Primary cultures of mouse small intestinal epithelial cells using the dissociating enzyme type I collagenase and hyaluronidase

The epithelium is a highly dynamic system, which plays a crucial role in the homeostasis of the intestinal tract. However, studies on the physiological and pathophysiological functions of intestinal epithelial cells (IECs) have been hampered due to lack of normal epithelial cell models. In the present study, we established a reproducible method for primary culture of mouse IECs, which were isolated from the viable small intestinal crypts of murine fetuses (on embryonic day 19), using type I collagenase and hyaluronidase in a short span of time (≤20 min). With this method, continuously growing mouse IECs, which can be subcultured over a number of passages, were obtained. The obtained cell lines formed a tight cobblestone-like arrangement, displayed long and slender microvilli, expressed characteristic markers (cytokeratin 18 and Notch-1), and generated increasing transepithelial electrical resistance and low paracellular permeability during in vitro culture. The cells also had enzymatic activities of alkaline phosphatase and sucrase-isomaltase, and secreted various cytokines (IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1), responding to the stimulation of Escherichia coli. These results show that the primary-cultured mouse IECs obtained by the method established here had the morphological and immunological characteristics of IECs. This culture system can be a beneficial in vitro model for studies on mucosal immunology and toxicology.

Morphological and Immunohistochemical Identification of Villous M Cells in the Small Intestine of Newborn Piglets / Identificación Morfológica e Inmunohistoquímica de los Epiteliocitos Microplegados de las Vellosidades en el Intestino Delgado de Lechones Recién Nacidos

Microfold (M) cells act as antigen-sampling sites for initiating antigen specific mucosal immune responses, but they may also provide a gateway for enteropathogen entry. In this study we demonstrated villous M cells by morphological and immunohistochemical methods to be present in the three regions of the small intestine from newborn piglets. Immunohistochemical analysis, using anti- cytokeratin 18 (CK18) primary antibodies, showed a gradually decreased density of M cells from the lower crypt epithelium to the upper villous epithelium. The proportion of villous M cells was greater in the ileum than in the duodenum or the mid-jejunum. Ultrastructural observation revealed that villous M cells were mainly columnar in shape in the duodenum and the mid-jejunum, and appeared as more pocket-like structure in the ileum. These villous M cells exhibited short and irregular microvilli, rich vesicles and reduced glycocalyx, but lacked the lymphocyte-containing basolateral invagination. Our results support evidence that M cells can develop in the small intestinal villous epithelium of newborn piglets, implying that villous M cells may begin developing in the pig's small intestine during fetal stages, which depends neither on the influence of the mucosal lymphoid tissue nor the antigen from the intestinal lumen stimulation. In addition, the variable morphology and heterogeneity distribution of villous M cells in the three regions of the small intestine may be indicative of its different functional properties. This information extent our understanding of the diversity of M cells and provides important basic knowledge for further research on the actual role of villous M cells in neonate.

Los epiteliocitos microplegados (células M) actúan como receptores de antígeno para iniciar la respuesta inmune específica de las mucosas, pero también pueden proporcionar una puerta de entrada para enteropatógenos. En este estudio, se demostró por métodos morfológicos e inmunohistoquímicos que los epiteliocitos microplegados de las vellosidades están presentes en las tres regiones del intestino delgado de lechones recién nacidos. Se utilizaron anticuerpos primarios de citoqueratina 18 (CK18) para el análisis inmunohistoquímico, el cual mostró una disminución gradual de la densidad de los epiteliocitos microplegados desde el epitelio de las criptas inferiores hasta el epitelio de las vellosidades superiores. La proporción de los epiteliocitos microplegados, fue mayor en el íleon que el duodeno o yeyuno medio. La observación ultraestructural reveló que los epiteliocitos microplegados fueron principalmente de forma columnar en el duodeno y el yeyuno medio. Además, mostraron microvellosidades cortas e irregulares, muchas vesículas y glucocáliz reducidos, pero carecían de invaginaciones basolaterales contenedoras de linfocitos. Nuestros resultados apoyan la evidencia de que los epiteliocitos microplegados pueden desarrollarse en el epitelio de las vellosidades intestinales de los lechones recién nacidos, lo que implica que estas células pueden comenzar a desarrollarse en el intestino delgado del cerdo durante las etapas fetales, y no dependen ni de la influencia del tejido linfoide de las mucosas ni del antígeno para la estimulación del lumen intestinal. Además, la morfología y heterogeneidad de distribución de los epiteliocitos microplegados en las tres regiones del intestino delgado pueden ser indicativas de sus diferentes propiedades funcionales. Esta información mejora nuestra comprensión de la diversidad de los epiteliocitos microplegados y proporciona conocimientos básicos importantes para la investigación sobre el papel de los epiteliocitos microplegados en las vellosidades del neonato.

