, the diverse GCs elaborated and secreted in the intestine are associated with specific functions in relation to their physiological significance, with special reference to their role in lubrication, buffering effect and prevention of proteolytic damage to the epithelium together with other biological processes, such as osmoregulation and ion exchange. The lectinhistochemical analysis of the intestinal mucosa reveals the presence of terminal residues of glucose, mannose and galactose. In conclusion, this study has shown that GCs synthesized in the intestine of C. paleatus exhibit a high level of histochemical complexity and that the lectin binding pattern of the intestinal mucosa is characteristic of each species and the variations are related with the multiple functions performed by the mucus in the digestive tract. The information generated here may be a relevant biological tool for comparing and analyzing the possible glycosidic changes in the intestinal mucus under different conditions, such as changes in diet or different pathological stages. Rev. Biol. Trop. 64 (1): 327-340. Epub 2016 March 01.

ResumenEl pez neotropical Corydoras paleatus, de respiración aérea de tipo facultativa, utiliza el sector caudal del intestino para el intercambio gaseoso. En América del Sur, los peces con respiración aérea se encuentran en las aguas dulceacuícolas tropicales y subtropicales, donde la probabilidad de hipoxia es alta. El objetivo de este trabajo fue caracterizar mediante técnicas histoquímicas tradicionales y de lectinhistoquímica el patrón de carbohidratos de la mucosa intestinal. Para ello se utilizaron muestras de intestino de 25 ejemplares sanos adultos recolectados en la provincia de Buenos Aires (Argentina). Las muestras fueron fijadas en formol amortiguado al 10 % y se procesaron para su inclusión en parafina. Posteriormente, los cortes fueron incubados con una batería de lectinas biotiniladas. Se utilizó el sistema de marcado con estreptavidina-biotina (LSAB) para su detección, diaminobencidina como cromógeno y hematoxilina como colorante de contraste. Para localizar y diferenciar los glicoconjugados (GCs) de las células caliciformes, se utilizaron las siguientes técnicas histoquímicas: PAS, PAS*S, PAPS, KOH/PA*S, PA/Bh/KOH/PAS, KOH/PA*/Bh/PAS, Azul Alcian y Azul de Toluidina a diferentes pHs. Microscópicamente, se observa la estructura general del intestino de los vertebrados y el epitelio intestinal presenta todos los tipos celulares característicos de esta región. El sector craneal del intestino de este teleósteo, es el sitio de digestión y absorción, y posee una estructura similar a la de otros grupos de peces. En cambio, los enterocitos de la porción caudal, son células cúbicas bajas, entre ellos se observan células caliciformes y capilares sanguíneas que llegan hasta el lumen de la mucosa. Por fuera del epitelio, se observa una lámina propia-submucosa muy desarrollada compuesta por tejido conectivo, altamente vascularizada que no presenta glándulas. De acuerdo con las técnicas histoquímicas, los diversos GCs elaborados y secretados por la mucosa intestinal se encuentran asociados con funciones específicas de importancia fisiológica, como su rol en la lubricación, su efecto amortiguador y la prevención de daños proteolíticos del epitelio junto con otros procesos biológicos, tales como la osmorregulación y el intercambio iónico. El análisis lectinhistoquímico de la mucosa intestinal revela la presencia de residuos terminales de glucosa, manosa y galactosa. En conclusión, en este estudio se demuestra que los GCs sintetizados en el intestino de C. paleatus muestran un alto nivel de complejidad histoquímica y que el patrón de unión de lectina de la mucosa intestinal es característico para cada especie y las variaciones se hallan relacionadas con las múltiples funciones realizadas por el mucus en el tracto digestivo. La información brindada en este trabajo es una herramienta de relevancia biológica para comparar y analizar los posibles cambios glicosídicos del mucus intestinal bajo diferentes condiciones como los cambios en la dieta o diferentes estados patológicos.

Tissue content of sulfomucins and sialomucins in the colonic mucosa, without fecal stream, undergoing daily intervention with sucralfate

PURPOSE: To measure the content of acidic mucin, sialomucin, and sulfomucins in the colonic mucosa without fecal stream submit to intervention with sucralfate (SCF). METHODS: Thirty-six rats were submitted to a right colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, SCF at 1.0 g/kg/day or 2.0 g/kg/day. Colitis was diagnosed by histological analysis. Acid mucins were determined with the Alcian-Blue and sulfomucin and sialomucin by high iron diamine-alcian blue (HID-AB) techniques. The mucins were quantified by computer-assisted image analysis. Mann-Whitney and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). RESULTS: SCF enemas decreased the inflammation score and was related to the concentration used and time of the intervention. SCF at both concentrations increased the content of acid mucin, which was related to the concentration used and to the improvement in the inflammatory score. There was an increase in the content of sulfomucins and sialomucins in SCF groups. SCF increased sulfomucins from 2 weeks of intervention, which was not related to the dose or time of application. The increase in sialomucin content was related to the time and dose used in the intervention. CONCLUSION: Sucralfate increased the content of acidic mucins, primarily at the expense of sialomucin, which was affected by the dose and time of intervention. .

