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Tissue and cell-specific patterns of expression of rat liver and intestinal fatty acid binding protein during development and in experimental colonic and small intestinal adenocarcinomas.

BACKGROUND: Mammalian small intestinal and colonic epithelium express members of a multigene family of hydrophobic ligand binding proteins of which liver and intestinal fatty acid binding protein represent among the most abundant intestinal gene products. These proteins are expressed in a cell- and region-specific manner and emerge in a temporally distinctive pattern. EXPERIMENTAL DESIGN: We have studied expression of these genes in the small intestine and colon of rats treated with 1,2-dimethylhydrazine since these animals develop a predictable pattern of both small and large bowel cancers. Studies were also undertaken in fetal and neonatal small intestine and colon. RESULTS: There was a 10- and 50-fold decrease, respectively, in mRNA abundance for intestinal and liver fatty acid binding protein in RNA from colon cancers compared with either uninvolved or control RNA. Immunocytochemical analysis revealed decreased staining for both proteins within their normal distribution together with ectopic clusters of cells reactive for liver fatty acid binding protein within colonic tumors. A more striking mosaic of immunocytochemical staining for both liver and intestinal fatty acid binding protein was found in small intestinal adenocarcinomas. Similar mosaic patterns of immunocytochemical staining were transiently detectable in rat fetal small intestine and neonatal colon. CONCLUSIONS: The region- and cell-specific expression of these genes, which may be linked temporally to events in intestinal differentiation, are subject to disruption in a cell-specific manner in the transformed, or dedifferentiated, phenotype.

1,2-Dimethylhydrazine↗

Intestinal permeability as a measure of small intestinal mucosal integrity: correlation with jejunal biopsy.

The permeability of the small intestine was measured and jejunal biopsy performed in 39 children with gastrointestinal disorders. Intestinal permeability was measured using orally administered mannitol and lactulose as probe molecules in an isotonic solution (274 mOs/l), and the results were expressed as the ratio of the urinary excretion of the two sugars over 5 h. Urine samples were analysed for mannitol and lactulose content by high performance liquid chromatography. Children with small intestinal mucosal damage, irrespective of the cause, had a significantly lower (P less than 0.001) mannitol excretion (mean recovery 1.21% of ingested dose) than those with a normal mucosa (mean recovery 5.3%), while lactulose excretion did not differ (P greater than 0.05). The mean value of the lactulose:mannitol urinary excretion ratios was significantly higher (P less than 0.001) in subjects with an abnormal mucosa (0.98) compared to those with a normal mucosa (0.2). Using the mean plus two standard deviations of the normal mucosa group to define the upper limit of normal, all lactulose: mannitol excretion ratios from the abnormal mucosa group were above this limit. The results of this study show that the sugar permeability test is a sensitive, non-invasive screening test for jejunal mucosal damage in children and shows good correlation with jejunal biopsy results.

Adolescent↗

Intestinal adaptation after extensive resection of the small intestine and prolonged administration of parenteral nutrition.

After a superior mesenteric thrombosis in a 77 year old man, all but 24 centimeters of the jejunum were resected and anastomosed to the remaining left colon. After being maintained on parenteral nutrition on a 24 hour basis for six weeks, the patient was placed on overnight parenteral nutrition in which he received 2 liters of parenteral nutrition solution containing about 2,200 calories. Over a period of nine months, hypertrophy, lengthening and dilatation took place so that the patient was almost able to support himself by mouth. We believe that this is the first patient in whom documented lengthening of the small intestine has been reported. The factors entering into the hypertrophy and dilatation are discussed. Many patients in this age group are denied therapy because of the consequences of massive enterectomy. With adaptation using prolonged parenteral nutrition, it should be possible for most patients to sustain themselves completely by mouth. Patients with massive necrosis of the small intestine should not be denied therapy even if only 1 foot of small intestine remains. This is likely to hypertrophy in length until parenteral nutrition is no longer necessary.

Adaptation, Physiological↗

Characterization of a putative receptor for intestinal trefoil factor in rat small intestine: identification by in situ binding and ligand blotting.

