Effects of deoxycorticosterone acetate on the electrical properties of rat large intestine: segmental differences

The functional heterogeneity of different segments of the rat large intestine was investigated by means of transepithelial potential difference (PD), short-circuit current (Isc) and transepithelial resistance (Rt) measurements in control rats and after deoxycorticosterone acetate (DOCA) pretreatment. Rt and PD were low in caecum and proximal colon but higher in the distal colon and rectum. Isc was highest in the distal colon, lower in the caecum, proximal colon, and rectum. None of the electrical properties was sensitive to amiloride in control conditions. DOCA increased PD and Isc in the caecum, distal colon and rectum but had no effect in the proximal colon. The increase of the Isc after DOCA in the distal colon and rectum was reached by induction of the amiloride-sensitive Isc associated with reduction of the amiloride-insensitive Isc. The effect of DOCA could be completely prevented by concurrent spironolactone treatment. The results suggest that the epithelia of the proximal parts of the large intestine are "leaky" whereas those of the distal colon and rectum are relatively "tight". It is concluded that there is a marked quantitative and qualitative segmental heterogeneity along the rat large intestine.     

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.     

Rates of passage of digesta and water absorption along the larg intestines of sheep, cows and pigs.

In 5 cows, the mean length of the large intestine was only slightly greater than that of 21 sheep and 3 pigs. At about one-fifth of the way along the large intestine of the sheep and cows, corresponding to the end of the proximal colon and start of the spiral colon, there were marked reductions in the amount of digesta present and in the lumen diameter. In pigs these parameters decreased gradually along the large intestine. In all parts of the large intestine, the cows had more water in the digesta than did the sheep or the pigs. Nevertheless, the decrease in water content between the caecum and the rectum was similar for the cows and the sheep but less in the pigs. The rate of passage of digesta increased gradually along the large intestine of the pigs, but in the cows and sheep the rate was least in the caecum and proximal colon and greatest in the spiral colon. The total retention time for digesta in the large intestine was approximately 30, 20, and 9 h in the pigs, sheep, and cows respectively. The rate of water absorption from the large intestine was most rapid in the cows and slowest in the pigs. Differences in faecal water content between the species were not due to differences in retention times in the large intestine.     

Feeding of soy protein isolate to rats during pregnancy and lactation suppresses formation of aberrant crypt foci in their progeny's colons: interaction of diet with fetal alcohol exposure

Soy protein isolate (SPI) in the diet may inhibit colon tumorigenesis. We examined azoxymethane (AOM)-induced aberrant crypt foci (ACF) in male rats in relation to lifetime, pre-weaning, or post-weaning dietary exposure to SPI and also within the context of fetal alcohol exposure. Pregnant Sprague Dawley rats were fed AIN-93G diets containing casein (20%, the control diet) or SPI (20%) as the sole protein source starting on gestation day 4 (GD 4). Progeny were weaned on postnatal day (PND) 21 to the same diet as their dams and were fed this diet until termination of the experiment at PND 138. Rats received AOM on PND 89 and 96. Lifetime (GD 4 to PND 138) feeding of SPI led to reduced frequency of ACF with 4 or more crypts in the distal colon. Progeny of dams fed SPI only during pregnancy and lactation or progeny fed SPI only after weaning exhibited similarly reduced frequency of large ACF in distal colon. Number of epithelial cells, in the distal colon, undergoing apoptosis was unaffected by diet. SPI reduced weight gain and adiposity, but these were not correlated with fewer numbers of large ACF. Lifetime SPI exposure similarly inhibited development of large ACF in Sprague Dawley rats whose dams were exposed to ethanol during pregnancy. In summary, feeding of SPI to rat dams during pregnancy and lactation suppresses numbers of large ACF in their progeny, implying a long-term or permanent change elicited by the maternal diet. Moreover, results support the use of ACF as an intermediate endpoint for elucidating effects of SPI and its biochemical constituents in colon cancer prevention in rats.     