CDX-2, MUC-2 and B-catenin as intestinal markers in pure mucinous carcinoma of the breast

BACKGROUND: Pure mucinous adenocarcinoma of the breast is a rare entity characterized by the production of variable amounts of mucin comprising 1% to 6% of breast carcinomas. Some mucinous adenocarcinomas have shown expression of intestinal differentiation markers such as MUC-2. This study examines the expression of intestinal differentiation markers in this type of breast carcinoma. RESULTS: Twenty-two cases of pure mucinous adenocarcinoma of the breast were assessed. Immunochemistry was performed for beta-catenin, CDX-2 and MUC-2. All cases were positive for B-catenin. MUC-2 positivity was observed in all cases; 63. 6% were 3 plus positive. All cases were negative for CDX-2. CONCLUSIONS: These results suggest that mucinous breast carcinomas express some markers of intestinal differentiation, such as MUC-2 and beta-catenin; however, future studies with a larger series of cases and using molecular techniques that help affirm these results are needed.

Ulcerative Colitis and Immunoglobulin G4

BACKGROUND/AIMS: Ulcerative colitis (UC) is sometimes associated with autoimmune pancreatitis (AIP). Infiltration of immunoglobulin G4 (IgG4)-positive plasma cells is sometimes detected in the colonic mucosa of AIP or UC patients. This study aimed to clarify the relation between UC and IgG4. METHODS: Associations with UC were reviewed in 85 AIP patients. IgG4 immunostaining was performed on biopsy specimens from the colonic mucosa of 14 AIP and 32 UC patients. RESULTS: UC was confirmed in two cases (type 1 AIP, n=1; suspected type 2 AIP, n=1). Abundant infiltration of IgG4-positive plasma cells in the colonic mucosa was detected in the case of suspected type 2 AIP with UC and two cases of type 1 AIP without colitis. Abundant infiltration of IgG4-positive plasma cells was detected in 10 UC cases (IgG4-present, 31%). Although 72% of IgG4-absent UC patients showed mild disease activity, 70% of IgG4-present patients showed moderate to severe disease activity (p<0.05). CONCLUSIONS: UC is sometimes associated with AIP, but it seems that UC is not a manifestation of IgG4-related disease. Infiltration of IgG4-positive plasma cells is sometimes detectable in the colonic mucosa of UC patients and is associated with disease activity.

NM23 protein expression in colorectal carcinoma using TMA (tissue microarray): association with metastases and survival / Expressão da proteína NM23 no carcinoma colorretal preparado em TMA (tissue microarray): associação com metástases e sobrevivência

CONTEXT: NM23, a metastasis suppressor gene, may be associated with prognosis in patients with colorectal carcinoma. OBJECTIVE: To analyze NM23 expression and its association with the presence of lymph node and liver metastases and survival in patients operated on for colorectal carcinoma. METHODS: One hundred thirty patients operated on for colorectal carcinoma were investigated. Tissue microarray blocks containing neoplastic tissue and tumor-adjacent non-neoplastic mucosa were obtained and analyzed by immunohistochemical staining using a monoclonal anti-NM23 antibody. Immunohistochemical expression was assessed using a semiquantitative scoring method, counting the percentage of stained cells. The results were compared regarding morphological and histological characteristics of the colorectal carcinoma, presence of lymph node and liver metastases, tumor staging, and patient survival. Statistical analysis was performed using the Mann-Whitney test, the Kruskal-Wallis test and Fisher's exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. RESULTS: NM23 expression was higher in colorectal carcinoma tissue than in adjacent non-neoplastic mucosa (P<0.0001). NM23 protein expression did not correlate with degree of cell differentiation (P = 0.57), vascular invasion (P = 0.85), lymphatic invasion (P = 0.41), perineural infiltration (P = 0.46), staging (P = 0.19), lymph node metastases (P = 0.08), or liver metastases (P = 0.59). Disease-free survival showed significant association (P = 0.01) with the intensity of NM23 protein immunohistochemical expression in colorectal carcinoma tissue, whereas overall survival showed no association with NM23 protein expression (P = 0.13). CONCLUSIONS: NM23 protein expression was higher in neoplastic colorectal carcinoma tissue than in adjacent non-neoplastic mucosa, showing no correlation with morphological aspects, presence of lymph node or liver metastases, colorectal carcinoma staging, or overall survival. Disease-free survival was higher in patients with increased NM23 expression.