Intestinal trefoil factor (ITF), a small peptide secreted by intestinal goblet cells, maintains mucosal integrity and promotes epithelial wound healing. Although ITF has been cloned, the detailed mechanism by which ITF interacts with intestinal epithelium remains elusive. In the present study, we expressed mature rat ITF (rITF) with pXa (a prokaryotic expression vector) in Escherichia coli to generate a biotinylated rITF fusion protein (bTag-ITF). By using bTag-ITF probe, we identified ITF binding cells in the crypts of the small intestine and mucous cells of the region of gastric glands. Using a ligand blotting technique, we further characterized a 50 kDa glycosylated protein from the membrane fraction of the small intestine, which bound to bTag-ITF. Our data suggest that this 50 kDa membrane glycoprotein is a putative receptor for ITF in the gastrointestinal tract.

Amino Acid Sequence↗

Glucose uptake in dilated small intestine.

BACKGROUND/PURPOSE: The development of dilated small intestine in patients with short bowel syndrome results in increased mucosal surface area. This study examines whether the incremental increase in surface area leads to a proportional increase in absorptive function of the small intestine. METHODS: Partial obstruction of the small intestine was created in rats by placing an intussusception valve in the proximal jejunum. Rats that underwent sham operations served as controls. One week postoperatively, the small intestine proximal and distal to the valve was removed. The intestinal diameter proximal and distal to the obstruction was measured. The rate of glucose uptake was measured by the everted sleeve technique. The results were analyzed by analysis of variance (ANOVA). RESULTS: The intestine proximal to the valve was significantly dilated and thickened when compared with the intestine distal to the valve. The wet mass per centimeter of the dilated segment was 2.5 times that of the control group (P<.001). The glucose uptake capacity of the dilated segment was slightly higher than that of the control group (540 v 420 nmol/min/cm, P<.05). However, the specific glucose uptake rate was reduced significantly in the intestine proximal to the valve (247 v 335 nmol/min/cm2, P<.01). CONCLUSIONS: Although the partial obstruction of small intestine resulted in a substantial increase in the intestinal surface area, the absorptive capacity of the dilated intestine per unit surface area was decreased significantly. This translated ultimately into a slight increase in the overall functional absorptive capacity of glucose in the small intestine. These results suggest that dilated small intestine may not enhance mucosal absorption.

Analysis of Variance↗

'Metachronous' adenocarcinoma of the small intestine.

Adenocarcinoma of the small intestine accounts for less than 1% of primary gastrointestinal malignancies (1). Small intestine contains 75% of the length of the gastrointestinal tract with 90% of the surface mucosal area, and yet carcinoma is rare. Symptoms of small bowel adenocarcinoma are vague and non-specific, and this region is relatively inaccessible which together contributes to their late diagnosis and poor prognosis. The authors report a case of two primary adenocarcinomas of the small intestine in the same patient.

Adenocarcinoma↗

The ontogeny of canine small intestinal submucosa regenerated bladder.

PURPOSE: Small intestinal submucosa has previously been shown to promote regeneration of transitional epithelium, smooth muscle and peripheral nerves in rat and dog bladders. The origin of these regenerated components is presently unknown. This study attempts to define the origin of vascular, smooth muscle and peripheral nerve regeneration. MATERIALS AND METHODS: A total of 22 adult male dogs weighing 25 to 30 kg. underwent partial cystectomy and immediate augmentation with a small intestinal submucosa patch graft. The small intestinal submucosa graft-native bladder interface was marked with permanent marking sutures for future reference. Small intestinal submucosa regenerated bladders were harvested at 2, 3, 4, 6, 8 and 10 weeks after augmentation. The tissue was then studied with routine histology and immunohistochemistry using factor VIII, smooth muscle specific actin (1A4) and neurofilament staining. RESULTS: Results demonstrated that epithelialization of the graft surface was complete by 3 to 4 weeks with normal transitional histology. In the early periods neovascularization was prominent throughout the entire graft, as shown by factor VIII staining. Later more mature vessels were noted. Early in muscle formation sheets of elongated spindle cells extended into the graft from the incised native bladder at both surgical margins and ran parallel to the mucosal surface. At 4 weeks this spindle cell proliferation completely traversed the graft. Trichrome stained sections of the 4-week-old grafts showed no evidence of muscle differentiation and the spindle cells appeared to be fibroblasts. However, these cells stained positive for smooth muscle specific actin (1A4), indicating myogenic potential. Between weeks 4 and 6 the spindle cells became more haphazardly arranged and were separated by loose interstitium. By weeks 8 to 10 there was distinct smooth muscle bundle formation within these areas of proliferating myocytes. Neural regeneration appeared to coincide with smooth muscle development. Early neurofilament positive cells were noted predominantly at the graft-native bladder interface. At 4 weeks neurofilament positive cells were present throughout the graft and by 10 weeks nerve trunks composed of several nerve fibers were identified in association with newly formed smooth muscle bundles. CONCLUSIONS: Small intestinal submucosa serves as a platform for bladder regeneration. Neovascularization smooth muscle and neural regeneration appear to occur through pannus ingrowth from the graft-native bladder interface. Smooth muscle regeneration seems to begin with the maturation of myofibroblasts, which migrate into the graft as early as 2 weeks after augmentation, and it progresses to the formation of distinct smooth muscle bundles by 10 weeks.