Relationship between the spike activities of the small and large intestines

Experiments were performed using chronically implanted electrodes on the dog smooth muscle wall of the stomach and of the small and large intestines. Electrical activity of the muscle wall was recorded before and after feeding. When reaching the terminal ileum the active part of the migrating myoelectrical complex (MMC) continuously induced bursts of spike potentials superimposed on the slow waves. This electrical activity spread to the ascending colon. We also showed the existence of a spike activity on the terminal ileum independent of the MMC (appearing during the phase 1) and propagating to the colon. A relationship between the spike activities of the small and large intestines was also present after feeding. Beside the well-known gastro-colic reflex, we observed an increase in the spike activity of the terminal ileum and ascending colon between the 4th-5th hours after feeding. This probably corresponds to the arrival of the first portions of contents, evacuated from the arrival of the first portions of contents, evacuated from the stomach, and of the last portions of small intestinal contents. In conclusion, there is a relationship between the spike activities of the small and large intestines in starving animals and after feeding, and the terminal ileum plays a substantial role in this relationship.     

Zinc deficiency is associated with suppression of colonocyte proliferation in the distal large bowel of rats

As zinc status may influence susceptibility to colon cancer, we examined the effect of dietary zinc deficiency on the proliferation of epithelial cells (colonocytes) in the large bowel of rats. When compared to feed-restricted rats, those with zinc deficiency showed a significant reduction in proliferation in the distal colon as assessed by accumulated metaphase arrest and crypt cell production rates in vivo. Zinc deficiency had no apparent effect on thymidine kinase activity in colonocytes but was accompanied by minor changes in fecal mass and fecal pH. In rats, zinc deficiency is associated with a reduction in the rate of proliferation of colonocytes in the distal colon.     

V gamma 9V delta 2 T cell response to colon carcinoma cells

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vgamma9Vdelta2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-alpha and IFN-gamma secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vgamma9Vdelta2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vgamma9Vdelta2 T cells of various origins, and Vgamma9Vdelta2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vgamma9Vdelta2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vgamma9Vdelta2 T cell agonists in immunotherapies targeting colon tumors.     

Dimensions of the intestine, diet and faecal water loss in some African antelope

Measurements of the intestine of 16 species of antelope were allometrically analysed. Moisture content of the digesta showed greatest interspecific variation in the spiral and distal colon. Small intestine length showed positive allometry, its circumference showed negative allometry, resulting in a surface area which was isometric or related to metabolic rate. Dimensions of the villi were non-allometric. Grazers had lower surface areas of the small intestine than intermediates/concentrate selectors owing to smaller circumferences. In the large intestine, the dimensions of the distal colon showed the greatest interspecific variation. Lengths of the large intestine showed positive allometry except for the caecum which was isometric. Circumferences and areas of the large intestine were isometric or related to metabolic rate. Species producing dry dung had larger lengths and areas of the large intestine but a smaller circumference of the proximal and spiral colon and also showed greater drying of digesta per unit area than those producing wetter dung. Grazers had smaller lengths, circumferences and areas of the large intestine than intermediates/concentrate selectors.     

Prevention of colon carcinogenesis and carcinoma metastasis by orally administered bovine lactoferrin in animals

Bovine lactoferrin (bLF), a milk protein known to have bacteriostatic properties was examined for its preventive effects on colon and other organ carcinogenesis and experimental metastasis. (Experiment 1) The influence on colon carcinogenesis was investigated in male rats treated with azoxymethane (AOM), then received 2 or 0.2% bLF for 36 weeks. Significant reduction in the incidence (27% and 46% of the control, respectively) and number of adenocarcinomas of the large intestine was observed. (Experiment 2) In BALB/c mice bearing subcutaneous (s.c.) implants of colon carcinoma 26 (Co 26Lu). bLF demonstrated significant inhibition of spontaneous lung metastasis (approximately 43% of the control). Number of cytotoxic asialoGM1+ and CD8+ cells in white blood cells increased (171% and 122% of control, respectively) after treatment. Results of those experiments indicate that bLF remarkably prevents colon carcinogenesis and lung metastasis of colon carcinoma cells, possibly due to increasing cytotoxic cells in the peripheral blood.     

