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Primary culture of the enteric nervous system from neonatal hamster intestine. Selection of vasoactive intestinal polypeptide-containing neurons.

The enteric nervous system is a major division of the autonomic nervous system and is responsible for the regulation of gastrointestinal function. The objective of the present study was to develop a simple and effective technique for isolating and culturing neurons of the enteric nervous system that would permit characterization of their development and regulatory peptide content. This was accomplished using a dispersed intestinal cell preparation cultured under conditions designed to support the growth and differentiation of neurons and neuroendocrine cells. Newborn hamster intestine was digested in 0.1% collagenase, mechanically dispersed, and cultured in RPMI 1640 supplemented with 2.5% serum and other additives. Phase and bright-field microscopy demonstrated neuronal cells and fibers after the second day in culture. This was confirmed by immunohistochemistry using antibodies directed against neurofilament and vasoactive intestinal polypeptide. Acetic acid extracts of the culture indicated that during the first 4 days of the culture the content of vasoactive intestinal polypeptide increased, whereas the content of substance P, mammalian bombesin, and neurotensin declined. High-performance liquid chromatography and fast protein liquid chromatography confirmed that the immunoreactive vasoactive intestinal polypeptide coeluted with synthetic and iodinated forms of the peptide. This study describes a technique for primary culture of intestinal tissue that supports the survival of enteric neurons and permits analysis of the development and synthetic and secretory characteristics of the enteric nervous system.

Animals↗

Immunoreactive trypsinogen levels in pediatric patients with intestinal failure awaiting intestinal transplantation.

The aim of this study was to evaluate pancreatic function in total parenteral nutrition (TPN)-dependent children with permanent intestinal failure by measuring immunoreactive trypsinogen (IRT) levels. Between 1992 and 1996, 105 pediatric patients with permanent intestinal failure were referred to the Children's Hospital of Pittsburgh for small intestinal transplant evaluation. Serum samples were available from 55 of them. Ten suffered from intestinal pseudo-obstruction or microvillus inclusion disease, while 45 had short bowel syndrome (SBS). IRT levels were significantly higher (p < 0.001) in SBS patients (89.4 +/- 9.2 ng mL) compared to controls (43.4 +/- 5.6 ng/ nL) without liver, gastrointestinal, or kidney disease. IRT levels did not correlate with liver injury, length of bowel, or the cause of SBS. Five of 20 patients who underwent intestinal transplantation developed pancreatitis during a median post-operative follow up 15.4 months later. IRT levels failed to predict who would develop pancreatitis post-transplant. The data suggest that elevated plasma IRT levels are common among children with intestinal failure, but fail to identify patients at risk for pancreatitis post-transplant.

Adolescent↗

Acute intestinal obstruction due to intramural haemorrhage in small intestine in a patient with severe haemophilia A and inhibitor.

Patients with severe haemophilia A usually present with joint, gastrointestinal and urinary tract haemorrhage. Bleeding elsewhere is often precipitated by pre-existing pathology or trauma. We report a patient with severe haemophilia A, who presented with symptoms of acute intestinal obstruction. He has a factor VIII inhibitor and receives recombinant factor VIIa on demand at home. The CT scan of abdomen showed dilated small intestine with fluid filled loops and a long segment in the jejunum with marked transmural thickening. There was no other pathology in the small intestine. These appearances were consistent with intramural haemorrhage in the small intestine as the cause of acute obstruction. He was managed conservatively with recombinant factor VIIa and this resulted in resolution of his symptoms. This case highlights an unusual presentation of bleeding in a haemophilia patient. Intestinal obstruction due to haemorrhage in the small intestinal wall is extremely rare and only previously reported in a few haemophilia patients. It also highlights the effectiveness of conservative management with recombinant factor VIIa as opposed to immediate exploratory surgery.

Acute Disease↗

On the mode of action of the sympathetic fibres on intestinal fluid transport: evidence for the existence of a glucose-stimulated secretory nervous pathway in the intestinal wall.