Evaluation of a canine small intestinal submucosal xenograft and polypropylene mesh as bioscaffolds in an abdominal full-thickness resection model of growing rats

We evaluated the biological scaffold properties of canine small intestinal submucosa (SIS) compared to a those of polypropylene mesh in growing rats with full-thickness abdominal defects. SIS is used to repair musculoskeletal tissue while promoting cell migration and supporting tissue regeneration. Polypropylene mesh is a non-resorbable synthetic material that can endure mechanical tension. Canine SIS was obtained from donor German shepherds, and its porous collagen fiber structure was identified using scanning electron microscopy (SEM). A 2.50-cm2 section of canine SIS (SIS group) or mesh (mesh group) was implanted in Sprague-Dawley rats. At 1, 2, 4, 12, and 24 weeks after surgery, the implants were histopathologically examined and tensile load was tested. One month after surgery, CD68+ macrophage numbers in the SIS group were increased, but the number of CD8+ T cells in this group declined more rapidly than that in rats treated with the mesh. In the SIS group, few adhesions and well-developed autologous abdominal muscle infiltration into the SIS collagen fibers were observed. No significant differences in the tensile load test results were found between the SIS and mesh groups at 24 weeks. Canine SIS may therefore be a suitable replacement for artificial biological scaffolds in small animals.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60 percent of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

Mucosal mast cell responses in rats (Rattus norvegicus) experimentally infected with Centrocestus caninus.

Mucosal mast cell (MMC) responses and worm recovery rates in rats experimentally infected with Centrocestus caninus were investigated. Metacercariae of C. caninus, procured from goldfish, Carassius auratus, were orally administered to twenty-five male rats (300 metacercariae each rat). The infected rats were sacrificed on days 3, 7, 14, 21 and 28 post-infection (PI) along with the control rats. Worm recovery was performed from each part of small intestine. To investigate MMC, duodenal, jejunal and ileal paraffinized-tissue sections were processed and stained with 1% alcian blue and 0.5% safranin-O. The average worm recovery rates were 42.8, 37.7, 21.2, 12.5 and 3.7% on days 3, 7, 14, 21 and 28 PI, respectively. The majority of the worms (98.9%) were collected from the duodenum and jejunum. The MMC numbers in the infected rats were significantly higher than those of the controls (p<0.05). A peak level was observed on days 14 PI and the numbers gradually decreased thereafter. The results reveal that MMC plays an important role in the expulsion of C. caninus from the host intestine. A more precise description of the role the MMC plays in helminth expulsion is still needed to understand the mechanism of host defense against intestinal helminthic infection, along with other effector cells, such as goblet cells.

Tumores del estroma gastrointestinal (GIST): revisión de la literatura / Gastrointestinal stromal tumors (gist): literature review

Los tumores del estroma gastrointestinal representan un tipo infrecuente de tumor de origen mesenquimático, con origen en las células intersticiales de Cajal. Se caracterizan por expresar un receptor de membrana mutante con actividad tirosina quinasa anormal (c-kit CD117) que condiciona su activación permanente y una proliferación celular no controlada. Pueden encontrarse a cualquier nivel del tracto gastrointestinal, pero son más frecuentes en estómago e intestino delgado. Frecuentemente cursan de forma asintomática, constituyendo un hallazgo endoscópico o radiológico. La enfermedad localizada habitualmente es de buen pronóstico tras su resección quirúrgica, mientras que la sobrevida es baja en etapas avanzadas, con escasa respuesta a la quimioterapia convencional. El surgimiento del imatinib, un inhibidor de la tirosina quinasa, ha representado un verdadero hito, dado su eficacia en el control de la enfermedad irresecable o metastásica, permitiendo un importante aumento en la sobrevida de estos pacientes.