Colonic epithelium reactive monoclonal antibodies

Summary We have produced a small library of colonic mucosa and colorectal carcinoma reactive monoclonal antibodies (MoAbs) by immunizations with extracts of human colon cancer tissue and a human colon cancer cell line. Hybridoma supernatants were tested on (normal and neoplastic) human tissues by immunoperoxidase methods to evaluate organ or tissue specificity. Initial biochemical characterization of the target antigens was performed by gelpermeation chromatography, Western blotting and competition assays.Based upon the immunoreactivity patterns and the characteristics of the antigen four groups of MoAbs could be distinguished. The first group concerns the antibodies PAR-LAM 3, 9 and 10. These antibodies react with an 87 kDa protein moiety in high molecular weight (2–5×10⁶ Da) glycoproteins. In intestinal and colon mucosa these antibodies showed diffuse binding with goblet cells. In colon carcinoma decreased reactivity with these MoAbs was found.The second group consists of antibodies PARLAM 8, 12 and 13. These antibodies react with large (>5×10⁶ Da) glycoproteins, most likely with carbohydrate epitopes. By immunohistochemistry in normal colon mucosa the antibodies all show granular supranuclear reactivity with goblet cells. These antibodies show increased reactivity with colon adenomas and adenocarcinomas.A third group is formed by PARLAM 2, which also reacts with a large (>5×10⁶ Da) glycoprotein, showing a granular distribution in goblet cells. In colon carcinomas more extensive expression is found than in normal colonic mucosa. Finally, the fourth group consists of PARLAM 11, which also reacts with a large (>5×10⁶ Da) glycoprotein, located in the brush border of colonic columnar cells.These antibodies might be useful tools for the analysis of the expression of mucin related glycoproteins in normal, preneoplastic and neoplastic colon mucosa.Supported by grant RL 82-1 of the Netherlands Cancer Foundation, K.W.F.     

Effect of increased intake of dietary animal fat and fat energy on oxidative damage, mutation frequency, DNA adduct level and DNA repair in rat colon and liver

The effect of high dietary intake of animal fat and an increased fat energy intake on colon and liver genotoxicity and on markers of oxidative damage and antioxidative defence in colon, liver and plasma was investigated in Big Blue rats. The rats were fed ad libitum with semi-synthetic feed supplemented with 0, 3, 10 or 30% w/w lard. After 3 weeks, the mutation frequency, DNA repair gene expression, DNA damage and oxidative markers were determined in liver, colon and plasma. The mutation frequency of the lambda gene cII did not increase with increased fat or energy intake in colon or liver. The DNA-adduct level measured by 32P-postlabelling decreased in both liver and colon with increased fat intake. In liver, this was accompanied by a 2-fold increase of the mRNA level of nucleotide excision repair (NER) gene ERCC1. In colon, a non-statistically significant increase in the ERCC1 mRNA levels was observed. Intake of lard fat resulted in increased ascorbate synthesis and affected markers of oxidative damage to proteins in liver cytosol, but not in plasma. The effect was observed at all lard doses and was not dose-dependent. However, no evidence of increased oxidative DNA damage was found in liver, colon, or urine. Thus, lard intake at the expense of other nutrients and a large increase in the fat energy consumption affects the redox state locally in the liver cytosol, but does not induce DNA-damage, systemic oxidative stress or a dose-dependent increase in mutation frequency in rat colon or liver.     

UNC5B receptor deletion exacerbates DSS‐induced colitis in mice by increasing epithelial cell apoptosis

The netrin-1 administration or overexpression is known to protect colon from acute colitis. However, the receptor that mediates netrin-1 protective activities in the colon during colitis remains unknown. We tested the hypothesis that UNC5B receptor is a critical mediator of protective function of netrin-1 in dextran sodium sulfate (DSS)-induced colitis using mice with partial deletion of UNC5B receptor. DSS colitis was performed in mice with partial genetic UNC5B deficiency (UNC5B^+/− mice) or wild-type mice to examine the role of endogenous UNC5B. These studies were supported by in vitro models of DSS-induced apoptosis in human colon epithelial cells. WT mice developed colitis in response to DSS feeding as indicated by reduction in bw, reduction in colon length and increase in colon weight. These changes were exacerbated in heterozygous UNC5B knockout mice treated with DSS. Periodic Acid-Schiff stained section shows damages in colon epithelium and mononuclear cell infiltration in WT mice, which was further increased in UNC5B heterozygous knockout mice. This was associated with large increase in inflammatory mediators such as cytokine and chemokine expression and extensive apoptosis of epithelial cells in heterozygous knockout mice as compared to WT mice. Overexpression of UNC5B human colon epithelial cells suppressed DSS-induced apoptosis and caspase-3 activity. Moreover, DSS induced large amount of netrin-1 and shRNA mediated knockdown of netrin-1 induction exacerbated DSS-induced epithelial cell apoptosis. Our results suggest that UNC5B is a critical mediator of cell survival in response to stress in colon.     

