First report of deep crypt secretory cells in ascending colon in chinchilla (Chinchilla lanigera): a histological and histochemical study / Primer reporte de células secretoras de criptas profundas en colon ascendente en chinchilla (Chinchilla lanigera): un estudio histológico e histoquímico

SUMMARY: The normal morphology of the colon differs among mammal species.The ascending colon presents several types of cells, responsible for carrying different functions for this organ. Among them, the mucus-secreting cells ensure the integrity of the mucosa, local defense, protection against different external factors, inflammatory diseases, cancer, etc. The ascending colon from 5 adult male chinchillas were processed for paraffin embedding and stained with three methods: Goldner's trichrome, PAS reaction, and Alcian blue staining procedure. The results showed that the structure of the ascending colon is similar to the one described in other species, i.e. mucosa, submucosa, muscularis externa, and serosa. Regarding the mucus-secreting cells present in the deeper part of the mucosal crypts (deep crypt secretory or DCS cells) turned out to be different not only morphologically from the surface goblet cells but also regarding the type of mucus synthesized. DCS cells have a multivacuolated, faintly stained cytoplasm with moderately PAS-positive reaction and intensely positive reaction to Alcian blue stain. The mean surface of DCS cells was 521.6 μm2 as compared to 437.9 μm2 for goblet cells (p<0.05). In conclusion, our study describes for the first time in chinchilla (Chinchilla lanigera) the presence of formerly known non-goblet or vacuolated cells, and recently entitled DCS cells in the glandular epithelium of the colon. The understanding of morphological peculiarities in chinchilla may serve as a good basis to understand the pathophysiology of various conditions that may arise.

RESUMEN: La morfología normal del colon es diferente entre las especies de mamíferos. El colon ascendente presenta varios tipos de células, encargadas de llevar varias funciones a este órgano. Entre ellos, las células secretoras aseguran la integridad de la mucosa, defensa local, protección frente a diferentes factores externos, enfermedades inflamatorias, cáncer, etc. Se procesaron para su inclusión en parafina el colon ascendente de 5 chinchillas machos adultos y se tiñeron con tres métodos: tricrómico de Goldner, reacción PAS y Azul de Alcian. Los resultados mostraron que la estructura de del colon ascendente es similar a la descrita en otras especies, es decir, mucosa, submucosa, muscular externa y serosa. Las células secretoras de la mucosa presente en la parte más profunda de las criptas mucosas (células secretoras de la cripta profunda o células DCS) resultaron ser diferentes morfológicamente de las células caliciformes superficiales, con citoplasma levemente teñido con reacción PAS positiva moderada y reacción intensamente positiva a Azul de Alcian. La superficie media de las células DCS fue de 521,6 μm2 en comparación con 437,9 μm2 de las células caliciformes (p <0,05). En conclusión, nuestro estudio describe por primera vez en chinchilla (Chinchilla lanigera) la presencia de células no caliciformes o vacuoladas anteriormente conocidas, y recientemente denominadas células DCS en el epitelio glandular del colon. La comprensión de las peculiaridades morfológicas de la chinchilla puede servir como una buena base para comprender la fisiopatología de las diversas afecciones.

The Adenomatous Polyposis Coli (APC) mutation spectra in different anatomical regions of the large intestine in colorectal cancer.

The enhanced incidence of colorectal cancer (CRC) in the U.S.A. has been linked to promutagens, such as heterocyclic aromatic amines, in the western diet that are produced by high temperature cooking of meat. However, a prior analysis of driver nonsense mutations in the Adenomatous Polyposis Coli (APC) gene, which is mutated in 75% of human CRC, indicated that the C·G → A·T transversions produced by this class of mutagens were not enriched but actually lower than what would be statistically anticipated. Moreover, the APC mutation patterns in the U.S.A. vs. China were indistinguishable despite differences in diet. In the present study, we have dissected the APC mutation pattern in tumors that arise in the different anatomical regions of the large intestine. The results show that the nonsense mutation pattern in APC differ in the different regions and that there is a statistically significant increase in C·G → A·T transversions in the rectum vs. the other regions, albeit, the percent of C·G → A·T mutations still remains lower than predicted based on random mutagenesis.