Animals↗

Is glutamine a unique fuel for small intestinal cells?

Although the small intestinal mucosa is designed to transport large quantities of all nutrients to the blood, the primary nutrients utilized by the enterocyte for growth and/or maintenance are quite restricted. The major fuels for the small intestinal mucosa are amino acids (glutamine, glutamate, aspartate), whereas glucose and fatty acids are of much less importance. Many of the experiments have been performed during growth or maintenance of mucosa in small rodents, especially the rat, a model in which the adaptation of the intestinal mucosa, at least to fasting, is quite different than in humans. A special role has been suggested for glutamine as a small intestinal fuel, compared with glutamate and aspartate, but the available data do not support this view. Clinical trials of glutamine supplementation suggest that, if glutamine has a role, it may be related to functions other than those relating to small intestinal function.

Editorial↗

Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine.

Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology. Screening of antibodies from a hybridoma library led to the identification of an acyl-CoA synthetase 5-specific monoclonal antibody. Protein synthesis, mRNA expression, and the enzyme activity of acyl-CoA synthetase 5 were studied by several methods in human small intestinal tissues with Crohn's disease or coeliac disease, respectively. Acyl-CoA synthetase 5 mRNA and protein levels were substantially reduced in injured small intestinal mucosa. Moreover, impaired synthesis of the acyl-CoA synthetase 5 protein was reflected by a decrease in intramucosal enzyme activity. Subtle changes of the acyl-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine.

Adult↗

Characterization of human small intestinal cytochromes P-450.

Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P-450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. The CYP composition of enterocytes could thus affect the potential toxicity or therapeutic efficacy of xenobiotics by modifying systemic uptake. We have characterized human enterocyte CYP composition to enable assessment of its functional roles. An isolation method for enterocytes from human small intestine was developed using EDTA buffer-mediated elution. Villous enterocytes were isolated in high yield, separated from crypt cells. Reverse transcriptase-polymerase chain reaction of total RNA from enterocytes revealed that CYP1A1, 1B1, 2C, 2D6, 2E1, 3A4, and 3A5 mRNA were expressed, but only CYP2C and 3A4 were detectable by Western immunoblotting in enterocyte microsomes from 10 human small intestines, whereas CYP1A1 was weakly detectable in two of eight intestines tested. Microsomal protein content decreased markedly along the small intestine from the duodenum to the ileum, whereas total CYP content and CYP3A4 erythromycin N-demethylase activity increased slightly in progressing from the duodenum to the jejunum and then decreased markedly toward the ileum. Levels of CYP3A4 and 2C protein did not decrease in concert as a function of length along the intestine distally. Maximal CYP content for the 10 intestines varied from 0.06 to 0.18 nmol/mg microsomal protein and maximal CYP3A4 erythromycin N-demethylase activity varied from 0.30 to 0.76 nmol/min/mg microsomal protein. In conclusion, CYP3A4 is the major form of CYP expressed in human small intestine enterocytes, CYP3A5 expression was not detected, CYP2C and, in some intestines, CYP1A1 were expressed. The highest metabolic activity occurred in the proximal intestine.

