The tankyrase inhibitor G007-LK inhibits small intestine LGR5+ stem cell proliferation without altering tissue morphology

Abstract Background The WNT pathway regulates intestinal stem cells and is frequently disrupted in intestinal adenomas. The pathway contains several potential biotargets for interference, including the poly-ADP ribosyltransferase enzymes tankyrase1 and 2. LGR5 is a known WNT pathway target gene and marker of intestinal stem cells. The LGR5+ stem cells are located in the crypt base and capable of regenerating all intestinal epithelial cell lineages. Results We treated Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice with the tankyrase inhibitor G007-LK for up to 3 weeks to assess the effect on duodenal stem cell homeostasis and on the integrity of intestinal epithelium. At the administered doses, G007-LK treatment inhibited WNT signalling in LGR5+ stem cells and reduced the number and distribution of cells traced from duodenal LGR5+ stem cells. However, the gross morphology of the duodenum remained unaltered and G007-LK-treated mice showed no signs of weight loss or any other visible morphological changes. The inhibitory effect on LGR5+ stem cell proliferation was reversible. Conclusion We show that the tankyrase inhibitor G007-LK is well tolerated by the mice, although proliferation of the LGR5+ intestinal stem cells was inhibited. Our observations suggest the presence of a tankyrase inhibitor-resistant cell population in the duodenum, able to rescue tissue integrity in the presence of G007-LK-mediated inhibition of the WNT signalling dependent LGR5+ intestinal epithelial stem cells.

Quantitative immunohistochemical analysis of duodenal ghrelin cells after sleeve gastrectomy in Wistar rats / Análise imunoistoquímica quantitativa das células de grelina duodenais após gastrectomia vertical em ratos Wistar

PURPOSE: Sleeve gastrectomy (SG) removes substantial part of the gastric mucosa, which produces ghrelin. This reduction is expected to force other organs, such as the duodenum, to compensate by increasing the number of ghrelin-producing cells. The purpose of this study was to evaluate whether this response occurs. METHODS: Twelve adult male, Wistar rats underwent SG and were reoperated 30 or 60 days after the initial surgery. During the second surgery, a segment of the duodenum was resected to count ghrelin cells using immunohistochemistry. In six animals, SG was not performed, and the duodenal segment served as a control for ghrelin cell counts. The ghrelin cell index (GCI), which is the number of ghrelin cells divided by the number of villi in each segment, was measured and used in statistical analysis by one-way analysis of variance (ANOVA). RESULTS:There were increases in the absolute numbers of cells 30 and 60 days after SG, but statistical analysis by ANOVA showed no significant difference between the groups. CONCLUSION: A compensatory increase in the number of duodenal immunopositive ghrelin cells did not occur as a response to sleeve gastrectomy.

OBJETIVO: A gastrectomia vertical (GV) remove a maior parte das células produtoras de grelina. Esta redução poderia induzir o duodeno a produzir mais células de grelina de forma compensadora. O objetivo deste trabalho foi estudar se esta compensação ocorre. MÉTODOS: Doze ratos Wistar, machos, foram submetidos à GV e reoperados 30 e 60 dias depois (grupos 30D e 60D) quando um segmento de duodeno foi ressecado para contagem de células de grelina por imunoistoquímica. Em seis animais não foi realizada a GV e um segmento de duodeno foi ressecado para contagem de células de grelina por imunoistoquímica (grupo controle). O índice de células de grelina (ICG), que é o número de células imunopositivas para grelina dividido pelo número de vilosidades do segmento foi calculado e utilizado na análise estatística pelo teste da análise de variância (ANOVA). RESULTADOS: Houve aumento no número absoluto de células 30 e 60 dias depois da gastrectomia vertical, mas a análise estatística por ANOVA não mostrou diferenças significantes entre os grupos. CONCLUSÃO: Não foi observado aumento compensatório no número de células de grelina duodenais após a gastroplastia vertical.

Distribution of serotonin-immunoreactive enterochromaffin cells in the gastrointestinal tract of the least shrew (Cryptotis parva) / Distribución de las células enterocromafines serotonina-inmunorreactiva del tracto gastrointestinal de la musaraña enana (Cryptotis parva)

Serotonin is an important neurotransmitter in the central (CNS) and peripheral (PNS) nervous systems. It is involved in a variety of physiological processes both in the gut and in the CNS. The present study examined the distribution of serotonin containing enterochromaffin cells in the gastrointestinal tract (GIT) of a vomit competent species, the least shrew. These cells were easily recognized by their globular granules stained with the H&E and serotonin immune-positive stain. The immunoreactive enterochromaffin cells (IERCs) were mainly confined to the basal portion of the glandular epithelium and were distributed throughout the shrew stomach, small and large intestine. None was found to be associated with the mucosal epithelial lining. Moreover, their distribution and count varied in different regions of the GIT suggesting specific functions for these regions. The highest concentration of IERCs was found in the colon followed by the Jejunum. Appreciable numbers of IERCs were found in the stomach especially at the esophageo-gastric junction. The gastric location of the IERCs was mainly in the basal portion of the gland. However, some IERCs were associated with the parietal cells of the stomach. Two types of IERCs were observed: One with globular secretory granules in their apical portion of the cytoplasm which were located within the glandular epithelial cells facing the glandular lumen which release their secretions into the lumen; and the second were basally located, facing the lamina propria of the mucosa. Their secretory granules were not distinct in shape, and are most probably paracrine in their mode of secretions...

