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PubMed1999

Purification of a new intestinal anti-proliferative factor from normal human small intestine.

Previous studies suggest that intestinal cell proliferation may be controlled by endogenous mitosis inhibitors. We describe here the isolation of a protein named intestinal anti-proliferative factor (IAF) from human small intestine. Successive DEAE anion exchange, isoelectric focusing and gel filtration chromatographies led to a purified anti-proliferative protein fraction used to produce antibodies. Using these antibodies as affinity chromatography ligand, IAF was purified from human small intestine cytosolic fraction. IAF was a potent inhibitor of adenocarcinoma colon cells (HT-29 D4 line) DNA synthesis and proliferation with 50% inhibition observed at picomolar concentrations. Analyzed on SDS/PAGE under reducing conditions, this protein migrates with an apparent molecular mass of 120 kDa and amino acid sequence of two internal peptides displays no homology with another listed protein. Cell cycle studies showed that the growth inhibitory effect was maximal between mid G1 and early S phases. Moreover, flow cytometry studies demonstrated that IAF inhibited the progression of HT-29 D4 cells from G1 to S phase. Northern blot analysis using a dipeptidyl peptidase i.v. probe revealed that the growth arrest mediated by IAF was not linked to differentiation processes. By Western blotting with polyclonal antibodies against IAF, we found that this protein was not detected in differentiated colonic carcinoma. Our results suggest that IAF might regulate intestinal cell proliferation.

Antineoplastic Agents↗
PubMed1990

[The pneumodynamic barium x-ray study of small intestine adhesions (a preliminary report)].

Small-intestinal adhesions are not very rare, but on conventional X-ray examination are frequently missed because of their poor semiotics. By the method of pneumodynamic-barium X-ray examination, which allows to obtain high-quality double-contrast image, in addition to the better visualized classical symptoms--exostotic rebounds, diverticulum-like deformities, peristaltic spasms, spastic contractions etc, one may evince also the following new criteria: when inflated the intestines do not freely increase their volume, as is normal, are not unplaited along the entire abdomen, but remain amassed in a definite zone (usually in the left half); On external pressure the inflated loops do not move, as do the ones without adhesions, but remain fixed, so that their structure remains unchanged. The intestinal loops are crooked with acute angles; when in a normally inflated state, they are not crooked, but long. A frequent finding in small intestinal adhesions are the V-shaped figures in whose bottom there is barium meal over which air passes. A study of the small-intestinal cannon contraction reveals insufficiency in the hydrodynamics of the intestinal passage, marked by delayed evacuation, in spite of the strong peristalsis. This method allows a more precise elucidation than the conventional X-ray examination of the abdominal status in small-intestinal adhesions and better orientates the surgeon to choose conservative or operative treatment.

Abdominal Neoplasms↗
PubMed2002

Primary malignant melanoma of the small intestine: report of a case.

The small intestine is the most common site of gastrointestinal (GI) metastases from cutaneous malignant melanoma; however, primary malignant melanoma originating in the small intestine is extremely rare. We report the case of a 72-year-old man found to have a primary malignant melanoma in the ileum. The patient presented with anorexia, weight loss, diffuse colicky abdominal pain, and episodic rectal bleeding. A preoperative diagnosis of a small intestinal tumor was based on the findings of enteroclysis and computed tomography scanning. This diagnosis was confirmed at laparotomy and an enterectomy was performed. Histopathological examination of the resected specimen clarified the exact nature of the lesion, confirming the diagnosis of melanoma. A thorough postoperative investigation did not reveal a primary lesion in the skin, anus, oculus, or any other location. Thus, we diagnosed this tumor as a primary lesion. One year after his operation, the patient remains well without any evidence of recurrence. Primary malignant melanoma of the small intestine is an extremely rare lesion, which must be differentiated from other intestinal tumors.

Aged↗
PubMed2000

Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology.

PURPOSE: Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS: Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS: Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS: Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.

Child↗
PubMed2005

Morphologic evaluation of regenerated small bowel by small intestinal submucosa.