Adolescent↗

Distribution of muscarinic receptor subtypes in rat small intestine.

Despite its great promise, small intestinal transplantation in some patients is complicated by difficult postoperative management. The reasons for this are complex. In a rat model of small intestinal transplantation, frequencies of migrating myoelectric complexes during fasting are reduced in ileal isografts and muscarinic receptor density is decreased. We hypothesized that the distribution of muscarinic 1 receptors localized to enteric neurons is altered after small intestinal transplantation. Distal small intestine was orthotopically transplanted in Lewis-to-Lewis donor-recipient combinations. At 3 months, transplanted and normal ileum was obtained to prepare membrane fractions. [N-methyl-3H]Scopolamine served as ligand, while scopolamine methylbromide, pirenzepine, and methoctramine were used in competitive homologous and heterologous displacement experiments. Receptor subtype models were examined by nonlinear regression analysis. In normal and transplanted ileum, heterologous displacement was consistent with three site models (P < 0.05). In normals, the muscarinic 1 receptor subtype was most abundant, with a relative distribution of 69 to 78%. There was a relative distribution of 13 to 16% for muscarinic 3 receptor subtype. After transplantation, the muscarinic 1 subtype decreased to a mean of 45% but the muscarinic 3 subtype increased to a mean of 42%. Using pirenzepine, mean pKD values were not different between the two groups. It is concluded that the decrease in muscarinic 1 receptor subtype after transplantation could be related to neuronal cell loss or to downregulation of the expression of muscarinic 1 receptors. The results did not support defective posttranslational processing of receptor proteins.

Animals↗

Quantitative analysis of lactase-phlorizin hydrolase expression in the absorptive enterocytes of newborn rat small intestine.

At birth, the mammalian small intestine displays regional differences in morphology as well as complex proximal-to-distal (horizontal) patterns of protein distribution. Lactase-phlorizin hydrolase (LPH), an enterocyte-specific disaccharidase crucial for the digestion of lactose in milk, reveals a characteristic horizontal pattern of expression at birth. However, it is not certain whether this topographic pattern is due to variations in epithelial structure along the length of the small intestine or to regional differences in the transcription of the LPH gene. In order to understand the mechanisms that regulate the regionalization of LPH at birth, we characterized the epithelial structure along the horizontal axis using stereologic techniques and correlated these data with the patterns of lactase activity and LPH mRNA abundance in the small intestine of unsuckled, newborn rats. Epithelial volume and microvillar surface area per unit of intestinal length decreased three-to fourfold from duodenum to distal ileum. In contrast, lactase activity and LPH mRNA abundance were highest in proximal jejunum and lowest in the most proximal and distal ends of the small intestine. Mean lactase activity per cell in proximal duodenum, proximal jejunum, and distal ileum was estimated at 12.0, 26.7, and 5.6 nU/absorptive enterocyte, respectively, and paralleled the concentration of LPH mRNA in the same segments: 20, 45, and 15 molecules of LPH mRNA/absorptive enterocyte. Our data indicate that horizontal gradients of lactase activity in the newborn rat intestine do not depend on epithelial organization or on enteral factors, since the horizontal gradient is established before suckling. Each absorptive enterocyte along the small intestine expresses lactase activity in a position-dependent manner which is controlled at the level of mRNA abundance.

Absorption↗

Intestinal permeability and function in dogs with small intestinal bacterial overgrowth.

