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ABSTRACT: Analysis of human intestinal epithelial regeneration has been limited. This study has used a novel SCID mouse-human model to test the hypothesis that distinct stages of human intestinal epithelial regeneration are accompanied by differential expression of growth regulatory genes. Disaggregated epithelium, which included crypt cell aggregates, was isolated from human fetal small intestine and transplanted subcutaneously in SCID mice. This method induced a coordinated regeneration response and enabled temporal separation of cell populations at different stages of histogenesis and cytodifferentiation. Graft epithelium was identified using a specific anti-human monoclonal antibody (MAb 5D3) against cytokeratins 8 and 18. Functional epithelial lineages were identified by appropriate markers. Growth regulatory genes relevant to proliferation and apoptosis, including Bcl-2, p53 and Ki67, were assayed at different stages of regeneration. During early regeneration, Bcl-2, p53, and Ki67 were expressed throughout the epithelial compartment. On completion of regeneration, these genes were expressed only in crypt epithelium and were absent from villi. This study has established a novel SCID mouse-human model of intestinal epithelial regeneration. During early regeneration, increased Bcl-2 and Ki67 expression may indicate suppression of apoptosis and enhanced proliferation respectively, consistent with expansion of the stem cell fraction. The p53 gene may influence pathways of differentiation during regeneration, analogous to its role during development.
The Journal of Pathology 02/1999; 187(2):229-36. · 6.32 Impact Factor
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The American Journal of Gastroenterology 02/1999; 94(1):3-5. · 7.28 Impact Factor
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ABSTRACT: Cyclin D3 promotes cell cycle progression but its expression and prognostic significance in human colorectal cancer is unknown. This study assayed cyclin D3 expression against cell cycle phase fraction and Duke's stage in 35 fresh human primary colorectal cancers. DNA content, cell cycle phase fraction and cyclin D3 expression were assessed by flow cytometry in disaggregated tumors. Cyclin D3 expression and S-phase fraction were independently related to Duke's stage. In Duke's stage C tumors, a higher proportion of cells expressed cyclin D3 (14.4 vs. 8.8%, mean; p < 0.05 by Mann-Whitney U test) and were in DNA synthesis (S) phase (21.1 vs. 9.7%, mean; p < 0.05 by Mann-Whitney U test). Neoplastic deregulation of cyclin D3 expression may provide a selective growth advantage which is related to stage in human colorectal cancer.
Oncology 01/1999; 56(1):66-72. · 2.27 Impact Factor
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ABSTRACT: The cytotoxicity of the antifolate inhibitors of de novo purine biosynthesis, lometrexol (LTX) and LY309887, can be abolished by hypoxanthine (HPX) salvage. The nucleoside transport inhibitor, dipyridamole (DP) can prevent HPX rescue from LTX growth inhibition in a cell line-specific manner. The studies described here have shown that, excluding colon and hematological malignancies, DP prevents HPX rescue from LTX growth inhibition in approximately one-third of cell lines with otherwise limited tissue specificity. The clinical dose-limiting toxicities of antipurine antifolates are to the bone marrow and gastrointestinal tract. In vitro models of these normal tissues were established, and the effect of DP on HPX rescue from LY309887 treatment was studied. Growth inhibition assays are not feasible in these primary cultures; therefore, an alternative assay, cellular ATP depletion, was validated in four tumor cell lines as a marker of de novo and salvage purine synthesis. In LY309887-treated cells, DP prevented HPX-mediated maintenance of ATP levels only in cell lines in which DP inhibited HPX rescue from antifolate cytotoxicity. Hence, ATP depletion is a reliable indicator of sensitivity of HPX transport to DP when direct cell growth measurement is impractical. In primary cultures of human hematopoetic progenitor cells and mouse small intestine, coincubation with HPX prevented LY309887-mediated ATP depletion, which was not blocked by DP. These data suggest that DP would not prevent HPX rescue from antipurine antifolate growth inhibition in sensitive normal tissues, whereas activity against certain solid human tumors would be maintained.
