Isolation of epithelial cells, villi and crypts from small intestine of pigeons (Columba livia)

The isolation of viable enterocytes, villi and crypts from the small intestine of a feral bird (Columba livia) is important for performing physiological experiments in ecologically relevant processes of membrane transport. The effectiveness of mechanical disruption, enzymatic digestion and chelating agents were compared. The objectives were to isolate enterocytes, villi and crypts from the small intestine of young pigeons; to evaluate the viability of the isolated intestinal epithelial cells isolated; and to verify the integrity of enterocytes by biochemical features. Enzymatic and mechanical methods yielded both elongated columnar and spherical cells. With the chelating method villi and crypts were obtained. All methods produced a high yield of intestinal epithelial cells with about 50% viability. Brush border enzymes (sucrase-isomaltase and alkaline phosphatase) activities were high and, as reported in chickens, they did not differ along the intestinal villus-crypt axis. Although the three methods have good viabilities, the enzymatic technique gives the best yield in cell number, while the chelating method provides the highest populations of morphologically distinctive villi and crypts.(AU)

Cell proliferation of the ileum intestinal mucosa of diabetic rats treated with ascorbic acid

The objective of this work was to evaluate the effect of the ascorbic acid supplementation on the cellular proliferation on the ileum mucosa of diabetic rats. Fifteen 90-days rats were divided in the groups: control, diabetic and diabetic supplemented with ascorbic acid (DA). Two hours prior the sacrifice, they were injected with Vincristin. Semi-seriate histological cuts stained with HE were accomplished. About 2500 crypt cells from the intestinal mucosa were counted in order to obtain the metaphasic indexes. The height and depth of 30 villi and 30 crypts were measured for each animal, respectively. The metaphasic indexes showed no significant changes when we compared the three groups: 20.2 +/- 0.7 (control), 18 +/- 1.9 (diabetic) and 17 +/- 1.4 (DA) (p > 0.05). The values obtained from the crypts measurement were 221.2 +/- 8.5 (control), 225.3 +/- 9.5 (diabetic) and 222 +/- 34 (DA). The villi of the control, diabetic and DA animals presented the following results: 301.7 +/- 25.33, 304.8 +/- 25.63 and 322.1 +/- 45.77 respectively. The morphometric data were not different statistically (p > 0.05). Summing up, the present work showed that there was no alteration in the cellular proliferation of the ileum of diabetic-induced rats supplemented with ascorbic acid.(AU)

Clinical utility of counting intraepithelial lymphocytes in celiac disease intestinal mucosa / Utilidad clínica del recuento de linfocitos intraepiteliales en la mucosa intestinal para el diagnóstico de enfermedad celíaca

BACKGROUND/AIMS: Our aims were to establish the clinical utility of assessing the intraepithelial lymphocyte (IEL) density in intestinal biopsies from a large series of individuals and to determine the best threshold discriminating celiac disease (CD) patients and controls in two populations with different pre-test prevalence. METHODS: We prospectively performed intestinal biopsy and CD-related serology in 349 subjects undergoing upper GI endoscopy. While 116 had symptoms suggestive of a small bowel disorder (high prevalence), 233 individuals were randomly selected from patients referred to endoscopy because upper GIsymptoms (low prevalence). Diagnosis of CD was based on the concordance of classical histological features and a positive CD serology. RESULTS: While 58 patients had a newly diagnosed CD (52 in the high and 6 in the low prevalence groups), 291 subjects did not meet diagnostic criteria of the disorder. Patients had a highly significant greater IEL density than controls (p < 0.00001). Based on the ROC curve, a count of 22.8 IEL/100 epithelial cells had the highest performance for diagnosing CD in the overall population and for subjects in the high pre-test probability subgroup and 22.5% was ,he best cut-off for those diagnosed in the low risk population (area under the curves: 0.979, 0.979 and 0.993, respectively). An abnormal CD serology confirmed the diagnosis of CD in all the four patients with counts below 22.8%. CONCLUSIONS: Our study confirms that an IEL density of 22.8% is an adequate threshold to discriminate CD patients and controls in individuals irrespective of the prevalence of the disorder.(AU)