CONTEXTO: O NM23, denominado de gene supressor de metástases, pode estar relacionado com o prognóstico em doentes com carcinoma colorretal. OBJETIVOS: Analisar a expressão do marcador tumoral NM23 relacionando-a com a presença de metástases linfonodais e hepáticas e com a sobrevivência dos doentes operados por carcinoma colorretal. MÉTODO: Cento e trinta doentes operados por carcinoma colorretal foram analisados. Blocos de "tissue microarray" foram obtidos com tecido neoplásico e com mucosa não neoplásica adjacente ao tumor e submetidos ao estudo imunoistoquímico com o anticorpo monoclonal NM23. A imunoexpressão foi avaliada por método semiquantitativo, com contagem do percentual de células coradas. Os resultados encontrados foram relacionados com as características morfológicas e histopatológicas do carcinoma colorretal, presença de metástases linfonodais e hepáticas, estádio e sobrevivência dos doentes. O estudo estatístico foi realizado com os testes de Mann-Whitney, Kruskal-Wallis e exato de Fisher. A análise da sobrevivência foi calculada pelo método de Kaplan-Meier e pelo teste de long-rank. RESULTADOS: A expressão do marcador NM23 foi maior no tecido do carcinoma colorretal do que na mucosa não-neoplásica adjacente (P<0,0001). A expressão da proteína NM23 não apresentou relação com o grau de diferenciação celular (P = 0,57), invasão vascular (P = 0,85), invasão linfática (P = 0,41), infiltração perineural (P = 0,46), estádio (P = 0,19), metástases linfonodais (P = 0,08) ou metástases hepáticas (P = 0,59). A sobrevivência livre de doença mostrou relação significante (P = 0,01) com a intensidade de imunoexpressão da proteína NM23 no tecido do carcinoma colorretal, e a sobrevivência global não mostrou relação com a expressão da proteína NM23 (P = 0,13). CONCLUSÕES: A expressão da proteína NM23 foi mais intensa no tecido neoplásico do carcinoma colorretal do que na mucosa não-neoplásica adjacente. A expressão da proteína NM23 não se relacionou com os aspectos morfológicos, presença de metástases linfonodais ou hepáticas, estádio do carcinoma colorretal ou com a sobrevivência global. A sobrevivência livre de doença foi maior nos doentes com expressão aumentada do gene supressor de metástases NM23.

Quantification by computerized morphometry of tissue levels of sulfomucins and sialomucins in diversion colitis in rats / Quantificação por morfometria computadorizada dos níveis teciduais de sulfomucinas e sialomucinas na colite de exclusão em ratos

PURPOSE: To quantify the intensity of sulfomucin and sialomucin expression in the colon mucosa, by means of computer-assisted image processing, comparing segments with and without fecal stream and correlating with the duration of fecal transit exclusion. METHODS: Forty-five Wistar rats were subjected to diversion of the fecal stream in the left colon by means of constructing a proximal colostomy and distal mucosal fistula. They were distributed randomly into three experimental groups of 15 animals, of which 10 were subjected to colon diversion (experimental subgroup) and five were only subjected to laparotomy, without colon diversion (control subgroup). The three experimental groups were formed according to the sacrifice date, which was to be performed six weeks after the surgical procedure (Group A), 12 weeks (Group B) and 18 weeks (Group C). The sulfomucin and sialomucin expression in the colon mucosa was evaluated using the histochemical technique of high iron diamine-alcian blue (HID-AB). The tissue expression was quantified for each animal, in the segments with and without fecal stream, at a location where there were four complete contiguous crypts in two random fields, with the aid of the computer-assisted image analysis software. The final value was taken to be the mean reading from the two fields selected, in the segments with and without fecal stream. To compare the expressions of the two mucin subtypes in the segments with and without fecal stream, the paired Student t test was used. To analyze variance according to duration of exclusion, ANOVA with the Newman-Keuls post-test was used, setting the significance level at 5 percent (p<0.05). RESULTS: There were significant reductions in tissue sulfomucin and sialomucin content in the colon without fecal stream, independent of the duration of exclusion considered. There was increased tissue sulfomucin content and decreased tissue sialomucin in the segments without fecal stream, ...