BACKGROUND/PURPOSE: Previous studies have shown small intestinal submucosa (SIS) can be used as biodegradable scaffolds in tissue engineering small intestine. The purpose of this study is to evaluate the regeneration of neointestine and its morphology using SIS. METHODS: A 2-cm tubular SIS graft from Sprague Dawley rat donors was interposed in the middle of a 6-cm ileal Thiry-Vella loop of Lewis rats, which was used to construct an ileostomy. The grafts were harvested at each of the time points ranging from 2 weeks to half a year after implantation, and native small intestine and grafts were investigated for morphology using histology and immunohistochemistry. RESULTS: At the early postoperative period, SIS grafts were colonized by numerous inflammatory cells. A mucosal epithelial layer began to line the luminal surface of the graft by 4 weeks, and by 12 weeks, the luminal surface was covered completely by a layer of neomucosa. Neomucosa with typical small bowel morphology was characterized by a columnar epithelial cell layer with goblet cells, Paneth cells, absorptive enterocytes, and enteroendocrine cells. Significant differences between neomucosa by 12 weeks and 24 weeks in the measurements of mucosal thickness, villus height, and crypt depth were found. The outer walls of SIS grafts were composed of distinct bundles of well-formed smooth muscle-like cells with some fibrovascular tissue. CONCLUSIONS: This initial study suggests that tissue engineering neointestine using SIS can develop structural features of the normal intestine. Small intestinal submucosa might be a viable material in the creation of neointestine for patients suffering short bowel syndrome.

Animals↗
PubMed1999

Vasoactive intestinal peptide suppresses migrating myoelectric complex of rat small intestine independent of nitric oxide.

The involvement of nitric oxide (NO) in the biological response to vasoactive intestinal peptide (VIP) on the migrating myoelectric complex (MMC) of small bowel and systemic arterial blood pressure was investigated in the rat. Animals were supplied with bipolar electrodes for electromyography of the small intestine and blood pressure was assessed by a pressure transducer connected to a carotid artery. In the first session, Nomega-nitro-L-arginine (L-NNA) was administered intravenously at 1, 2, 4 and 20 mg kg(-1). Effects of L-NNA at 1 and 20 mg kg(-1) were also studied after L-arginine 300 mg kg(-1). In the second session, intravenous infusion of VIP 500 pmol kg(-1) min(-1) was administered before and after L-NNA at 1 and 20 mg kg(-1). L-NNA at increasing doses stimulated myoelectric spiking of the small bowel until at 4 mg kg(-1) the MMC was disrupted and irregular spiking induced. Neither at 1 nor 20 mg kg(-1) did L-NNA affect the inhibitory motility response or decrease of blood pressure induced by VIP at a dose of 500 pmol kg(-1) min(-1). Our results show that effects of VIP on motility of the small intestine and systemic arterial blood pressure are direct and not dependent on NO as a common final link.

Animals↗
PubMed1982

Effects of intraluminal pressure on regional blood flow in obstructed and unobstructed small intestines in the rat.

Small bowel circulation in the rat was studied with the microsphere technique before and after 10 minutes of elevated intraintestinal pressure. A distal small bowel loop consisting of 10 vascular arcades was used. The specimens were inflated with nitrogen gas. Regional blood flow in both obstructed and unobstructed small bowels was significantly diminished by an intraintestinal pressure of 40 mm Hg. A pressure of 20 mm Hg impaired circulation only in the obstructed small intestine, whereas the unobstructed intestine did not show a similar decrease. These findings suggest that moderate intraluminal pressures can imply a potential hazard to the vulnerable capillary bed of an obstructed intestine. Hence, it is suggested that intraoperative decompression should be performed to avoid this postoperative threat to bowel viability.

Animals↗
PubMed1980

Transport of sugars and amino acids across guinea pig small intestine.

Using guinea pig small intestine for measurements of influx across the brush border membrane (Jmc), unidirectional transmural fluxes (Jms,Jsm), and steady-state epithelial uptake [A]c, the characteristics of the mutual inhibition were examined. The mutual inhibition of Jmc was in the range of 9--16%, and appeared to be sodium dependent, Jms and [A]c were inhibited by at least 40%, and Jsm was unaffected. All the results can be explained in terms of therheogenic version of the sodium gradient hypothesis. Whereas only the mutual inhibition of Jmc can also be explained in terms of allosteric interactions between the binding sites for sugars and amino acids.

Animals↗
PubMed2005

Physiologic properties of small intestine submucosa.