Introducción: El recuento elevado de linfocitos intraepiteliales (LIEs) es un rasgo destacado aunque inespecífico de la enteropatía de la enfermedad celíaca (EC). Un recuento mayor a 40 LIEs/100 células epiteliales ha sido considerado por mucho tiempo esencial para el diagnóstico. Sin embargo, estudios recientes con escaso número de muestras han cuestionado este valor de corte. Objetivos: Determinar el rango normal de LIEs en biopsias intestinales y establecer su capacidad diagnóstica de EC en dos poblaciones con diferente prevalencia. Métodos: Realizamos prospectivamente biopsias de duodeno distal y serología para EC en 349 pacientesconsecutivos a quienes se les realizó una videoendoscopia digestiva alta. El grupo A consistió en 116 pacientes derivados a biopsia intestinal por síntomas sugestivos de malabsorción (considerados de alta prevalencia de EC) y el grupo B consistió en 233 pacientes randomizados entre quienes fueron derivados a endoscopía alta por síntomas gastrointestinales no sugestivos de EC (baja prevalencia de EC). El diagnóstico de EC se basó en criterios histológicos clásicos y serología positiva. Resultados: Cincuenta y ocho pacientes tuvieron EC (52 en el grupo de alto riesgo y 6 en el de baja prevalencia) y 291 individuos no tuvieron criterios de la enfermedad. Los pacientes tuvieron una densidad de LIEs significativamente mayor que los controles (p<0.00001). Basado en las curvas ROC, el conteo de 22.8 LIEs/100 células epiteliales tuvo la mejor sensibilidad y especificidad para el diagnóstico de EC en la población general y entre los sujetos con alta probabilidad y 22.5% fue el mejor valor de corte para la población de bajo riesgo (áreas bajo las curvas: 0.979, 0.979 y 0.993, respectivamente). Todos aquellos pacientes celíacos con recuento de LIEs por debajo de 22% (n=4), tuvieron serología positiva para EC. El clásico valor de 40% tuvo una sensibilidad del 55%. Conclusiones: Nuestro estudio confirma que una...(AU)

Role of mast cells in gastrointestinal mucosal defense

The purpose of this review, based on studies from our laboratory as well as from others, is to summarize salient features of mast cell immunobiology and to describe their associations with gastrointestinal mucosal defense. Gastrointestinal mast cells are involved in many pathologic effects, such as food hypersensitivity. On the other hand, they also play a protective role in defense against parasitic and microbial infections. Thus, they have both positive and negative effects, but presently the mechanisms that control the balance of these various effects are poorly known. It has been suggested that stabilization of mast cells may be a key mechanism to protect the gastrointestinal tract from injury. Few molecules are known to possess both mast cell stabilizing and gastrointestinal cytoprotective activity. These include zinc compounds, sodium cromoglycate, FPL 52694, ketotifen, aloe vera, certain flavonoids such as quercetin, some sulfated proteoglycans such as chondroitin sulfate and dehydroleucodine. Dehydroleucodine, a sesquiterpene lactone isolated from Artemisia douglasiana Besser, exhibits anti-inflammatory and gastrointestinal cytoprotective action. The lactone stimulates mucus production, and inhibits histamine and serotonin release from intestinal mast cells. The lactone could act as a selective mast cell stabilizer by releasing cytoprotective factors and inhibiting pro-inflammatory mast cell mediators. (AU)

Células enteroendocrinas intraepiteliales en ciego y apéndice de conejos sensibilizados con ovoalbumina / Intraepithelial enteroendocrine cells in cecum and appendix from ovalbumin sensitized rabbits

Las células enteroendócrinas se relacionan con la motilidad, secreción y absorción de nutrientes. Actualmente está en estudio su relación con la respuesta inmune. El ciego y el apéndice del conejo, contienen en su luz bacterias y nutrientes en distintas etapas de digestión, con potencial acción antígenica. En el presente trabajo se evalúan las modificaciones en número y distribución de las células enteroendócrinas intraepiteliales en ciego y apéndice cecal de conejos sensibilizados con ovoalbúmina (OVA). Se utilizaron conejos neozelandeses adultos divididos en dos grupos, G1: grupo control (n=10). G2: sensibilizados vía intraperitoneal con OVA (n=10). Muestra de ciego y apéndice se fijaron en formol buffer al 10 por ciento marcándose las células enteroendócrinas con anticuerpo monoclonal anti-cromogranina A. En cada animal se contaron 400 campos microscópicos a mayor aumento, refiriéndose el número de células enteroendócrinas cada 100 enterocitos. En ciego se consideró epitelio superficial y criptal, mientras que en apéndice, epitelio superficial, criptas superficiales y criptas profundas. En epitelio superficial de ciego se hallaron 1,6 CEI/100 enterocitos en epitelio superficial y 3/100 en criptas. En G2 6 CEI/100 en epitelio superficial y 12/100 en criptas. La diferencia entre G1 y G2 fue estadísticamente significativa (p<0,05). En apéndice el epitelio superficial del G1 mostró 5,2/100 mientras que en G2 fue de 5.4/100 (no significativo). Las criptas superficiales evidenciaron 8,5/100 (G1) y 11.3/100 (G2) (p<0,05). En criptas profundas 4,9/100 (G1) y 8,5/100 (G2) (p<0,05). En los animales sensibilizados se detectó aumento significativo en la cantidad de células enteroendócrinas en ambos órganos. Dicho incremento podría atribuirse a un aumento de los gránulos intracitoplasmáticos o a la diferenciación de células generatrices a células APUD, como respuesta a la sensibilización. (AU)