The effect of sympathetic nerve stimulation or close i.a. infusion of noradrenaline on net fluid transport was investigated on anesthetized cats. In the presence of glucose in the solution perfusing the intestinal lumen the adrenergic mechanisms increased net fluid absorption in normal intestines. Substituting glucose with mannitol in the perfusate abolished this effect of adrenergic stimulation on the net fluid uptake. Furthermore, the effect of noradrenaline on net fluid transport in normal or choleraic intestines was abolished by tetrodotoxin (TTX), a nerve conductivity blocking agent. This suggests that the sympathetic influence is dependent on intraluminal glucose and that noradrenaline exerts its effect mainly via nerves. TTX significantly increased fluid uptake from normal intestines perfused with an isotonic electrolyte solution containing glucose while a considerably smaller effect was seen in intestinal segments perfused with a solution with mannitol. Based on these findings it is proposed that glucose in some way activates intramural nervous reflex(es) in the intestinal wall. According to this hypothesis the enhancement of fluid transport induced by adrenergic stimuli is explained by an inhibition of the glucose-activated nervous secretion.

Animals↗

Segmental intestinal transporters and metabolic enzymes on intestinal drug absorption.

Recently, a physiologically-based, segregated flow model that incorporates separate intestinal tissue and flow to both a nonabsorptive and an absorptive outermost layer (enterocytes) was shown to better describe the observations on route-dependent morphine glucuronidation in the rat small intestine than a traditional physiologically-based model. These theoretical models were expanded, as the segmental segregated flow model and the segmental traditional model, to view the intestine as three segments of equal lengths receiving equal flows to accommodate heterogeneities in segmental transporter and metabolic functions. The influence of heterogeneity in absorptive, exsorptive, and metabolic functions on drug clearance, bioavailability (F), and metabolite formation after intravenous and oral dosing was examined for the intestine when the tissue was the only organ of removal. Simulations were performed for first-order conditions, when drug partitioned readily (flow-limited distribution) or less readily (membrane-limited distribution) into intestinal tissue, and for different gastrointestinal transit times. The intestinal clearance was found to be inversely related to the rate constant for absorption of a drug that was subjected to secretion and was positively correlated with the metabolic and secretory intrinsic clearances. F was positively correlated with the absorption rate constant but was inversely related to the metabolic and secretory intrinsic clearances. The gastrointestinal transit time decreased metabolite formation, increased clearance, and decreased F. The simulations further showed that a descending metabolic intrinsic clearance yielded a lower F and an ascending segmental distribution of metabolic intrinsic clearance yielded a higher F.

Algorithms↗

Intestinal tumors of rats by gastric or intestinal administration of cycad extract and cycasin.

Sprague-Dawley rats were given gastric intubation of cycad extract (group 1), rectal infusion of cycasin (group 2), or rectal indusion of cycad extract after external colostomy at 1/3 proximal portion of the large intestine (group 3). In group 1, intestinal tumors developed in any portion of the intestinal tract ranging from the duodenum to the rectum. In group 2, tumors developed in mucosa of the large intestine. In group 3, however, tumors arose from both sites of intestinal mucosa which were in contact and not in contact directly with the cycad extract infused. Possible hypothesis for intestinal tumor development by cycad extract and cycasin was presented.

Adenocarcinoma↗

Cloning of intestinal phospholipase A2 from intestinal epithelial RNA by differential display PCR.

Differential display polymerase chain reaction (DD-PCR) is a powerful technique for comparing gene expression between cell types, or between stages of development or differentiation. Differentially expressed genes may be cloned and analysed further. Here we extend the use of DD-PCR to analyse differences in gene expression between two complex epithelia: that of the small intestine and of the large intestine. The aim of this study was to identify genes expressed preferentially in Paneth cells. Paneth cells are secretory epithelial cells putatively involved in host defense and regulation of crypt cell proliferation and are found at the base of the small intestinal crypts adjacent to the stem cell zone. Of 34 clones that were analysed, partial sequencing identified two clones related to known Paneth cell products: a homologue of secretory phospholipase A2 (clone B1) and a homologue of a neutrophil defensin (clone C5). B1 was strongly expressed in Paneth cells, as demonstrated by in-situ hybridization. B1 was also expressed at a lower level in the large intestinal epithelium. A full length B1 cDNA clone was isolated and sequenced, and shown to be highly homologous to type II secretory phospholipase A2 genes, and almost identical to the enhancing factor gene and the putative gene for the MOM-1 locus. B1 expression is limited to the intestinal tract, and we propose that it be designated intestinal phospholipase A2, or i-PLA2. The method we describe is well suited to the rapid identification of genes expressed exclusively or predominantly in Paneth cells.