Mutation detection in KRAS Exon 1 by constant denaturant capillary electrophoresis in 96 parallel capillaries

Mutations in KRAS exon 1 oncogene are frequently found in colon carcinomas. A correlation between the mutated KRAS and the prognosis and outcome of treatment of colon cancer patients was reported in the literature. The object of our work was to establish a high-throughput method with high sensitivity to enable screening of tumor mutation status of KRAS exon 1 in large groups of colon cancer patients. KRAS exon 1 sequences from DNA isolated from 191 sporadic colon cancers were PCR amplified using one primer labeled with fluorescein and a second primer extended by a GC-clamp. After PCR amplification samples were subjected to automated 96-array constant denaturant capillary electrophoresis using a modified MegaBACE 1000 sequencing instrument. Mutant samples were identified by characteristic peak patterns. The sensitivity of detection of a mutant allele in a background of the wild-type alleles was 0.3%. Using the 96-array instrument a typical screening of 191 samples for KRAS mutation status could be performed within 2 h. A KRAS exon 1 mutation was found in 66 of 191 (34.6%) of the samples. The 96-array constant denaturant capillary electrophoresis provides an opportunity for the high-sensitivity screening of large cancer populations for KRAS exon 1 mutations.     

An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants

The colon mucus layers minimize the contact between the luminal flora and the epithelial cells, and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side, and mucus thickness was measured using a micropipette. Mucus thickness, adhesion, and growth rate were recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. In mouse ileum, the mucus could be removed by gentle aspiration, whereas in colon ～40 μm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 μm/h) and mouse (100 μm/h) colon but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 μm, mouse colon: Δ130 μm, human colon: Δ140 μm). In conclusion, while retaining key properties from the mucus system in vivo, this setup also allows for studies of the highly dynamic mucus system under well-controlled conditions.     

Prospective survey of veterinary practitioners’ primary assessment of equine colic: clinical features,diagnoses, and treatment of 120 cases of large colon impaction

Background

Large colon impactions are a common cause of colic in the horse. There are no scientific reports on the clinical presentation, diagnostic tests and treatments used in first opinion practice for large colon impaction cases. The aim of this study was to describe the presentation, diagnostic approach and treatment at the primary assessment of horses with large colon impactions.

Methods

Data were collected prospectively from veterinary practitioners on the primary assessment of equine colic cases over a 12 month period. Inclusion criteria were a diagnosis of primary large colon impaction and positive findings on rectal examination. Data recorded for each case included history, signalment, clinical and diagnostic findings, treatment on primary assessment and final case outcome. Case outcomes were categorised into three groups: simple medical (resolved with single treatment), complicated medical (resolved with multiple medical treatments) and critical (required surgery, were euthanased or died). Univariable analysis using one-way ANOVA and Tukey’s post-hoc test, Kruskal Wallis with Dunn’s post-hoc test and Chi squared analysis were used to compare between different outcome categories.

Results

1032 colic cases were submitted by veterinary practitioners: 120 cases met the inclusion criteria for large colon impaction. Fifty three percent of cases were categorised as simple medical, 36.6% as complicated medical, and 9.2% as critical. Most cases (42.1%) occurred during the winter. Fifty nine percent of horses had had a recent change in management, 43% of horses were not ridden, and 12.5% had a recent / current musculoskeletal injury. Mean heart rate was 43bpm (range 26-88) and most cases showed mild signs of pain (67.5%) and reduced gut sounds (76%). Heart rate was significantly increased and gut sounds significantly decreased in critical compared to simple medical cases (p<0.05). Fifty different treatment combinations were used, with NSAIDs (93%) and oral fluids (71%) being administered most often.

Conclusions

Large colon impactions typically presented with mild signs of colic; heart rate and gut sounds were the most useful parameters to distinguish between simple and critical cases at the primary assessment. The findings of seasonal incidence and associated management factors are consistent with other studies. Veterinary practitioners currently use a wide range of different treatment combinations for large colon impactions.

     

Identification of insulin receptors on the mucosal surface of colon epithelial cells