Feeding manipulation elicits different proliferative responses in the gastrointestinal tract of suckling and weanling rats

Food deprivation has been found to stimulate cell proliferation in the gastric mucosa of suckling rats, whereas the weanling period has been reported to be unresponsive in terms of proliferative activity. In the present study we analyze regional differences in the effect of milk or food deprivation on cell proliferation of the epithelia of the esophagus and of five segments of small intestine in suckling, weanling and newly weaned Wistar rats of both sexes. DNA synthesis was determined using tritiated thymidine to obtain labeling indices (LI); crypt depth and villus height were also determined. Milk deprivation decreased LI by 50 percent in the esophagus (from 15 to 8.35 percent) and small intestine (from 40 to 20 percent) of 14-days-old rats. In 18-days-old rats, milk and food deprivation decreased LI in the esophagus (from 13 to 5 percent) and in the distal segments of the small intestine (from 36-40 to 24-32 percent). In contrast, the LI of the epithelia of the esophagus (5 percent) and of all small intestine segments (around 30 percent) of 22-day-old rats were not modified by food deprivation. Crypt depth did not change after treatment (80 to 120 mum in 14- and 22-day-old rats, respectively). Villus height decreased in some small intestine segments of unfed 14- (from 400 to 300 mum) and 18-day-old rats (from 480 to 360 mum). The results show that, contrary to the stomach response, milk deprivation inhibited cell proliferation in the esophagus and small intestine of suckling rats, demonstrating the regional variability of each segment of the gastrointestinal tract in suckling rats. In newly weaned rats, food deprivation did not alter the proliferation of these epithelia, similarly to the stomach, indicating that weanling is a period marked by the insensitivity of gastrointestinal epithelia to dietary alterations.

Isolation and characterization of intraepithelial lymphocytes from rat small intestine.

A modified procedure for isolation of intraepithelial lymphocytes (IEL) from rat small intestine was developed which makes use of a protease inhibitor-phenyl methyl sulfonyl fluoride (PMSF) in the isolation medium. Yield and viability of IEL isolated in presence of PMSF were significantly higher as compared to those isolated in absence of PMSF. IEL isolated in presence PMSF demonstrated a significantly higher proliferative response to concanavalin A (con A) compared to those isolated in absence of PMSF. The natural killer cell activity of IEL population isolated in presence of PMSF using YAC-1 lymphoma cells as targets was also significantly higher compared to those isolated in absence of PMSF. Phenotypic analysis using monoclonal antibodies specific for rat T lymphocyte subsets revealed that majority of IEL were cytotoxic/suppressor phenotype with a minor subset of helper/inducer phenotype. The method described yielded a population of IEL which is suitable for further functional studies in vitro.

Proliferaçäo celular do epitélio intestinal: mecanismos de adaptaçäo e controle após ressecçäo extensa do intestino delgado / Intestinal epithelium cell proliferation: mechanisms of adaptation and control after extensive resection of small intestine

O epitélio da mucosa do intestino delgado é altamente dinâmico com diversidade de populaçoes celulares que requerem complexa integraçao de processos de proliferaçao e diferenciaçao celular. Os conceitos globais de organizaçao cinética da mucosa do intestino delgado estao bem entendidos, embora muitos aspectos ainda necessitem ser esclarecidos. Diversos fatores estao envolvidos no controle da proliferaçao epitelial da mucosa intestinal, entre eles os fatores luminais e humorais, a matriz extra-celular e os mecanismos intra-celulares. Esta revisao discute a proliferaçao celular epitelial no intestino delgado e o papel dos mecanismos de seu controle após ressecçao intestinal extensa.

Hormonas gastrointestinales / Gastrointestinal hormones

En los últimos años se ha incrementado el conocimiento acerca del papel fisiológico y fisiopatogénico de algunas hormonas gastro-intestinales que ya eran conocidas desde hace algún tiempo. Asimismo, se han descubierto nuevos péptidos biológicamente activos en el tracto digestivo. Algunas hormonas que antes se pensaba que estaban restringidas al aparato digestivo se han localizado en el sistema nervioso central y algunos péptidos localizados en el sistema nervioso central y algunos péptidos localizados en las terminaciones nerviosas de los plexos del tubo digestivo son activos participantes de los procesos fisiológicos digestivos. Esto ha hecho el panorama más complejo, de tal manera que podemos hablar en forma genérica de "endocrinología gastrointestinal". En el presente artículo, discutiremos acerca de los conceptos generales de la fisiopatología hormonal gastro-intestinal y, en forma individual, del papel que juegan algunas de esas hormonas sobre los procesos biológicos normales y anormales, enfatizando la importancia del diagnóstico bioquímico de dichas alteraciones.