La serotonina es un importante neurotransmisor del sistema nervioso central (SNC) y periférico (SNP). Está implicado en una variedad de procesos fisiológicos, tanto en el intestino y el SNC. El presente estudio examinó la distribución de la serotonina contenida en las células enterocromafines del tracto gastrointestinal (TGI) de una especie competente al vómito, la musaraña enana. Estas células se reconocen fácilmente por sus gránulos globulares teñidas con H-E y la inmuno-tinción positiva para serotonina. Las células enterocromafines inmunorreactivas (CEI) se limitan principalmente a la parte basal del epitelio glandular y se distribuyeron por todo el estómago, intestino delgado e intestino grueso de la musaraña. Ninguna célula se encontró asociada al revestimiento epitelial mucoso. Además, su distribución y el recuento varió en diferentes regiones del TGI sugiriendo funciones específicas de estas regiones. La mayor concentración de CEI se encuentran en el colon seguido por el yeyuno. Números apreciables de CEI se encontraron en el estómago, especialmente en la unión esofago-gástrica. La ubicación de las CEI gástricas fue principalmente en la porción basal de la glándula. Sin embargo, algunas CEI se asociaron con las células parietales del estómago. Dos tipos de CEI se observaron, una con gránulos secretores globulares en su porción apical del citoplasma que se encuentra dentro de las células epiteliales glandulares que enfrenta el lumen glandular que liberan sus secreciones en el lumen, y el segundo se encuentra basalmente, frente a la lámina propia de la mucosa. Sus gránulos secretores no fueron diferentes en forma, y probablemente son más paracrinas en su modo de secreción...

Effects of the ascorbic acid supplementation on NADH-diaphorase myenteric neurons in the duodenum of diabetic rats

We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate theneuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6 ) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.

Evaluation of the areas of neuronal cell bodies and nuclei in the myenteric plexus of the duodenum of adult rats

This study compared the areas of cell body and nucleus profiles of the myenteric neurons in the antimesenteric and intermediate regions of the duodenum of adult rats. Five male rats were used. The duodenum was removed and dissected to whole-mount preparations, which were stained by the Giemsa technique. The areas of cell body and nucleus profiles of 100 neurons, 50 from each region, of each animal, were assessed with image analyser. Based on the global mean +/- SD of the areas of cell body profiles, neurons were labelled as small, medium or large. It was observed that the neurons did not differ significantly in size or incidence between the antimesenteric and intermediate regions. However, the nuclei of the small and medium neurons were significantly smaller in the latter region. It is discussed that the smaller nuclear size could be related to the cell bodies being slightly smaller on this region and to a possible smaller biosynthetic activity which would influence nuclear size.

Accion del extracto de higado de ratones parcialmente hepatectomizados sobre la actividad mitotica de los enterocitos del raton joven / Action of liver extract from partially hepatectomized mice on the mitotic activity of young mouse enterocytes

En un trabajo anterior demonstramos que el plasma de retones adultos obtenido 36 horas post hepatectomía parcial, ejerce un efecto inhibitorio en la actividad mitótica de los enterocitos crípticos duodenales del ratón joven. En el presente trabajo se analiza la posibilidad de que dicho efecto se origine en algún factor del hígado regenerante. Para ello se estudia la acción del extracto de hígado de ratones adultos (90 días) obtenido 36 horas después de su hepatectomía parcial (70 por ciento), sobre la actividad mitótica de los enterocitos de ratones jóvenes, analizando 3 niveles celulares de las criptas duodenales. Se emplearon 36 ratones hembra de la cepa C3H/S de 27 días de edad la mitad de los cuales recibió, a las 16:00 horas, una inyección intraperitoneal de solución fisiológica, y restantes extracto hepático (0,01 ml/g). Lotes de 8 animales de cada grupo se sacrificaron a las 08:00/16, 12:00/20 y 16:00/24 (hora del día/horas post tratamiento) previa inyección de colchicina (2mug/g) 4 horas antes. Los resultados, expresados como metafases colchicínas por mil núcleos, demuenstran que la actividad mitótica, en los animales tratados con extracto, es significativamente menor con respecto a los testigos. El efecto inhibidor se manifesta en los niveles celulares de 1 a 4 y de 5 a 12 células de las criptas analizadas. En el nivel superior, de 13 a 20 células, no se aprecia ninguna modificación de la actividad proliferativa. Esta inhibición de la actividad mitótica de los enterocitos de las zonas basal y media de las criptas duodenales es probablemente debido a factores hepáticos difusibles.