Animals↗

Using porcine small intestinal submucosa in intestinal regeneration.

Small intestinal submucosa (SIS) is an unusual tissue that promotes constructive tissue remodeling when applied as a xenogeneic material. The aim of our experimental study was to assess its effectiveness in intestinal regeneration. Twenty white New Zealand rabbits were anesthetized and underwent celiotomy. A 6-cm antimesenteric incision was created at the jejunal segment. An elliptical SIS graft measuring 6 cm long and 2 cm wide was sutured to the jejunal defect as a patch graft. Thirteen living rabbits were divided into groups of three and the grafts were harvested at postoperative weeks 2, 4, and 6. The obtained specimens were evaluated for gross and histologic appearance. In morphometric examination, in the 2, 4, and 6 weeks groups, the diameters of grafted intestines were larger than preoperatively by 50%, 25%, and 25% respectively; also the grafts had contracted to 0%, 25%, and 50% of their original sizes respectively. At the end of 2 weeks, the grafts were intact without evidence of epithelial regeneration. By 4 weeks, intestinal tissue regeneration was started, and epithelial coverage of the grafts was detected. The grafts were covered with a complete intestinal mucosa at 6 weeks. Remarkable regeneration marked fibroplasia, angiogenesis, and mild mononuclear cell infiltration had also occurred throughout the grafts at 6 weeks. Porcine SIS appeared an effective biodegradable scaffold, facilitating regeneration of intestinal tissue. These results suggest that SIS may be useful to increase the mucosal surface of intestine and may provide a new substance for short gut syndrome in the future.

Animals↗

Canine intestinal contents vs. simulated media for the assessment of solubility of two weak bases in the human small intestinal contents.

PURPOSE: This study was conducted to assess the relative usefulness of canine intestinal contents and simulated media in the prediction of solubility of two weak bases (dipyridamole and ketoconazole) in fasted and fed human intestinal aspirates that were collected under conditions simulating those in bioavailability/bioequivalence studies. METHODS: After administration of 250 mL of water or 500 mL of Ensure plus [both containing 10 mg/mL polyethylene glycol (PEG) 4000 as nonabsorbable marker], intestinal aspirates were collected from the fourth part of the duodenum of 12 healthy adults and from the mid-jejunum of four Labradors. Pooled samples were analyzed for PEG, pH, buffer capacity, osmolality, surface tension, pepsin, total carbohydrates, total protein content, bile salts, phospholipids, and neutral lipids. The shake-flask method was used to measure the solubility of dipyridamole and ketoconazole in pooled human and canine intestinal contents and in fasted-state-simulating intestinal fluid (FaSSIF) and fed-state-simulating intestinal fluid (FeSSIF) containing various bile salts and pH-buffering agents. RESULTS: For both compounds, solubility in canine contents may be predictive of human intralumenal solubility in the fasting state but not in the fed state. The poor agreement of results in canine and human aspirates can be attributed to the higher bile salt content in canine bile. Solubility in FaSSIF containing a mixture of bile salts from crude bile predicted satisfactorily the intralumenal solubility of both drugs in the fasted state in humans. Solubility in FeSSIF, regardless of the identity of bile salts or of the buffering species, deviated from intralumenal values in the fed human aspirates by up to 40%. This was attributed to the lack of lipolytic products in FeSSIF, the higher bile salt content of FeSSIF, and the lower pH of FeSSIF. CONCLUSIONS: FaSSIF containing a mixture of bile salts from crude bile, and FeSSIF containing lipolytic products and, perhaps, having lower bile salt content but slightly higher pH, should be more useful than canine intestinal aspirates for predicting intralumenal solubilities in humans.