Efeito da fitohemaglutinina na proliferaçäo celular do epitélio do intestino delgado de ratos / Effect of phytohemagglutinins in cell proliferation of the epithelium of the small intestine of rats

Vinte ratos albinos, foram divididos em dois grupos de dez: grupo e, que recebeu dieta sólida contendo 4% de fitohemaglutinina (FHG) ativa e água "ad libitum" e grupo P controle pareado iso-calórico, que recebeu a mesma dieta sólida porém com fitohemaglutinina inativada pelo calor e água "ad livitum". Diariamente os animais foram pesados, as quantidades de raçäo e água foram anotados. No 14§ de experimento, os animais foram sacrificados e colhidos fragmentos de jejuno e íleo para estudo morfocinético. Os resultados mostraram que: a ingestäo hídrica foi semelhante em ambos os grupos estudados e o grupo E ganhou menos peso corporal do que o grupo P. As populaçöes de enterócitos das vilosidades jejunais do grupo E, foram menores em termos estatísticos quando comparadas ao grupo P, e o contrário se deu no íleo, onde o grupo E foi maior que o P. A altura da vilosidade do grupo E foi semelhante ao do grupo P, mas o íleo do grupo E foi maior do que o do grupo P. As profundidades, as populaçöes e as taxas de produçäo celular nas criptas jejunais do grupo E foram significativamente maiores que no grupo P. Concluindo, os achados no presente estudo fornecem evidências de que a ingestäo de fitohemaglutinina lesa a mucosa jejunal proxima, diminuindo a populaçäo celular da vilosidade e estimulando a hiperplasia da cripta, desenvolvendo adaptaçäo localizada. Este modelo adaptativo é semelhante ao que acontece na doença celíaca. Esta lesäo proximal estimula a hiperplasia da unidade cripta-vilosidade do epitéileal, desenvolvendo adaptaçäo à distância. Estas adaptaçöes aconteceram em animais que ingeriram fitohemaglutinina, mesmo na vigência de desnutriçäo multicarencial

Effect of raw winged bean [Psophocarpus tetragonolobus (L.) DC] tuber lectin on gastrointestinal tract of growing rats.

The fraction containing high hemagglutinating activity was prepared from raw winged bean tubers and orally administered to growing rats. The food intake and body weights of these rats decreased as the level of lectin increased and significant lectin activity was detected in the faeces extracted from these rats which is anti-genically similar to the native lectin preparation. Microscopic examination has revealed morphological changes in the intestinal epithelial cells. The binding action of the lectin to the mucosal epithelia of the gastrointestinal tract is indicative of the deleterious effects caused by the winged bean tuber lectin.

effect of methotrexate [MTX] on the small intestine of the mouse: V-cytochemical study

Cytochemical analysis of the normal mouse intestine reveals the presence of a high concentration of proteins, RNA and DNA, carbohydrates and lipids. The Golgi apparatus zone gives a PAS-positive reaction confirming its role in the secretion of mucopolysaccharides, particularly in goblet cells and cell membranes. The brush border is strongly PAS-positve. The crypt lumen contains diffuse acid mucopolysaccharides and glycogen granules. After MTX-treatment, all cytochemical reactions show a decrease in the amount of metabolites studied [total proteins, nucleic acids, carbohydrates, fats]. Of all these substances, proteins show the most detectable decrease in amount than control. The decrease is more pronounced in crypts than in villi. We conclude that MTX produces an imbalance in the absorptive power of cells and/or in the coordinate synthesis of DNA, RNA and protein resulting in a suppression in their synthesis. The amount of these substances shows variability [decrease then increase] depending on the physiological state of the cells. After recovery, the DNA content increased and consequently RNA and proteins also increased and reached the control levels

effect of methotrexate on the small intestine of the mouse: VI-mitotic activity

The normal jejunal crypt epithelium shows numerous mitoses. Mitotic counts show that the percentage of crypt cells undergoing mitosis is in the average of 2.92% and goblet cell counts about 6%. Newly formed cells resulting from mitosis in crypts of Lieberkuhn migrate along the villi and are extruded at the tips of villi. MTX induced marked reduction of crypt mitoses in all experimental groups associated with occasional abnormal mitotic figures [chromosomal abnormalities]. MTX inhibition of mitosis most probably occurs in the S-phase and not in the metaphase stage. The reduction in mitotic counts is evident within 2 h of a single sublethal dose, and remains depressed until 6 h. During the period of mitotic reduction, many small spherical DNA-containing inclusion bodies appear in the cytoplasm of crypt cells, indicating the early death of these cells. After 12 h in GI and after 5 weekly doses in GIV, mitosis increases to peak values which are greater than in controls. The existence of waves of increased activity suggests, synchronization in a portion of the crypt cell population which is not destroyed by MTX. There is no definite correlation between the number of mitoses and the cumulative doses of MTX. The duration of mitotic inhibition correlates with the duration of MTX action on DNA synthesis, and is dose and timeinterval dependent. This inhibition, though sometimes marked, is transient, and the epithelial defects are rapidly repaired following resumption of DNA synthesis, after cessation of treatment