Ultrastructural and histochemical study on the Paneth cells in the rat ascending colon.

Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell-like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle-associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron-dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, ß-defensin-1 and -2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC-specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell-like cells rich in mucus granules to PLC-like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon.

Dietary bovine lactoferrin alters mucosal and systemic immune cell responses in neonatal piglets.

Lactoferrin (LF) is a multifunctional immune protein found at high concentrations in human milk. Herein, the effect of dietary bovine LF (bLF) on mucosal and systemic immune development was investigated. Colostrum-deprived piglets were fed formula containing 130 [control (Ctrl)], 367 (LF1), or 1300 (LF3) mg of bLF/(kg body weight · d). To provide passive immunity, sow serum was provided orally during the first 36 h of life. Blood, spleen, mesenteric lymph node (MLN), and ascending colon (Asc) contents were collected on day 7 (n = 10-14/group) and day 14 (n = 10-12/group). Immune cell populations were quantified by flow cytometry and immunoglobulins (Igs) were measured by ELISA. Additionally, immune cells were isolated from spleen and MLNs (n = 7/group) on day 7 and stimulated ex vivo with phytohemagglutinin or lipopolysaccharide (LPS) ± LF for 72 h. Secreted cytokine concentrations were quantified by multiplex assay. Lymphocyte populations [cluster determinant (CD)4, CD8, and natural killer cells] developed normally and were unaffected by dietary bLF. LF3 piglets tended to have 1.4 to 2 times more serum IgG than Ctrl piglets (P = 0.07) or LF1 piglets (P = 0.03), but IgA in Asc contents was unaffected by bLF. Asc IgA was 4 times higher on day 14 than day 7. Spleen cells from LF3 piglets produced 2 times more interleukin (IL)-10 and tumor necrosis factor (TNF)-α ex vivo than those from Ctrl or LF1 piglets. MLN cells from LF1 and LF3 piglets produced 40% more IL-10 and tended to produce 40% more IL-6 (P = 0.05) than those from Ctrl piglets. However, ex vivo bLF did not affect the cytokine response of spleen or MLN cells to LPS. In summary, dietary bLF alters the capacity of MLN and spleen immune cells to respond to stimulation, supporting a role for LF in the initiation of protective immune responses in these immunologically challenged neonates.

Relaxin exerts two opposite effects on mechanical activity and nitric oxide synthase expression in the mouse colon.

The hormone relaxin exerts a variety of functions on the smooth muscle of reproductive and nonreproductive organs, most of which occur through a nitric oxide (NO)-mediated mechanism. In the stomach and ileum, relaxin causes muscle relaxation by modulating the activity and expression of different nitric oxide synthase (NOS) isoforms region-dependently. Nothing is known on the effects of relaxin in the colon, the gut region expressing the highest number of neuronal (n) NOSß-immunoreactive neurons and mainly involved in motor symptoms of pregnancy and menstrual cycle. Therefore, we studied the effects of relaxin exposure in the mouse proximal colon in vitro evaluating muscle mechanical activity and NOS isoform expression. The functional experiments showed that relaxin decreases muscle tone and increases amplitude of spontaneous contractions; the immunohistochemical results showed that relaxin increases nNOSß and endothelial (e) NOS expression in the neurons and decreases nNOSα and eNOS expression in the smooth muscle cells (SMC). We hypothesized that, in the colon, relaxin primarily increases the activity and expression of nNOSß and eNOS in the neurons, causing a reduction of the muscle tone. The downregulation of nNOSα and eNOS expression in the SMC associated with increased muscle contractility could be the consequence of continuous exposue of these cells to the NO of neuronal origin. These findings may help to better understand the physiology of NO in the gastrointestinal tract and the role that the "relaxin-NO" system plays in motor disorders such as functional bowel disease.