OBJETIVO: Quantificar, por meio de processamento de imagem assistida por computador, a intensidade de expressão de sulfomucinas e sialomucinas na mucosa cólica comparando segmentos com e sem trânsito e relacionando-a ao tempo de exclusão de trânsito fecal. MÉTODOS: Quarenta e cinco ratos Wistar machos foram submetidos à derivação do trânsito no cólon esquerdo pela confecção de colostomia proximal e fístula mucosa distal. Foram divididos de forma randomizada em três grupos experimentais de 15 animais, nos quais 10 foram submetidos à derivação do trânsito cólico (subgrupo experimental) e cinco somente a laparotomia exploradora sem desvio do trânsito fecal (subgrupo controle). Os três grupos experimentais foram formados segundo o sacrifício ter sido realizado em seis (grupo A), 12 (Grupo B) e 18 semanas (Grupo C). A avaliação da expressão de sulfomucinas e sialomucinas na mucosa cólica foi realizada pela técnica histoquímica da diamina de ferro alto alcian-blue (HID-AB). A quantificação da expressão tecidual foi determinada, para cada animal, nos segmentos com e sem trânsito, em local onde existiam quatro criptas contíguas e íntegras em dois campos aleatórios com auxílio de programa de análise de imagem assistida por computador. Adotou-se como valor final a média das leituras dos dois campos selecionados, nos segmentos providos e desprovidos de trânsito fecal. Na comparação entre a expressão dos dois subtipos de mucinas nos segmentos com e sem trânsito fecal utilizou-se o teste t de Student pareado. Para análise de variância segundo o tempo de exclusão utilizou-se o teste de ANOVA com o pós-teste de Newmann-Keuls, estabelecendo-se nível de significância de 5 por cento (p<0,05). RESULTADOS: Houve redução significante no conteúdo tecidual de sulfomucinas e sialomucinas no cólon desprovido de trânsito fecal, independente do tempo de exclusão considerado. Houve aumento no conteúdo tecidual de sulfomucinas e diminuição de sialomucinas nos segmentos ...

Alterations in the intestinal wall due to protein malnutrition in rats: Evaluation of the rupture strength and the tissue's collagen / Alterações da parede intestinal na carência de proteína no rato: Avaliação da força de ruptura e do colágeno tecidual

PURPOSE: To study the effect of protein malnutrition on the intestinal wall of rats by evaluating alterations in the rupture force and dosing tissue collagen in the ileum and distal colon. METHODS: One hundred and twenty rats, that had an average weight of 100g, were used. They received water and a standard diet with 20 percent protein during 7 days for adaptation to the diet itself and to environmental conditions. After that period, the animals were randomly distributed in two groups of 60 rats each: Group 1 - the animals received a control diet with 20 percent casein for 21 days; Group 2 - hypoprotein diet with 2 percent casein for 21 days. After the adaptation period, 12 animals of each group were sacrificed at 5 moments: the beginning of experimental period (M0), 4° day (M1), 7° day (M2), 14° day (M3) and 21° day (M4). The diet to the other rats was maintained until the last sacrifice. The following variables were evaluated: body weight, blood albumin rate, tissue's hydroxyproline, hydroxyproline/total protein ratio and rupture strength in the intestinal wall of the ileum and the distal colon. RESULTS: It was observed that the rupture strength in the ileum segment and distal colon was lower in malnourished animals (Group 2); the loss of mechanical resistance was higher in the distal colon segment than in the ileum probably due to the smaller concentration of tissue collagen in the distal colon. CONCLUSION: Protein malnutrition induces the loss of mechanical resistance of the ileum and distal colon and may be associated with a smaller percentage of collagenous tissue formation in the intestinal wall.

OBJETIVO: Avaliar o efeito da desnutrição protéica na parede intestinal do rato através da medida de força de ruptura e dosagem do colágeno tecidual no íleo e cólon distal. MÉTODOS: Foram utilizados 120 ratos, pesando em média 100g, que receberam durante 07 dias uma dieta padrão, contendo 20 por cento de caseína para adaptação dos animais as condições do biotério. Após esse período os animais foram divididos em dois grupos de 60, o controle denominado grupo um que recebeu a dieta padrão, e o grupo teste denominado grupo dois, que recebeu dieta hipoprotéica contendo 2 por cento de caseína. Os dois grupos receberam suas respectivas dietas por um período de 21 dias. Após esse período iniciou-se o sacrifício seqüencial dos animais em ambos os grupos, em número de 12 animais em cada momento, correspondendo ao dia Zero (MO), 4º dia (M1), 7º dia (M2), 14º dia (M3), e 21º dia (M4) sendo mantida a mesma dieta até o final do sacrifício. Em cada momento foram avaliados o peso corpóreo, albumina sanguínea, hidroxiprolina tecidual, relação hidroxiprolina/proteína tecidual e a força de ruptura no segmento ileal e cólico dos animais. RESULTADOS: Observou-se que a força de ruptura do segmento ileal e do cólon distal foi menor nos animais desnutridos (Grupo 2). A perda da resistência mecânica foi maior no segmento do cólon distal do que no segmento ileal, provavelmente pela menor concentração do colágeno tecidual no cólon distal. CONCLUSÃO: A desnutrição protéica induz a diminuição da resistência mecânica no íleo e no cólon distal associado a diminuição do colágeno tecidual na parede intestinal.