Poblacion intraepitelial de linfocitos T en ileon de conejos sensibilizados con ovoalbumina / Intraepithelial T cell population in ileum from ovalbumin sensitized rabbit

Se caracterizó la población de linfocitos T intraepiteliales del íleon terminal de conejos neozelandeses sensibilizados com ovoalbúmina por vía intraperitoneal y desafiados por vía oral. Los nivels de IgE anti-OVA obtenidos durante la sensibilización se evaluaron mediante el test de anafilaxia cutánea pasiva (PCA) positiva para una dilución de 1/160. La respuesta anafilácticta en el intestino luego del desafío oral fue confirmada por la aparición de edema en la vellosidad y marcada degranulación mastocitaria. Se analizaron en cada tejido, células T totales (CD5+), subpoblación de linfocitos T (CD4+) y células que expresaron MHC II R-RQ (marcador de activación celular). Las células positivas se contaron en 30 vellosidades por animal, presentándose los valores como número de células positivas cada 1000 núcleos de células absortivas. El número de células CD5+ en el grupo experimental fue de 122.5/1000 núcleos. Las que expresaron marcadores de activación (MHC II R-RQ) mostraron un incremento: 32.2 en el grupo experimental 8 horas luego del desafío comparado com el grupo control: 12.6 células positivas/1000 núcleos (p<0.05) y dentro del grupo experimental entre los sacrificados a la hora y a las 8 horas (9.3 vs. 32.2). p<0.05. Se demuestra un incremento de células T activadas en el epitelio del íleon terminal en el grupo experimental sensibilizado con ovoalbúmina 8 horas después del desafío oral. (AU)

Influence of pirenzepine on colonic serotonin changes induced by short chaim fatty acid

En este trabajo hemos la influencia de un ácido graso de cadena corta (acetato) sobre el número de células enterocromafines (EC) conteniendo serotonina (5HT) a dos diferentes pH (pH 6.9, estímulo absortivo y pH 2.9 estímulo secretor) infundido durante una hora en el colon. El número de células EC disminuye significativament con una solución infundida a pH 2.9, especialmente en el ciego. La acción de la pirencepina en prevenir esta reducción demuestra que el mecanismo se efectúa parcialmente a través de receptores colinérgicos. Por parte, se observa una disminución de la liberación de 5HT, a través de un mecanismo colinérgico, como lo indica la inhibición observada con la droga antimuscarínica (AU)

Gut mucosal immunostimulation by lactic acid bacteria

The beneficial properties of lactic acid bacteria (LAB) on human health have been frequently demonstrated. The interaction of LAB with the lymphoid cells associated to the gut to activate the mucosal immune system and the mechanisms by which they can exert an adjuvant effect is still unclear, as well as if this property is common for all the LAB. We studied the influence of the oral administration of different geneous of LAB such as Lactobacillus casei, L. acidophilus, L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. plantarum, Lactococcus lactis and Streptococcus thermophilus. We determined if the LAB assayed were able to stimulate the specific, the non-specific immune response (inflammatory response), or both. We demonstrated that all the bacteria assayed were able to increase the number of IgA producing cells associated to the lamina propria of small intestine. This effect was dose dependent. The increase in IgA+ producing cells was not always correlated with an increase in the CD4+ T cell number, indicating that some LAB assayed only induced clonal expansion of B cells triggered to produce IgA. Most of them, induced an increase in the number of cells involved in the inflammatory immune response. CD8+ T cell were diminished or not affected, with exception of L. plantarum that induced an increase at low dose. This fact would mean that LAB are unable to induce cytotoxicity mechanisms. We demonstrated the importance in the selection of LAB to be used as gut mucosal adjuvant. The different behaviours observed among them on the gut mucosal immune response, specially those that induce inflammatory immune response, show that not all the LAB can be used as oral adjuvant and that the beneficial effect of them can not generalized to genous or specie. The immunoadjuvant capacity would be a property of the strain assayed.(AU)