Heterogeneity in enterohemorrhagic Escherichia coli O157:H7 fecal shedding in cattle is related to Escherichia coli O157:H7 colonization of the small and large intestine.

In the last decade, Escherichia coli O157:H7 have emerged as important pathogens of the gastrointestinal tract of humans. Healthy cattle have been identified as the primary reservoir, however, the factors affecting heterogeneous E. coli O157:H7 fecal shedding are not fully understood. The aim of this study was to investigate the contribution of E. coli O157:H7 colonization of small and large intestinal sites to the heterogeneity of fecal shedding in cattle. There was a dose-dependant E. coli O157:H7 E318N colonization of duodenum, jejunum, ileum, cecum, ascending colon, spiral colon, descending colon, and the rectoanal junction in vitro with no difference in E. coli O157:H7 colonization of the rectoanal junction and other intestinal sites. There were 10-100 times greater E. coli O157:H7 colonization of intestinal sites from persistent shedding cattle compared with non persistent shedding cattle. Novel pathologies were associated with E. coli O157:H7 colonization sites in the small and large intestine. The first pathology, focal petechiae, was present throughout the intestinal tract of cattle that ceased shedding E. coli O157:H7 for 5-12 weeks or in the jejunum, ileum, cecum, and ascending colon of cattle shedding E. coli O157:H7 for 4-5 months. The second pathology, mucosal hemorrhages, was present in the same sites as the focal petechiae in cattle shedding for 5 months and these hemorrhages were in the final stages of repair. Several features of these hemorrhages support this conclusion including the brown appearance, low amount of classic E. coli O157:H7 induced A/E lesions, flattened epithelium, and blunted villi. Although mucosal hemorrhages were present in the jejunum, ileum, cecum, and ascending colon in cattle shedding for 4 months, many other pathologies were also present that were indicative of hemorrhagic enteritis as evidenced by the blood red appearance of hemorrhages, severe edema, and dark red erythema. Escherichia coli O157:H7 were associated with both pathologies suggesting it is the causative agent. The current study supports a relationship between the amount of E. coli O157:H7 colonization in intestinal sites and heterogeneous fecal shedding by cattle.

Quantity and distribution of eosinophils in the gastrointestinal tract of children.

There are a lack of data on the quantity and location of eosinophils in the gastrointestinal tract of healthy individuals. Accordingly, we examined gastrointestinal biopsies obtained during endoscopic evaluation of pediatric patients. Biopsies were previously interpreted as having no diagnostic abnormality. The presence of extracellular eosinophil constituents and the quantity of eosinophils in atopic versus nonatopic individuals was determined. In the esophagus, eosinophils were present in only 2.7% of high-power fields (hpf), with a mean value of 0.03+/-0.10 eosinophils/hpf (mean+/-standard deviation) and a maximum of 1 eosinophil/hpf. Examination of the antrum and fundus revealed similar numbers of eosinophils in the lamina propria (1.9+/-1.3 and 2.1+/-2.4 eosinophils/hpf, respectively), with no eosinophils observed in the surface epithelium. In the small intestine, there were 9.6+/-5.3 (maximum, 26 eosinophils/hpf) and 12.4+/-5.4 eosinophils/hpf (maximum, 28 eosinophils/hpf) in the intercryptal lamina propria of the duodenum and ileum, respectively. The number of eosinophils in the surface epithelium and crypt epithelium was minimal. In the large intestine, the highest concentration of eosinophils was observed in the cecum (20.3+/-8.2 eosinophils/hpf; maximum, 50 eosinophils/hpf), and there were lower concentrations in the transverse and sigmoid colon (16.3+/-5.6 and 8.3+/-5.9 eosinophils/hpf, respectively). The percentage of fields demonstrating extracellular eosinophil granules in all gastrointestinal segments was 70% to 93%, and extracellular granules were most numerous at the edge of the biopsy (P<0.05). Atopic and nonatopic patients had comparable numbers of eosinophils. These data establish baseline gastrointestinal eosinophil values in pediatric patients without apparent pathological disease.