Actividad mitotica de los enterocitos de las criptas duodenales en ratones portadores de un hepatocarcinoma / Mitotic activity of duodenal-crypt enterocytes in mice with hepatocarcinoma

La presencia de tumores injertados en el ratón generalmente provoca modificaciones de la proliferación de sus poblaciones celulares normales. En el presente trabajo se analiza la actividad mitótica (AM) de los enterocitos de las criptas duodenales del ratón injertado con el hepatocarcinoma ES12a. Se utilizan ratones C3H/S hembras de 28 días de edad, estandarizados para análisis de periodicidad, distribuidos en dos grupos: testigos y portadores del tumor injertado. Animales de ambos grupos se reparten en seis lotes [n = 6-10] que se sacrifican cada cuatro horas después de recibir una dosis de 2 mug/g de colchicina. Muestras de duodeno se fijan en formol tamponado al 10 por ciento y se procesan para la determinación de la actividad mitótica. Se controlan, por animal, los enterocitos correspondientes a 20 criptas, seccionadas longitudinalmente, registrando las células con metafase y su localización topográfica. A partir de estos valores se determinan los índices mitóticos (metafases colchicínicas x 1000 núcleos) de toda la cripta y de cada zona previamente establecida. Con estas cifras se define la X + SEM de cada lote. Las diferencias a comparar se analizan utilizando la prueba de "t"de Student. Los resultados demuestran que la presencia del tumor ES12a ejerce tanto un efecto inhibitorio sobre la AM de los enterocitos, como un desfasamiento de la curva circadiana de este parámetro de crecimiento.

Peculiar histopathological features of giardiasis in distal duodenal biopsies

Histological changes in 20 Giardia positive duodenal biopsies [Group A] were compared with 50, Giardia negative duodenal biopsies [Group B], taken during the same period. Stool examinations in Group B were negative for Giardia. Surface epithelium, villous and crypt architecture and cellular infiltrates were examined and compared between the groups. Atrophic changes in the villi were more common in Group A as compared to B [P<0.0001]. Intraepithelial neutrophil infiltration [P<0.001], infiltration of the lamina propria with plasma cells [P<0.5], and presence of eosinophils in the lamina propria [P<0.001] were significant findings in group A. Some of the changes were related to the density of Giardia colonization e.g., the goblet cell depletion [P<0.05] and the density of plasma cell infiltration in lamina propria [P<0.01]. Erosions and ulcerations were less commonly seen in group A. Thus we conclude that giardiasis manifests its peculiar features in the distal duodenal mucosa and a biopsy of this region is an important diagnostic tool for detection of this disease

Aspectos morfológicos, morfométricos e citoquímicos da parede duodenal de ratos com diferentes idades / Morphologic, morphometric and cytochemical aspects in the duodenal wall in rats with differents ages

Foram analisados as características morfológicas, morfométricas e citoquímicas ocorridas na parede duodenal de ratos com 3, 12 e 24 meses de idade. A conservaçao morfológica encontrou-se a existência de 2 padroes. O primeiro envolvendo animais de 3 a 12 meses e o segundo composto pelos animais de 24 meses. A morfometria evidenciou um aumento progressivo do número de células caliciformes, com envelhecimento do animal, e mais, o conteúdo de mucina destas células sofreu gradativa acidificaçao com decorrer da idade. Nao se observou alteraçao no índice mitótico nos 3 grupos de animais.

Aspectos morfológicos e histoquímica de mucinas das glândulas de Brünner e células caliciformes duodenais de macaco prego (Cebus apella) / Morphological aspects and mucins histochemistry of Brünner's glands and duodenal globlet cells of tufted capuchin monkey (Cebus apella)

Foram realizados estudos morfológicos e a histoquímica de mucinas das glândulas de Brünner e células caliciformes duodenais de macacos (Cebus apella) adultos, de ambos os sexos e em jejum. As glândulas duodenais säo túbulo-alveolares e seus ductos se abrem no fundo das glândulas intestinais ou diretamente na luz duodenal. As células caliciformes säo numerosas e situam-se entre outras células do epitélio intestinal. Histoquimicamente foi evidenciada presença de mucinas neutra nas glândulas de Brünner e seus ductos e, nas células caliciformes, mucinas neutras, sialomucinas e mucinas sulfatadas. tanto nas glândulas como nas células calciformes foi detectada a presença do radical alfa-amino. Os aspectos morfológicos e histoquímicos säo também comparados com aqueles já descritos em alguns primatas