Small intestinal bacterial overgrowth (SIBO) has been reported to occur commonly in dogs with signs of chronic intestinal disease. There are usually few intestinal histological changes, and it is uncertain to what extent bacteria cause mucosal damage. The aim of this study was to apply a differential sugar absorption test for intestinal permeability and function to the objective assessment of intestinal damage in dogs with SIBO. Studies were performed on 63 dogs with signs of chronic small and, or, large bowel disease, in which SIBO (greater than 10(5) total or greater than 10(4) anaerobic colony forming units/ml) was diagnosed by quantitative culture of duodenal juice obtained endoscopically. None of the dogs had evidence of intestinal pathogens, parasites, systemic disease or pancreatic insufficiency. differential sugar absorption was performed by determining the ratios of urinary recoveries of lactulose/rhamnose (L/R ratio, which reflects permeability) and D-xylose/3-O-methylglucose (X/G ratio, which reflects intestinal absorptive function) following oral administration. Dogs with SIBO comprised 28 different breeds, including 13 German shepherd dogs. SIBO was aerobic in 18/63 dogs (29 per cent), and anaerobic in 45/63 (71 per cent). Histological examination of duodenal biopsies showed no abnormalities in 75 per cent, and mild to moderate lymphocytic infiltrates in 25 per cent of the dogs. The L/R ratio was increased (greater than 0.12) in 52 per cent, and the X/G ratio reduced (less than 0.60) in 33 per cent of the dogs. Differential sugar absorption was repeated in 11 dogs after their four weeks of oral antibiotic therapy. The L/R ratio declined in all 11 dogs (mean +/- SD pre: 0.24 +/- 0.14; post: 0.16 +/- 0.11; P < 0.05), but changes in the X/G ratio were more variable. These findings show that SIBO is commonly associated with mucosal damage not detected on histological examination of intestinal biopsies, and that changes in intestinal permeability following oral antibiotics may be used to monitor response to treatment.

Administration, Oral↗

Premalignant conditions of the small intestine.

Cancer of the small intestine is rare compared with other sites in the gastrointestinal tract. Of the four major primary small-bowel tumors (adenocarcinomas, lymphomas, carcinoid, and leiomyosarcomas), adenocarcinomas and lymphomas are associated with diseases that seem to increase the risk of developing these malignancies. In the case of immunoproliferative small intestinal disease and celiac disease, both of which are thought to predispose patients to the development of primary lymphoma, treatment of the predisposing conditions seems to decrease the risk of developing subsequent malignancy. Recognition of the increased risk associated with other conditions, such as immunodeficiency syndromes, nodular lymphoid hyperplasia, Crohn's disease, the gastrointestinal polyposis syndromes, hereditary nonpolyposis colon cancer, neurofibromatosis, long-standing ileostomy, and urinary diversion procedures, may lead to early diagnosis and improved survival.

Acquired Immunodeficiency Syndrome↗

[The effects of interleukin-11 and epidermal growth factor on residual small intestine after experimental extensive small bowel resection].

BACKGROUND/PURPOSE: Interleukin 11 (IL-11) is a multifunctional cytokine derived from bone marrow, which has a trophic effect on small bowel epithelium. This study compares the effects of IL-11 with epidermal growth factor (EGF), a growth factor known to enhance small bowel adaptation. METHODS: Forty Sprague-Dawley rats (90-100 g) underwent an 85% mid-small bowel resection with primary anastomosis on day 0. Rats were divided into four treatment groups: controls (group I) received bovine serum albumin (BSA), group II received IL-11, 125 mg/kg subcutaneously (SC) twice daily, group III received EGF, 0.10 mg/g SC bid, and group IV received EGF and IL-11 in the above doses. Half of the animals (five per group) were killed on day 4 of therapy, and the rest on day 8. Animals were evaluated for weight, mucosal length, and bowel wall muscle thickness on days 4 and 8, and expression of proliferating cell nuclear antigen (PCNA) in intestinal crypt and smooth muscle cells on day 8. RESULTS: Body weight was similar at day 4 and 8. Mucosal thickness in groups II (IL-11) and IV (IL-11 and EGF) was significantly increased at days 4 and 8 compared with controls (group I) and EGF (group III, P < 0.001). Muscle thickness was significantly increased in the EGF and combined group IV compared with the BSA controls and IL-11 groups (P < 0.001). Thirty-two percent of the mucosal crypt cells in group I stained positive for PCNA, whereas 51%, 53%, and 60% stained positive in groups II (IL-11), III (EGF), and IV (IL-11 and EGF), respectively. In groups I and II, 2% and 1.7% of the myocytes stained positive for PCNA, whereas 11.2% and 5.2% in group III and IV. CONCLUSIONS: These data suggest that IL-11 has a trophic effect on small intestinal enterocytes, causing cell proliferation and increased mucosal thickness. EGF has a more generalized effect causing proliferation of both enterocytes and myocytes. IL-11, with or without EGF may be a useful adjunct in treatment of short bowel syndrome.