Animals↗

Short-term intestinal ischemia-reperfusion alters intestinal motility that can be preserved by xanthine oxidase inhibition.

While the effects of transient intestinal ischemia on mucosa have been well investigated, less is known about its effect on motor function. An experimental study was designed to investigate the effects of ischemia-reperfusion (I/R) on intestinal motility and intestinal muscular microcirculation. Wistar albino rats were divided into four groups: (1) baseline, (2) sham operation, (3) I/R, and (4) I/R with allopurinol pretreatment. Ischemia was induced by clamping the superior mesenteric artery (SMA) for 10 min. Gastroanal transit time (GATT) was measured with serial x-rays after instillation of barium sulfate to the stomach. Intestinal muscular microcirculation was evaluated by determining the number of carbon-perfused intestinal muscular microvessels (CPIMM). I/R prolonged GATT and decreased CPIMM significantly (P < 0.01). Pretreatment with allopurinol prevented prolongation of GATT and returned the number of CPIMM to the level of sham treatment (P < 0.01). In conclusion, reperfusion after 10 min of SMA ischemia alters intestinal motility. The no-reflow phenomenon plays an important role in this alteration of motility. Administration of allopurinol before reperfusion preserves intestinal motility by preventing the occurrence of no-reflow phenomenon.

Allopurinol↗

Intestinal transit time in children with intestinal neuronal malformations mimicking Hirschsprung's disease.

A total of 106 consecutive children with intestinal neuronal malformations were included in a prospective study. The intestinal transit time was assessed using a modification of Hinton's method. The results of transit time studies, the associated specific histochemical findings, therapeutic procedures, and the clinical course on follow-up assessments over a mean period of 2.4 years were analysed. The intestinal transit time was prolonged in all 53 patients with aganglionosis and in 37 (69.8%) out of 53 children with other intestinal malformations. Eight out of 16 children with IND type B had an abnormal transit time, 1 underwent anterior resection, and 2 had a temporary colostomy. In 7 out of 8 children with hypoganglionosis and 9 out of 10 children with a reduced parasympathetic tone the transit time was prolonged. A resection was performed in 7 and 2 of these children respectively. Both patients with heterotopia of the myenteric plexus had a prolonged bowel transit and parts of the large bowel had to be resected. Only 11 out of 17 children with heterotopia of the submucous plexus, dysganglionosis, or immature ganglia had a prolonged transit time, 2 underwent sphincteromyotomy. At follow-up, all patients with malformations other than aganglionosis stated that symptoms had improved and they were willing to tolerate their complaints. However, 25 reported on persistent constipation, 6 on overflow encopresis. All children who required surgery had a prolonged intestinal transit time, but also 21 (56.8%) of 37 children who were successfully treated without surgery. None of the 16 children with normal transit had to be operated. It is concluded that specific histochemical findings do not always correlate with delayed intestinal transport. The determination of the intestinal transit time represents an important tool to identify the clinical relevance of histochemical findings in the individual patient.

Child, Preschool↗

Is type III intestinal metaplasia an obligatory precancerous lesion in intestinal-type gastric carcinoma?

This retrospective study was performed to assess whether type III intestinal metaplasia is an obligatory precancerous lesion of intestinal-type gastric carcinoma and to determine its possible use as a marker of enhanced cancer risk. From 48 consecutive patients with gastric cancer who underwent a gastrectomy over a 3-year period (mean age 72.0 years; 29 M/19 F), at least two sections from antrum, corpus and tumour-surrounding mucosa were obtained for the examination of presence and subtypes of intestinal metaplasia (IM). It was found that 77.1% of the carcinomas were of the intestinal type and 22.9% of the diffuse type. The intestinal-type was more often found in males (P = 0.01); the mean age at diagnosis in this type was higher than in the diffuse cancer group (P = 0.004). There was a high prevalence of total IM in both the intestinal (75.7%) and diffuse group (88.9%). Type I IM was predominant in antrum and corpus of patients from both groups. Type III IM was only found among patients with intestinal-type carcinoma. However, its prevalence was rather low (26.3%). Therefore the absence of this lesion in patients with other risk factors cannot be used as an argument for lowering the degree of surveillance and its presence seems to be sufficient indication for long-term follow-up.