BACKGROUND: Porcine small intestine submucosa (SiS) has been introduced as a bioprosthesis in herniorrhaphy. This study evaluates in vivo properties of SiS that would affect clinical use. MATERIALS AND METHODS: Twelve pigs underwent implantation of SiS (perforated and nonperforated) on the peritoneal surface. Gross characteristics were evaluated and random samples harvested for histological study at 2 (n = 6) and 8 (n = 6) weeks. Collagen deposition was determined by polarized microscopy. Neovascularity (percent area blood vessels, %A(bv)) was determined by immunohistochemical staining with a polyclonal CD-31 antibody. RESULTS: Perforated SiS had a higher density of capillary ingrowth compared with nonperforated at both 2 (5.6%A(bv) versus 1.4%A(bv), P < 0.05) and 8 weeks (6.0%A(bv) versus 1.6%A(bv), P < 0.05). Compared with 2 weeks, 8-week SiS had a larger proportion of incorporation (25% versus 83%, P < 0.05) and new collagen deposition (50% versus 94%, P < 0.05). Significant contraction was observed in SiS 8 weeks after implantation (preimplant area 98 cm2 versus post-implant area 50 cm2, P < 0.05). CONCLUSION: SiS incorporated well 8 weeks after implantation, with deposition of new collagen. Perforated SiS demonstrated a more rapid and greater amount of neovascularity. The degree of contraction suggests that larger areas of SiS should be selected for herniorrhaphy than would be necessary if synthetic materials were used.

Absorbable Implants↗
PubMed1975

Electron probe analysis of calcium transport by small intestine.

Calcium transport in small intestine of rat and chick has been studied at the cellular level using the electron probe X-ray microanalyzer. Tissues were examined directly after removal as well as after incubation in a calcium solution. In both preparations, discrete calcium localizations were found associated with intracellular and extracellular goblet cell mucus. The in vitro preparations showed calcium in transit across the absorptive epithelium in discrete localizations. Although the primary path of transport was along lateral cell borders, some localizations were found in the cytoplasm in a supranuclear position. The effect of vitamin D depletion and repletion was to decrease and increase, respectively, the number of calcium localizations in transit across the epithelium. These results suggest that calcium is transported while in a sequestered form and indicate that goblet cell mucus plays a role in this transport process.

Aging↗
PubMed1999

Multiple familial gastrointestinal autonomic nerve tumors and small intestinal neuronal dysplasia.

Multiple small intestinal stromal tumors were removed from mother and natural daughter within 15 months of each other. Both had long histories of recurrent iron deficiency anemia and upper gastrointestinal bleeding. Light microscopy revealed that the tumors had arisen in conjunction with diffuse hyperplasia/ dysplasia of Auerbach's myenteric plexus. Immunohistochemistry generally did not show myogenic or paraganglionic phenotypes; CD34 was positive in most tumors. Electron microscopy confirmed the association with the abnormal Auerbach's plexus and showed the structure of gastrointestinal autonomic nerve tumors (GANTs). These findings provide information as to the origin and evolution of GANTs, and also have implications for the clinical management of these tumors which appear to occur more frequently than previously thought.

Aged↗
PubMed1978

Bacterial populations of the small intestine in uremia.

The small intestinal bacterial flora of 15 patients with chronic renal insufficiency was compared with that of subjects with blind loop synDROME. 9 patients were on regular hemodialysis with high protein intake and 6 (serum creatinine 7.5 to 12.5 mg/dl) were maintained on low protein diet. The chronic renal patients harbored a greatly increased microbial flora of both anaerobes and aerobes in the duodenum and jejunum, quantitatively comparable to those in blind loop subjects. The composition did not differ significantly in the two groups. Some organisms may have the potential to metabolize substrates which reach the intestinal lumen from the diet and bile, and perhaps to generate toxic metabolites that could contribute to uremic toxicity or malabsorption.

Aerobiosis↗
PubMed2001

Metabolic characterization of the major human small intestinal cytochrome p450s.

Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. CYP3A4 is the major form of CYP expressed in enterocytes and CYP2C is also expressed at a significant level. In this study, we further characterized the expression of CYP3A4 and CYP2C in human enterocytes and their interindividual variations by examining the metabolic activities from 10 individuals. CYP3A4 in human jejunum microsomes, as determined by 6beta-testosterone hydroxylase activity, varied from 0.36 to 2.46 nmol/min/mg. The apparent average K(m) and V(max) values from two representative individuals were 54 microM and 3.2 nmol/min/mg, respectively. CYP2C9 and CYP2C19 in human jejunum microsomes, as determined by diclofenac 4'-hydroxylase and mephenytoin 4'-hydroxylase activities, varied over an 18-fold range (7.3-129 pmol/min/mg) and 17-fold range (0.8-13.1 pmol/min/mg), respectively. The mean apparent K(m) for diclofenac 4'-hydroxylase was 9.9 microM , whereas the apparent mean K(m) for S-mephenytoin 4'-hydroxylase was 79.3 microM . The mean intrinsic clearance (V(max)/K(m)) was approximately 130-fold greater for diclofenac 4'-hydroxylase than for mephenytoin 4'-hydroxylase. The metabolic activities of CYP2C9 and CYP2C19 were confirmed by inhibition by sulfaphenazole for CYP2C9 and ticlopidine for CYP2C19. In addition, CYP2C9 activities did not correlate with CYP3A4 activities, while CYP2C19 activities had a significant but poor correlation with those of CYP3A4. Thus the major CYP activities in human enterocytes have large interindividual variabilities that are not strongly related.

Adult↗
PubMed2000

Early establishment of epithelial apoptosis in the developing human small intestine.

In the adult small intestine, the dynamic renewal of the epithelium is characterized by a sequence of cell production in the crypts, cell maturation and cell migration to the tip of villi, where apoptosis is undertaken. Little is known about enterocytic apoptosis during development. In man, intestinal architectural features and functions are acquired largely by mid-gestation (18-20 wks); the question whether the establishment of enterocytic apoptotic processes parallels or not the acquisition of other intestinal functional features remains open. In the present study, we approached this question by examining enterocytic apoptosis during development of the human jejunum (9-20 wks gestation), using the ISEL (in situ terminal uridine deoxynucleotidyl nick-end labelling) method. Between 9 and 17 wks, apoptotic enterocytes were not evidenced. However, beginning at the 18 wks stage, ISEL-positive enterocytes were regularly observed at the tip of villi. Since the Bcl-2 family of proteins constitutes a critical checkpoint in apoptosis, acting upstream of the apoptotic machinery, we investigated the expression of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1). By immunofluorescence, we found that all homologs analyzed were expressed by enterocytes between 9 and 20 wks. However, Bcl-2 homologs underwent a gradual compartmentalization of epithelial expression along the maturing crypt-villus axis, to establish gradients of expression by 18-20 wks. Western blot analyses indicated that the expression levels of Bcl-2 homologs were modulated during morphogenesis of the crypt-villus axis, in parallel to their gradual compartmentalization of expression. Altogether, these data suggest that regulatory mechanisms of human enterocytic apoptosis become established by mid-gestation (18-20 wks) and coincide with the maturation of the crypt-villus axis of cell proliferation, differentiation and renewal.

Apoptosis↗
PubMedDate not supplied

[Hydrolysis and absorption of lysozyme in the small intestine].

Preparations of chicken small intestine were used in the experiment in vitro simulating processes of membranous digestion (inverted intestinal segments) and absorption (inverted intestinal myasis). It was established that lysozyme was hydrolyzed on the internal mucosa surface regardless of its concentration in the gastro-intestinal tract, and only insignificant quantity of lysozyme (0.027%) penetrates the intestinal wall. The method of lysozyme determination through its action on the cellular wall of Micrococcus lisodeicticus, and highly efficient liquid chromatography were used to study the transport process. The data presented have evidenced that lysozyme is well hydrolyzed under the action of intestinal peptide hydrolyses, and only insignificant amounts of non-splitted lysozyme can penetrate the blood.

Animals↗
PubMed1983

The development of intraepithelial and Peyer's patch lymphocyte sub-types in the small intestine of newborn rats.