Brush-border membranes were isolated from the mucosal surface of rabbit proximal colon epithelial cells by a procedure involving Ca2+ precipitation. Ouabain-insensitive K+-phosphatase, a marker enzyme for the colon brush-border membrane, was enriched 17-fold by this technique, while no enrichment was observed in the activity of ouabain-sensitive K+-phosphatase, a marker for the basal-lateral membrane. Insulin binding studies revealed a dose-dependent inhibition of 125I-insulin binding with porcine insulin and approximately 4 X 10(-9) M insulin was required to produce 50% inhibition of 125I-insulin binding, while desoctapeptide insulin, insulin-like growth factor I, and A chain of insulin had less effect on 125I-insulin binding. This is the first demonstration of the existence of high-affinity insulin binding sites on the brush-border membrane of mammalian colon epithelial cells. Subsequent studies with the cross-linking agent disuccinimidyl suberate confirmed the presence of insulin binding sites in these membranes and autoradiography of polyacrylamide gels revealed that the binding subunit of the colon epithelial cell brush-border insulin receptor is similar in size to that observed in hepatic tissue. Interestingly, the insulin binding capacity/mg of protein of this preparation is high, suggesting that large numbers of insulin receptors are present in vivo on the mucosal surface of colon epithelial cells. The potential physiological role of these previously unrecognized insulin receptors is discussed.     

Induction of tumor-specific cytotoxic T lymphocytes and natural killer cells by tumor cells transfected with the interleukin-2 gene

To study the antitumor effect of local production of interleukin-2 (IL-2) from tumor cells, the poorly immunogenic murine colon cancer cells, colon26, was transfected with murine IL-2 cDNA in a bovine papilloma virus vector. IL-2 gene transfectants (mIL2+colon26) did not alter their growth rate compared with parental colon26 cells in vitro, but reduced their tumorigenicity in vivo. Immunization with mIL2+colon26 cells could induce protective immunity against parental colon26 cells. Following intravenous challenges, the colonies of lung metastasis were also inhibited. Moreover, inoculation of mIL2+ colon26 cells slowed the growth of challenged renal cell carcinoma cells, RenCa. Intraperitoneal inoculation of IL-2 gene transfectants generated a large number of peritoneal exudate cells and these cells had a highly cytolytic activity against colon26 and YAC-1. These results suggest that inoculation with IL-2 transfected tumor cells can stimulate not only cytotoxic T lymphocytes but also natural killer cells, and that these cells will act as antitumor effector cells in host animals.     

Active pyrimidine absorption by chicken colon

Pyrimidine absorption by chicken large intestine was investigated employing the everted sac and flux chamber techniques. 3H-labelled uracil was used as substrate. The small intestine and the colon unlike the caecum, transported uracil from the mucosal to the serosal surface against a concentration gradient in the everted sac experiments. Furthermore, there was a net transport of uracil from the mucosal to the serosal side of the colon and jejunum in the flux chamber experiments. Uracil transport by the everted colon sacs against a concentration gradient was inhibited when the purine hypoxanthine was present in the incubation medium. Uracil transport by the everted colon sacs was also inhibited under anaerobic conditions and when 2,4-dinitrophenol was present in the incubation medium. Replacing the Na+ ions of the incubation medium by Li+ ions also caused an inhibition of uracil transport. It is concluded from these results that uracil (and probably other pyrimidines) are absorbed from the chicken colon by a Na+ ion-dependent active transport process having also an affinity for purines.     

Differential influence of butyrate concentration on proximal and distal colonic mucosa in rats born germ-free and associated with a strain of Clostridium paraputrificum

In vivo influence of butyrate in colonic mucosa was studied using a model of gnotobiotic rats monoassociated with a Clostridium paraputrificum. Rats were fed a diet containing increasing amounts of non-digestible carbohydrates, the fermentation of which led to modulated amounts of butyrate in the large intestine. In the proximal colon, the increase in the butyrate concentration alters crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells. Conversely, in the distal colon, no relation was found between the increase in butyrate concentration and crypt depth or number of mucin-containing cells. In both the proximal and distal colon, the mitotic index remained unchanged. In conclusion, in vivo production of physiological quantities of butyrate had a trophic effect on proximal colonic mucosa, but did not influence the distal epithelium.     

Immune expulsion of the nematode Aspiculuris tetraptera from mice given primary and challenge infections.

The distribution of larval Aspiculuris tetraptera was studied in 4-week-old male and female CFLP mice. Whereas on days 10–12 the larvae were entirely confined to the anterior third of the colon, by day 14 larvae could be found throughout the colon. After day 17 the larvae were again restricted to the anterior colon. This change in distribution was co-incident with a loss of a large proportion of the worm burden, which occurred more consistently in female than in male mice.The degree of acquired immunity stimulated by various immunizing regimens was assessed by the survival of a challenge infection in experimental and control mice. It was found that a high level of immunity was achieved by exposure to a 19-day primary infection, a 36-day low-level infection and also by three 6-day infections, in each of which the larvae were removed by piperazine treatment immediately after the crypt phase.