Aged↗

[Analysis of intestinal motility by enteric sound recording system--clinical evaluation of aclatonium napadisilate and prostaglandine F2 alpha on intestinal movement].

In patients with so-called postoperative enteroplegia after laparotomy, effects of aclatonium napadisilate (abbreviated as TM-723 hereinafter) and prostaglandine F2 alpha (abbreviated as PGF2 alpha) on intestinal motility were determined using an enteric sound recording system (phonointestinography) and simultaneously the time required for breaking wind after operation was examined. TM-723 was administered at a dose of 50 mg from the intestinal fistula after dissolved in 5 ml of physiological saline, while PGF2 alpha was administered as an i.v. drip infusion at a dose of 2 mg dissolved in 500 ml of 5% glucose solution. Intestinal movement was determined at McBurney point using the enteric sound recording system developed at our department. The time required for breaking wind after operation was determined in three groups received TM-723, PGF2 alpha and placebo, respectively. No significant intergroup difference was observed. As to effect on intestinal motility, TM-723 increased intestinal motility when it was reduced in so-called postoperative enteroplegia phase. Its efficacy became higher with a decrease in motility. On the contrary, TM-723 rather tended to depress intestinal motility when it was increased. On the other hand, PGF2 alpha tended to increase intestinal motility regardless of motility in enteroplegia phase.

Acetylcholine↗

[Phylogenetic analysis of intestinal bacterium using the distribution of intestinal bacterium in cyprinidate fishes and 16S DNA sequence data set].

There is a complex- and multi-effect for interdependent survival between intestinal- microorganisms and hosts. The symbiosis or coevolution that results from this effect for interdependent survival is used to reveal the phylogenies of hosts as well as intestinal microorganisms. The symbiosis or coevolution between intestinal microorganisms and hosts has been generated by interactive natural selection occurred between them. The symbiosis information that has been formed by interactive natural selection during a long evolutionary process must be recorded in DNA sequences. According to this point of view,we analyzed the phylogeny of 9 intestinal bacteria genera using their contents in intestines of 8 Cyrinidate species. At the same time,we fetched the 16S rRNA gene DNA sequences of 43 intestinal bacteria species being included in these nine genera of six intestinal families from GeneBank and constructed phylogenetic trees by NJ and MP methods. The NJ tree and MP tree have the same topologic configuration and are identical with the classical phylogenetic tree. Both the trees of 16S rRNA gene separated 43 bacteria species into gram-negative bacteria group and the gram-positive bacteria group,which are the first branches. Each of the first branches (groups) made again 6 subbranches (subgroups) where each subbranch is a family.Especially,the subbranch (subgroup) of enterobacteriaceace made again four small branches as genus taxon. This tree also shows that bacilliform bacterium is distinct from each other in the NJ and MP trees. After all species on the tree are merged,the topological configuration of the unrooted tree of 16S gene is closed to that of the host range unrooted tree.However,the position of bacillus is greatly changed on both the unrooted trees. The difference can be found if we increase the examination level and extend the hosts examed.

English Abstract↗

[Estimation of efficiency of complex-method intestinal therapy using Nitella flexilis test-object for acute intestinal obstruction of various etiology].

The aim of our investigation was to define the presence of correlation between toxicity of intestinal contents and clinical and laboratory indices in the cases of ileus and peritonitis with the use of complex method of enteral therapy. Experiments have been carried out on internodal cells of Nitella Flexilis water plant. Efficiency of the given method (investigation of toxicity of the intestinal content with the use of test-object Nitella Flexilis) has been studied in 25 patients, who underwent correction of morpho-functional status of small intestine. Results of investigation have shown that changes in toxicity of intestinal content reliably reflect the nature and dynamics of the pathobiologic processes proceeding in GIS in treated patients suffering from acute intestinal impassability. Using the method of correction of morpho-functional status of small intestine decreases the role of "Intestinal factor" in formation of endogenous intoxication, which in turn results in early normalization of clinical and laboratory indices, favored process of early post-operational period, decreased rate of lethality and shortened rehabilitation period.