Morphological regional differences of epithelial cells along the midgut in Diatraea saccharalis Fabricius (Lepidoptera: Crambidae) larvae / Diferenças morfológicas regionais das células epiteliais ao longo do intestino médio de larvas de Diatraea saccharalis Fabricius (Lepidoptera: Crambidae)

The sugarcane borer, Diatraea saccharalis Fabricius, is a pest to sugarcane and many other crops. This work aims to characterize morphological variability in the epithelial cells (columnar, goblet and regenerative) along the midgut of D. saccharalis larvae. Fragments of the midgut (anterior, middle and posterior regions) were fixed and processed by light and scanning electron microscopy. There are both cytochemical and ultrastructural differences in the morphology of the epithelial cells, depending on their localization along the midgut. The apical surface of columnar cells shows an increase in both number and size of the apical protrusions from the anterior to the posterior midgut regions. There is an increase in the amount of PAS-positive (Periodic Acid-Schiff Reaction) granules detected in the cytoplasm of both the columnar and regenerative cells, from the anterior to the posterior region. The goblet cell apical surface is narrow in the anterior region, and enlarged in the posterior midgut; the chamber's cytoplasm extrusion are small and thin at the apical cavity surface, being thicker, longer and more numerous at the basal portion of the cavity. Our results suggest that the sugarcane borer midgut has two morphologically different regions, the anterior and the posterior; the middle region is a transitional region.

A broca da cana, Diatraea saccharalis Fabricius, é uma praga da cana-de-açúcar e outras plantações. O objetivo deste trabalho foi caracterizar variações morfológicas nas células epiteliais (colunares, caliciformes e regenerativas) ao longo do intestino médio de larvas de D. saccharalis. Fragmentos do intestino médio (anterior, mediano e posterior) foram fixados e processados para microscopia de luz e eletrônica de varredura. Existem diferenças morfológicas citoquímicas e ultra-estruturais nas células epiteliais, dependendo da sua localização no intestino médio. A superfície apical de algumas células colunares exibe projeções citoplasmáticas que aumentam em número e volume da região anterior para a posterior do intestino médio. Existe aumento dos grânulos PAS-positivos (Reação de Schiff) no citoplasma apical das células colunares e regenerativas, da região anterior para a posterior. A câmara das células caliciformes, na região anterior do intestino médio, mostra seu ápice estreito, enquanto que na posterior essa porção da câmara é alargada; as evaginações citoplasmáticas da câmara são pequenas e finas no ápice, sendo numerosas, longas e mais espessas na porção basal. Os resultados sugerem que o intestino médio da broca da cana apresenta duas regiões morfologicamente distintas, a anterior e posterior; a região mediana é uma região de transição.

Papel de las poliaminas en la alimentación: Importancia de las poliaminas en la alimentación infantil

Durante la década de los 90 del siglo pasado comenzaron los primeros estudios que señalaban a las poliaminas como importantes factores de crecimiento de la leche materna. Sin embargo, todavía se desconoce mucho sobre el papel que desempeñan en la alimentación infantil y cual sería la ingesta recomendada para este grupo poblacional. En los últimos años va siendo cada vez mayor la atención que muestra la comunidad científica internacional hacia las poliaminas, debido no sólo al importante papel que desempeñan en el metabolismo celular, sino por su posible participación en diversas patologías y durante el desarrollo del organismo. Sería recomendable, teniendo en cuenta que el contenido en poliaminas de las fórmulas infantiles es unas 10 veces menor al de la leche materna, profundizar más en este campo, con el fin de garantizar una correcta alimentación durante la etapa de lactancia.

Role of polyamines in diet. Importance of polyamines in infant nutrition. The first studies indicating polyamines as important growth factors in breast milk began during the nineties of last century. Nevertheless, it is still not well known the role they play in infant nutrition or what the recommended intake would be for this population group. In recent years, there has been increased attention of the international scientific community towards polyamines, not only due to the important role they play in the cellular metabolism, but also to their possible implication in some diseases and during the development of the human organism. Bearing in mind that the content in polyamines of the infant formula is around tenfold less than in breast milk, it would be recommended to gain insight into this theme in order to guarantee correct nutrition during lactation.