In vivo recording of colonic motility in conscious rats with deficiency of interstitial cells of Cajal, with special reference to the effects of nitric oxide on colonic motility.

BACKGROUND: We recorded in vivo colonic motility in rats with a deficiency of interstitial cells of Cajal (ICC) (Ws/Ws rats) and in wild-type rats (+/+ rats), with special reference to the effects of nitric oxide (NO) on colonic motility in both types of rats, in order to ascertain the role of ICC in colonic motility, and the relationship between NO and ICC in regard to colonic motility. METHODS: Miniature strain-gauge force transducers were sutured on the surface of the ascending and sigmoid colon of Ws/Ws rats and +/+ rats as controls. After 1 week and a fasting period of 24 h, colonic motility in +/+ and Ws/Ws rats was recorded. We also studied the effect of NO on colonic motility in both types of rats, by means of the administration of N-nitro-L-arginine methyl ester (L-NAME) or L-arginine. RESULTS: In +/+ rats, there were contractions with high amplitude and long duration in both the ascending and sigmoid colon. The number, amplitude, and duration of contractions in the ascending colon were 9.9/20 min, 6.1 g, and 22.7 s, respectively. These findings in the sigmoid colon were 5.2/20 min, 5.2 g, and 23.0 s, respectively. The number of contractions in the ascending and sigmoid colon in Ws/Ws rats (2.3 and 1.0/20 min) was significantly lower than that in +/+ rats (P < 0.05). The number of contractions in the ascending and sigmoid colon in +/+ rats (9.7 and 5.1/20 min before treatment) was significantly increased by L-NAME administration (28.7 and 13.9/40-60 min after treatment; P < 0.05), but that in Ws/Ws rats was not influenced. The number of contractions in the ascending and sigmoid colon in +/+ rats (10.2 and 5.2/20 min before treatment) was significantly decreased by L-arginine administration (3.6 and 2.1/40-60 min after treatment; P < 0.05), but that in Ws/Ws rats was not influenced. CONCLUSIONS: ICC must be related to the occurrence of a normal number of colonic contractions. NO may be involved in the inhibitory regulation of colonic motility, and the effect of NO on the occurrence of contractions appears to be mediated by ICC.

Effects of nifedipine and nickel on plateau potentials generated in submucosal interstitial cells distributed in the mouse proximal colon.

The effects of nifedipine and nickel ions (Ni2+), known inhibitors of L- and T-type voltage-gated Ca-channels respectively, were investigated on plateau potentials recorded from submucosal interstitial cells distributed in the mouse proximal colon. Plateau potentials were generated at a frequency of about 15 times min(-1) and were formed of two components. The primary component had an initial fast rate of rise with a transient potential (rate of rise, 130 mV/s; peak amplitude, 35 mV) and was followed by a secondary plateau component with a sustained potential (amplitude, 25 mV; duration, 2.6 s). Each cell from which recordings were made was injected with neurobiotin. Subsequent morphological examination with a confocal microscope indicated successful visualization of injected cells only in the presence of 18beta-glycylrhetinic acid (an inhibitor of gap junctional connections), suggesting that these cells were dye-coupled with surrounding cells. The cells injected with neurobiotin exhibited an oval-shaped cell body with bipolar processes and were distributed in the submucosal layer, suggesting that they were submucosal interstitial cells of Cajal (ICC-SM). The plateau potentials were not altered by 0.01 microM nifedipine, but were reduced in duration by 0.1 microM nifedipine, and abolished by 1 microM nifedipine. The rate of rise of plateau potentials, but not their amplitude, was reduced by Ni2+ (> 10 microM), with no significant alteration of the membrane potential. In the presence of 100 microM Ni2+, the plateau potentials were changed to a triangular form. Thus, the plateau potentials were formed by two types of voltage-gated channel current: the initial component was produced by a Ni2+-sensitive channel current and the plateau component by a nifedipine-sensitive current. The possible involvement of two different types of voltage-gated Ca2+-channels in the generation of submucosal pacemaker potentials was discussed.