Quantificaçåo de plasmócitos produtores de IgA, IgM e IgG em mucosa de intestito delgado de crianças- padronizaçåo do método / Density of IgA, IgM and IgG-producing cells of small bowell mucosa of children

A densidade numérica de plasmócitos produtores de IgA, IgM e IgG, na mucosa do intestino delgado, foi obtido através do estudo das biópsias realizadas em 6 crianças com idade de 15 meses a 7 anos. Duas delas, com imunoglobulina A sérica abaixo dos valores normais para a idade e sem história de diarréia. As outras 4, com diarréia crônica e dosagem de IgA sérica normal. Os fragmentos obtidos por biópsia com a cápsula de Crosby, tamanho pediátrico, foram fixados em formol a 5 por cento, ph 7,0 e lavados em sacarose a 30 por cento e, a seguir, congelados em hexano-nitrogênio líquido. Cuidados especiais foram tomados durante a orientaçåo e inclusåo do material para se conseguirem cortes perfeitamente perpendiculares à muscular da mucosa. Os cortes foram obtidos em criostato e processados para a demonstraçåo de plasmócitos produtores de IgA, IgM e IgG, pela técnica de imunoflorescência direta (Nairn, 1969; Fry & Wilkinson, 1963). A determinaçåo do número de cada categoria de plasmócitos foi conseguida através da aplicaçåo da fórmula morfométrica: N= 2n. Vp/A (id + 2t) do método II de Aherne (1967) para contagem de células. Para cada tipo de célula contendo imunoglobulina A, M e G foram efetuadas contagens, segundo dois procedimentos. No primeiro, as contagens individuais de IgA, IgM e IgG de cada caso foram efetuadas, em 200 campos, procedendo-se do seguinte modo: contava-se o número de células em campos microscópicos sucessivos, que eram obtidos pela varredura dos cortes da base para o topo (isto é da regiåo da muscular da mucosa até o topo do vilo). A faixa lateral seguinte do corte, a ser varrida, era medida a alguma distância da primeira, partindo a varredura do topo para a base. Neste procedimento nåo se anotava a procedência do campo, se da regiåo superior, média ou inferior da mucosa. Para cada caso, os resultados das contagens individuais de plasmócitos IgA, IgM e IgG apresentaram desvio padråo de elevado valor. Assim, para medidas feitas com 200 campos para células produtoras de IgA, o desvio padråo variava em cada um dos seis casos de dois terços do valor da média até um valor próximo da mesma. Através de testes estatísticos adequados, evidenciou-se que valores confiáveis do número de células por mm3 de lâmina própria de mucosa seriam obtidos somente com contagens de 100 a 200 campos microscópicos. No segundo procedimento de amostragem, planejado após a análise dos dados anteriormente obtidos, os cortes de mucosa foram arbitrariamente divididos em 3 segmentos: superior, médio e inferior. O segmento superior ia do topo de vilosidade até a extremidade inferior da regiåo que representava os dois terços superiores do vilo, estendendo-se o médio do limite superior assinalado até a raiz das glândulas intestinais de Lieberkühn e o inferior, do limite inferior acima até a muscular da mucosa. Verificou-se em todos os casos que a concentraçåo de células produtoras de IgA por mm3 de lâmina própria era menor na regiåo superior que na regiåo média, ocorrendo a maior concentraçåo de plasmócitos na regiåo inferior. Para células IgM e IgG, as maiores concentraçoes ocorriam em segmentos médios e inferior, havendo menor concentraçåo celular também ao nível do topo de vilosidade. O desvio padråo das contagens feitas neste segundo procedimento representava aproximadamente 1/5 1 1/13 avos do valor da média. Através de testes adequados, mostrou-se que resultados consistentes do número de plasmócitos produtores de IgA, IgM e IGG podiam ser obtidos em cada caso com contagens apenas de 10 campos da regiåo superior, 10 campos da regiåo média e 10 campos da regiåo inferior. Deve-se destacar que, apesar de se ter detectado, uma variaçåo segmentar da base para o topo, na concentraçåo numérica dos diferentes tipos de plasmócitos em crianças com deficiência parcial de IgA e naquelas com diarréia sem esta deficiência, tal fato nåo deve ser interpretado como indicativo de que este padråo de estratificaçåo tenha necessariamente ocorrência geral.