Multidrug resistance gene (MDR-1) expression in the colonic mucosa of patients with refractory ulcerative colitis / Expresión del gen de multirresistencia a drogas (MDR-1) en la mucosa colónica de pacientes con colitis ulcerosa refractaria

BACKGROUND: P-Glycoprotein (P-gp), a product of the MDR-1 gene, is a transmembrane efflux pump involved in drug transport, first described in cancer refractoriness. In the normal bowel P-gp is detectable on superficial epithelial cells, but has not been described in crypt epithelium. The role of P-gp and its intestinal expression in steroid-refractory ulcerative colitis (UC) are controversial. AIM: to compare P-gp immunostaining pattern in colonic epithelial cells of steroid-refractory versus steroid-responder UC patients. METHODS: P-gp was assessed by immunohistochemistry in rectal biopsies obtained from 19 patients with active UC, including pre-surgical samples from 11 refractory patients who underwent colectomy, and 8 responders. We devised a 5-point (0-4) score, according to the percentage of epithelial surface with positive immunostaining in the superficial and crypt epithelium (apical, lateral and cytoplasmic areas). RESULTS: Compared with responders, steroid-refractory patients had significantly higher immunostaining scores in the superficial epithelium, both in apical (2.8+/-0.5 versus 1.1+/-0.5, p=0.023) and cytoplasmic cellular areas (2.7+/-0.5 versus 1.2+/-0.5, p=0.032). Positive immunostaining of the superficial epithelium was frequently detected in refractory patients (apical: 9/11 cases, cytoplasmic: 10/11 cases) but was only observed in 4/8 responders. P-gp was also detected in similar areas of the crypt epithelium in 6/11 refractory patients, while it was infrequent in the group of 8 responders (1 apical 1 case, cytoplasmic 2 cases). Samples from the mucosa of normal ileal pouch-anal anastomoses obtained several years after the surgical procedure had a P-gp immunostaining pattern which was similar to that of rectal samples from patients with refractory UC. CONCLUSIONS: These results suggest a critical role of P-gp overexpression in steroid-refractory UC.

Antecedentes. La glicoproteína P (P-gp), un producto del gen MDR-1, es una bomba de eflujo transmembranainvolucrada en el transporte de drogas, descripta por primera vez en el cáncer refractario. En el intestino normal, P-gp se detecta sobre las célulasepiteliales superficiales, pero no se la ha descripto en el epitelio de las criptas. El papel de P-gp y su expresiónintestinal en la colitis ulcerosa (CU) refractaria a esteroides es controvertido. Objetivo. Comparar elpatrón de inmunotinción de P-gp en células epiteliales colónicas de pacientes con CU refractaria vs.respondedora a esteroides. Métodos. Se estudió P-gp por inmunohistoquímica en biopsias rectales obtenidasde 19 pacientes con CU activa, incluyendo muestras prequirúrgicas de 11 pacientes refractarios que fueronsometidos a una colectomía y muestras de 8 respondedores. Ideamos un score de 5 puntos (0-4), según elporcentaje de superficie epitelial con inmunotinción positiva en el epitelio superficial y críptico (áreas apical,lateral y citoplásmica). Resultados. Comparados con los respondedores, los pacientes refractarios a esteroides tenían scores de inmunotinción significativamente mayores en el epitelio superficial, tanto en lasáreas celulares apical (2.8+0.5 vs. 1.1+0.5, p=0.023) como citoplásmica (2.7+0.5 vs. 1.2+0.5, p=0.032). Se detectó frecuentemente inmunotinción positiva en el epitelio superficial en los pacientes refractarios (apical: 9/11 casos, citoplásmica: 10/11 casos), pero la misma se observó sólo en 4/8 respondedores. P-gp también sedetectó en áreas similares del epitelio de las criptas en 6/11 pacientes refractarios, en tanto que fue infrecuenteen el grupo de los 8 respondedores (1 caso en el área apical y 2 en la citoplásmica). Fuerón estudiadasbiopsias de la mucosa de la anastomosis pouch ileal - anal, obtenidas varios años después del procedimeinto quirúrgico, observándose un patrón de...

Polymorphonuclear leukocyte migration across intestinal epithelium induces epithelial cytokine expression

La migración de neutrófilos polimorfonucleares es un rasgo común de la inflamación activa, que precede la formación de abscesos. La contribución relativa de células epiteliales como fuente de quimioquinas en la infiltración de leucocitos durante la inflamación intestinal no ha sido estudiada. Para evaluar esta contribución nosotros diseñamos un modelo heterólogo de migración transepitelial, “in vitro”, haciendo uso de PMN de rata y células epiteliales de origen humano. Nosotros demostramos que neutrófilos polimorfonucleares luego de su activación quimiotáctica, inducen el incremento en los niveles de ARNm de IL-1ß, IL-8 en las células epiteliales de intestino, mientras que no afecta al ARNm de ENA-78. Estos resultados sugieren que quimiocinas y citoquinas sintetizados por la célula epitelial podrían jugar un papel en el mantenimiento de la respuesta inflamatoria.