Small intestinal intraepithelial (IEL) and Peyer's patch (PPL) lymphocytes of newborn rats have been studied in histological sections and in isolated cell suspensions. Initially IEL numbers were low compared with older animals, and fewer cells were labelled by the monoclonal antibody markers used. At birth, 41% of IEL expressed receptors for MRC OX8 (T suppressor marker) yet lacked receptors for W3/13 (pan-T marker) which was present on only 1% of these cells. IEL with receptors for W3/25 (T helper marker) were not seen until 2 weeks of age. Granulated cells (GIEL) accounted for only 19% of IEL at birth. The numbers and relative proportions of these lymphocyte sub-types were still not mature at the time of weaning. In the first week of life there was a short lived upsurge in numbers of GIEL and MRC OX8+ IEL. The relative distribution of PPL sub-types was mature 4 weeks after birth, but the morphology of this tissue did not appear to be well differentiated until 1 week later.

Animals↗
PubMed2005

Changes in chicken intestinal zinc exporter mRNA expression and small intestinal functionality following intra-amniotic zinc-methionine administration.

A 303-bp cDNA of intestinal zinc exporter (ZnT1) was isolated from chicken jejunum by reverse transcriptase-polymerase chain reaction and sequenced, and showed 42% homology to Homo sapiens and Rattus novergicus intestinal ZnT1 genes. This specific probe was used to examine the effect of zinc-methionine (ZnMet) administration on the mRNA expression of ZnT1 and on small intestinal development and functionality. In this study, ZnMet was injected into the naturally consumed amniotic fluid of 17-day-old chicken embryos. The ZnT1 gene showed an approximately 200% increase in its mRNA levels from 48 h post-ZnMet injection, as compared to the control. An analysis of the gene expression of the brush-border enzymes and transporters showed increased mRNA expression of sucrase isomaltase, leucine-aminopeptidase, sodium-glucose cotransporter and Na+K+ATPase transporter (Na+K+ATPase) from 48 h post-ZnMet injection, in comparison to controls. Significant increases (P<.05) in the biochemical activity of the brush-border enzymes and transporters, and in jejunal villus surface area were detected from day of hatch (96 h post-ZnMet injection) as compared to controls. These results suggest that ZnMet administration into prenatal intestine via injection into the amniotic fluid enhances intestinal development and improves its functionality.

Amino Acid Sequence↗
PubMedDate not supplied

Nicotinic and muscarinic modulation of 5-hydroxytryptamine (5-HT) release from porcine and canine small intestine.

Strips of porcine and canine small intestine were incubated in vitro and the release of 5-hydroxytryptamine (5-HT) was determined by HPLC with electrochemical detection. The spontaneous outflow of 5-HT from the porcine and canine small intestine largely reflects calcium-dependent 5-HT secretion from enterochromaffin cells which are under a spontaneous neuronal, excitatory input as indicated by the inhibitory effect (30-40%) of tetrodotoxin. In both species, nicotine enhanced the release of 5-HT in a concentration-dependent manner by a maximum of about 50% at 100 microM. This effect was blocked by the nicotine receptor antagonist hexamethonium, but not by the subtype-selective nicotine receptor antagonist alpha-bungarotoxin. The effect of nicotine was rapidly desensitized. The presence of tetrodotoxin abolished the effect of nicotine on 5-HT release in canine tissue but not in porcine tissue. The presence of the muscarine receptor antagonist scopolamine prevented the effect of nicotine on 5-HT release from canine tissue, but significantly enhanced 5-HT release from porcine tissue. The muscarine receptor agonist oxotremorine inhibited 5-HT release from porcine tissue, but increased 5-HT release from canine tissue. However, in the presence of tetrodotoxin, oxotremorine enhanced 5-HT release in tissue from both species. In conclusion, activation of nicotine receptors induce the release of 5-HT from porcine and canine small intestine. In the dog, the effect of nicotine is mediated via the release of acetylcholine which then stimulates 5-HT release via muscarine receptors on the enterochromaffin cells. In the pig, the stimulatory effect of nicotine appears to be located directly on the enterochromaffin cells. In addition, activation of neuronal muscarine receptors in the porcine small intestine induced the release of a previously unidentified neurotransmitter which inhibited 5-HT release. Nicotine, via cholinergic interneurons, also appears to induce the release of this inhibitory neurotransmitter which opposes the direct stimulatory action of nicotine on 5-HT release.

Acetylcholine↗