Animals↗

Effects of interleukin-11 and epidermal growth factor on residual small intestine after experimental massive small bowel resection.

Interleukin 11 (IL-11) is a multifunctional cytokine derived from bone marrow, which has a trophic effect on small bowel epithelium. This study compares the effects of IL-11 with epidermal growth factor (EGF), a growth factor known to enhance small bowel adaptation. Forty Sprague-Dawley rats (90-100g) underwent an 85% mid-small bowel resection with primary anastomosis on day 0. Rats were divided into four treatment groups: controls (group I) received bovine serum albumin (BSA), group II received IL-11, 125 microg/kg subcutaneously (SC) twice daily, group III received EGF, 0,10 microg/g SC bid, and group IV received EGF and IL-11 in the above doses. Half of the animals (five per group) were killed on day 4 of therapy, and the rest on day 8. Animals were evaluated for weight, mucosal length, and bowel wall muscle thickness on days 4 and 8, and expression of proliferating cell nuclear antigen (PCNA) in intestinal crypt and smooth muscle cells on day 8. Body weight was similar at day 4 and 8. Mucosal thickness in groups 11 (IL-11) and IV (IL-11 and EGF) was significantly increased at day 4 and 8 compared with controls (group I) and EGF (group III, P<.001). Muscle thickness was significantly increased in the EGF and combined group IV compared with the BSA controls and IL-11 groups (P < .001). Thirty-two percent of the mucosal crypt cells in group I stained positive for PCNA, whereas 51%, 53%, and 60% stained positive in groups II (IL-11), III (EGF), and IV (IL-11 and EGF), respectively. In groups I and II, 2% and 1.7% of the myocytes stained positive for PCNA, whereas 11.2% and 5.2% in group III and IV. These data suggest that IL-11 has a trophic effect on small intestinal enterocytes, causing cell proliferation and increased mucosal thickness. EGF has a more generalized effect causing proliferation of both enterocytes and myocytes. IL-11, with or without EGF may be a useful adjunct in treatment of short bowel syndrome.

Animals↗

Small intestinal submucosa as a small-caliber venous graft: a novel model for hepatocyte transplantation on synthetic biodegradable polymer scaffolds with direct access to the portal venous system.

BACKGROUND/PURPOSE: Hepatotrophic factors in the portal blood are critically important for the survival of heterotopically transplanted hepatocytes. Currently, no model exists for the implantation of hepatocytes on biodegradable polymer scaffolds with direct access to the portal blood. This study investigates the use of small intestinal submucosa (SIS) as a small-caliber venous conduit that may be used for the implantation of tissue-engineered liver. METHODS: SIS was prepared from segments of rat jejunum and implanted as a venous conduit between the portal vein and inferior vena cava in 26 heparinized Lewis rats. Venograms were performed periodically, and the grafts were harvested at various time-points and examined by scanning electron microscopy (SEM) and histology. Von Willebrand Factor (vWF) staining was performed to assess endothelialization. RESULTS: Five rats died of technical complications. Seventeen of 21 rats (81%) maintained patent grafts at the time of death up to 8 weeks. Venograms demonstrated patent grafts at 3 and 8 weeks. SEM results showed a smooth luminal surface with endothelial-like cells by 3 weeks. Histology demonstrated a confluent luminal endothelial monolayer, absence of thrombus, and neovascularization in the SIS graft. VWF staining results were positive, confirming the growth of endothelial cells on the luminal surface. In preliminary studies, implantation of hepatocytes seeded on biodegradable polymer tubes into the SIS graft demonstrated clusters of viable cells after 2 days. CONCLUSIONS: Rat SIS can be prepared readily, maintains high patency as a small-caliber venous graft, and may be a useful model for the transplantation of tissue-engineered liver with access to the portal circulation.

Animals↗