Polymorphonuclear neutrophil migration is a common feature of active inflammation that precedes the formation of abscesses. The relative contribution of epithelial cells as a source of chemokines in the recruitment of leukocytes during intestinal inflammation has not been studied. To evaluate this contribution, we have designed a heterologous “in vitro” model for transepithelial PMN migration, based on the use of rat neutrophils and human epithelial cells. We show that polymorphonuclear neutrophil, upon chemotactic activation, induces an increase in IL-1ß, IL-8 levels in intestinal epithelial cells, while not changed ENA-78 mRNA. These results suggest that chemokines and cytokines synthesized by intestinal epithelial cells could play a role in the maintenance of the inflammatory response.

Variability of spectra of laser-induced fluorescence of colonic mucosa: its significance for fluorescence detection of colonic neoplasia.

To determine the extent of a natural variability of the spectra of the autofluorescence and its significance for a reproducibility of different approaches typically used in studies on fluorescence detection of colonic lesions. Two independent series of experiments have been conducted during three years in the same laboratory. Macroscopic tissue specimens obtained during operations of patients with colonic cancers were studied in vitro. The tissues were excited using UV lines of c.w. He-Cd laser and pulsed nitrogen laser and the autofluorescence spectra were recorded for areas visually diagnosed as normal or pathologically changed mucosa. Natural variability of the autofluorescence spectra of colonic tissues seems to be most important factor limiting sensitivity and specificity of the diagnostic algorithms. The mean fluorescence spectra obtained for normal mucosa and its neoplastic lesions differ significantly but the differences are difficult to observe because of the high natural variability among the individual spectra. Further studies of biological basis of the colonic autofluorescence are necessary for a progress in the field of fluorescence detection of colonic neoplastic lesions.

Substance P in rectal mucosa of children with chronic constipation

Niveles de Substancia P fueron determinados por radioinmunoassay en la mucosa rectal de 17 niños con constipación, idiopática crónica y comparados con la de 9 ninõs sin constipación. En el grupo de niños con constipación, los niveles de Sustancia P fueron menores que aquellos de los controles: 47,6+-11 vs. 79,4+-11 pg/mg de peso húmero de tejido respectivamente (la diferencia no obtuvo significación estadística). Niveles de Sustancia P en la mucosa rectal de niños con soiling (11/17) no fueron diferentes de los niveles en niños constipados sin soiling (46,0+-13 vs. 50,5+-19). En niños con constipación, los niveles de Sustancia P no varían de acuerdo a la edad o la duración de síntomas. Los niveles de Sustancia P en la mucosa rectal de niños controles (sin constipación) fueron similares a aquellos que previamente observamos en adultos normales, y los niveles en niños constipados fueron intermediarios entre estos niveles normales y los de adultos constipados. Estas observaciones sugieren un problema de motilidad como factor importante en la patogenesis de la constipación crónica en niños.

Estudos imunohistoquímicos dos macrófagos na mucosa colônica em crianças na doença de Crohn e na retocolite ulcerativa inespecífica / Immunohistochemical study of macrophages in the colonic mucosa of children with Crohn's disease and ulcerative colitis

Sao quantificadas e analisadas as características histológicas dos macrófagos na mucosa colônica na doença de Crohn e na retocolite ulcerativa inespecífica. Foram estudados 12 pacientes com doença de Crohn 19 com retocolite ulcerativa inespecífica e 10 espécimes de mucosa retal representando o grupo controle, de acordo com o seguinte modelo: período I (PI) = pré-tratamento; período II (PII)= até dois anos de evoluçao e período III (PIII)= mais de dois anos de evoluçao. Os macrófagos foram identificados em mucosa colônica pelo monoclonal CD68 através do método de imunoperoxidase. A quantificaçao dos macrófagos foi feita através de análise de imagem computadorizada cromática, que expressa em porcentagem a área (mm2) ocupada pelas células CD68 positivas. A porcentagem da área ocupada pelos macrófagos se apresentava aumentada em ambas as doenças, em todos os períodos estudados, quando comparada com o grupo controle, porém sem diferença estatisticamente significante. A distribuiçao dos macrófagos dentro da mucosa do grupo controle foi subepitelial, enquanto na dos doentes atingiu toda a altura da mucosa, concentrando-se nas bases das úlceras e ao longo das fissuras. Na doença de Crohn as células CD68 positivas facilitaram a identificaçao dos microgranulomas, eventualmente despercebidos na coloraçao de hematoxilina-eosina. Embora nao houvesse diferença entre pacientes e controles quanto à área ocupada pelos macrófagos, a distribuiçao diferente pode sugerir a participaçao dos macrófagos na lesao destas duas doenças, embora nao permitam diagnóstico diferencial, possivelmente pela variabilidade dos valores. O CD68 nao identificou os diferentes estados funcionais dos macrófagos, mas a sua posiçao na mucosa sugere que com as fissuras e úlceras, sua funçao principal seria a fagocitose, e nos demais sítios a de célula apresentadora de antígenos e recrutadora de outras células inflamatórias.