Acute Disease↗

[Comparison of different morphometric methods to estimate the surface of the rat intestinal mucosa. Their relationship to intestinal function (author's transl)].

In contrast to the human small intestine with predominantly fingerlike villi, trapezoid forms are encountered in the small bowel of the rat. For this reason, it is doubtful whether it is sufficient to measure the villus height alone as a representative parameter for the assessment of the villus surface. In this study, the method developed by Clarke is employed to calculate the villus surface per unit area of intestine. The data obtained by this method are compared with those estimated planimetrically. Correlations were then established between the absorptive capacity and enzymatic activity of the intestinal mucosa on the one hand, and the villus height or the villus area obtained by each method on the other hand. Under our experimental conditions--a hyperregenerative transformation of the intestinal mucosa caused by chronic exposure to glucagon--the results indicate that the calculated villus surface per unit area of small intestine correlates better than the villus height with the functional parameters of the intestinal mucosa.

Animals↗

Intestinal secretagogues increase cytosolic free Ca2+ concentration and K+ conductance in a human intestinal epithelial cell line.

A human intestinal epithelial cell line (Intestine 407) is known to retain receptors for intestinal secretagogues such as acetylcholine (ACh), histamine, serotonin (5-HT) and vasoactive intestinal peptide (VIP). The cells were also found to possess separate receptors for secretin and ATP, the stimulation of which elicited transient hyperpolarizations coupled to decreased membrane resistances. These responses were reversed in polarity at the K+ equilibrium potential. The hyperpolarizing responses to six agonists were reversibly inhibited by quinine or quinidine. By means of Ca2(+)-selective microelectrodes, increases in the cytosolic free Ca2+ concentration were observed in response to individual secretagogues. The time course of Ca2+ responses coincided with that of hyperpolarizing responses. The responses to ACh and 5-HT were abolished by a reduction in the extracellular Ca2+ concentration down to pCa 7 or by application of Co2+. Thus, in Intestine 407 cells, not only the intestinal secretagogues, which are believed to act via increased cytosolic Ca2+ (ACh, 5-HT and histamine), but also those which elevate cyclic AMP (VIP, secretin and ATP) induce increases in cytosolic Ca2+, thereby activating the K+ conductance. It is likely that the origin of increased cytosolic Ca2+ is mainly extracellular for ACh- and 5-HT-induced responses, whereas histamine, VIP, secretin and ATP mobilize Ca2+ from the internal compartment.

Calcium↗

Diamine oxydase in rabbit small intestine: separations from a soluble monoamine oxidase, properties and pathophysiological significance in intestinal ischemia.

From all mammals investigated so far only in rabbits diamine oxidase could not be detected in any tissue except the gut. Thus this species was chosen for studying the physiological and pathophysiological function of this enzyme in the gastrointestinal tract. By gel filtration on Sephadex G 50 and G 200 the enzyme was purified 100-fold, separated from a soluble monoamine oxidase, and the properties of the two enzymes were determined. Diamine oxidase from rabbit small intestine deaminated putrecine (Km = 1.3 times 10(-4) M, pH-optimum 6.4-6.9) and histamine (Km = 8 times 10(-5) M, pH-optimum 7.5), but not serotonin, and was inhibited by aminoguanidine, but not by pargyline. Soluble monoamine oxidase from rabbit small intestine catabolized serotonin (Km = 1.8 times 10(-4) M, pH-optimum 8.8) but not putrescine and histamine, and was inhibited by pargyline, but not by aminoguanidine. Based on its properties in vitro intestinal diamine oxidase could inactivate the vasoactive biogenic amine histamine in vivo. To confirm this hypothesis, in rabbits the small intestine was damaged severely by inducing total intestinal ischemia, which occurs as mesenteric infarction also in human subjects and is accompanied by histamine release. Treatment with aminoguanidine and ischemia killed the animals 3-times faster than ischemia alone, which supported our hypothesis on a protective role of intestinal diamine oxidase against histamine.

Animals↗