Disaccharidase levels in normal epithelium of the small intestine of rats with iron-deficiency anemia

Iron-deficiency anemia is the nutritional deficiency most frequently occurring throughout the world, which manifests as a complex systemic disease involving all cells, affecting enzyme activities and modifying protein synthesis. In view of these considerations, the objective of the present study was to determine the effects of iron-deficiency anemia on disaccharidase and on the epithelial morphokinetics of the jejunal mucosa. Newly weaned male Wistar rats were divided into 4 groups of 10 animals each: C6w received a standard ration containing 36 mg elemental iron per Kg ration for 6 weeks; E6w received and iron-poor ration (5-8 mg/kg ration) for 6 weeks; C10w received an iron-rich ration (36 mg/kg ration) for 10 weeks; E10w received an iron-poor ration for 6 weeks and then an iron-rick ration (36 mg/kg) for an additional 4 weeks. Jejunal fragments were used to measure disaccharidase content and to study cell proliferation. The following results were obtained: 1) a significant reduction (P<0.001) of animal weight, hemoglobin (Hb), serum iron and total iron-binding capacity (TIBC) in groups E6w as compared to C6w; reversal of the alterations in Hb, serum iron and TIBC with iron repletion (E10w = C10w); animal weights continued to be significanly different in group E10w and C10w. 2) Sucrase and maltase levels were unchanged; total and specific lactase levels were significantly lower in group E6w and this reduction was reversed by iron repletion (E10w = C10w). 3) The cell proliferation parameters did not differ between groups. On the basis of these results, we conclude that lactase production was influenced by iron deficiency and that fact was not related to changes in cell population and proliferation in the intestinal mucosa.

Bauhinia purpurea agglutinin (BPA) binding sites in human gastrointestinal tract.

The binding of biotinylated BPA to parraffin sections of 18 normal gastrointestinal tract mucosa, 5 nonneoplastic polyps (NNP), 12 adenomas, and 59 carcinomas was studied by using avidinbiotin peroxidase complex (ABC) technique. In normal mucosa BPA appeared to bind both mucus and nonmucus glycoproteins but goblet cell mucus showed a decrease in binding and increase in binding of nonmucus glycoproteins as the cells lose their differentiation. BPA showed characteristic binding patterns in adenoma and carcinoma that differed from the pattern in normal mucosa. In normal mucosa linear binding to the apical cytoplasm in the columnar cells of the surface epithelium was observed, whereas in adenomas and carcinomas, in addition to the linear binding to the apical cytoplasm, diffuse cytoplasmic and granular deposits in the supranuclear, paranuclear or infranuclear zones were seen. Our findings suggest that BPA binding patterns in normal and neoplastic mucosa are related to the degree of cellular differentiation. In the process of malignant transformation the carbohydrate distribution undergoes progressive changes through the adenoma carcinoma sequence. These changes are related to the degree of dysplasia in adenomas and to the degree of differentiation in carcinomas.

Studies on nonesterified fatty acids in rat intestinal cell membranes.

Lipid composition of total membrane fractions prepared from scraped rat intestinal mucosa and isolated epithelial cells were compared. Membranes prepared from mucosa had four times higher nonesterified fatty acids (NEFA) as compared to the epithelial cell membranes. Cholesterol and phospholipid contents were similar in both the membrane preparations but triglyceride content was high and di- and monoglyceride were low in epithelial cell membranes as compared to the mucosal membranes. Inclusion of p-bromophenacyl bromide, a phospholipase inhibitor, in the intestinal lumen wash solution and homogenizing buffer did not reduce the NEFA content of the scraped mucosal membranes whereas inclusion of diethyl p-nitrophenyl phosphate, a lipase inhibitor reduced it by 40%. These results suggest that NEFA are normal constituent of intestinal cell membranes.