Clinical Cancer Research 12/1998; 4(11):2895-902. · 7.74 Impact Factor
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ABSTRACT: Functional dyspepsia is a chronic disorder of unknown aetiology. The lack of endoscopic abnormalities in patients with this disorder has led many physicians to believe that gastro-oesophageal reflux disease may be responsible for most symptoms. Our group has addressed this issue, by pathophysiological studies in a large cohort of Dundee patients with persistent dyspeptic symptoms. Peptic ulcer and gallstones were excluded in all patients by appropriate tests. Ambulatory pH monitoring showed oesophageal acid reflux that lay above the conventional diagnostic threshold in approximately 20% of patients. This subset was diagnosed as having gastro-oesophageal reflux disease. In the remainder, moderate or severe reflux-like symptoms were reported by approximately 44% patients, who were categorized as reflux-like functional dyspepsia. Reflux symptoms were mild or absent in 36% patients, who were categorized as non-reflux-like dyspepsia. While oesophageal pH profiles lay within the conventional normal range in both of these functional dyspepsia subgroups, patients with reflux-like functional dyspepsia had significantly greater acid exposure values, including total oesophageal acid exposure time, percentage time at a pH of less than 4.0, DeMeester scores and pain reflux event correlation. Hence patients with reflux-like functional dyspepsia have oesophageal acid exposure that lies below the diagnostic threshold for gastro-oesophageal reflux disease but exceeds that of patients with non-reflux dyspepsia. The high pain/reflux event correlation in reflux-like functional dyspepsia suggests that subthreshold oesophageal acid exposure may be associated with troublesome reflux symptoms.
Baillière s Clinical Gastroenterology 10/1998; 12(3):463-76.
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ABSTRACT: Irritable bowel syndrome is a common cause of abdominal pain and discomfort and may be related to disordered gastrointestinal motility. Our aim was to assess the effects of long-term treatment with a prokinetic agent, cisapride, on postprandial jejunal motility and symptoms in the irritable bowel syndrome (IBS).
Thirty-eight patients with IBS (constipation-predominant, n = 17; diarrhoea-predominant, n = 21) underwent 24-h ambulatory jejunal manometry before and after 12 week's treatment [cisapride, 5 mg three times daily (n = 19) or placebo (n = 19)].
In diarrhoea-predominant patients significant differences in contraction characteristics were observed between the cisapride and placebo groups. In cisapride-treated diarrhoea-predominant patients the mean contraction amplitude was higher (29.3 +/- 3.2 versus 24.9 +/- 2.6 mm Hg, cisapride versus placebo (P < 0.001); pretreatment, 25.7 +/- 6.0 mm Hg), the mean contraction duration longer (3.4 +/- 0.2 versus 3.0 +/- 0.2 sec, cisapride versus placebo (P < 0.001); pretreatment, 3.1 +/- 0.5 sec), and the mean contraction frequency lower (2.0 +/- 0.2 versus 2.5 +/- 0.4 cont./min, cisapride versus placebo (P < 0.001); pretreatment, 2.5 +/- 1.1 cont./min] than patients treated with placebo. No significant differences in jejunal motility were found in the constipation-predominant IBS group. Symptoms were assessed by using a visual analogue scale before and after treatment. Symptom scores relating to the severity of constipation were lower in cisapride-treated constipation-predominant IBS patients [score, 54 +/- 5 versus 67 +/- 14 mm, cisapride versus placebo (P < 0.05); pretreatment, 62 +/- 19 mm]. Diarrhoea-predominant IBS patients had a higher pain score after cisapride therapy [score, 55 +/- 15 versus 34 +/- 12 mm, cisapride versus placebo (P < 0.05); pretreatment, 67 +/- 19 mm].
Cisapride affects jejunal contraction characteristics and some symptoms in IBS.
Scandinavian Journal of Gastroenterology 07/1998; 33(6):605-11. · 2.02 Impact Factor
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ABSTRACT: The ontogeny of intestinal phase I and II xenobiotic metabolising enzymes and influence on susceptibility to genotoxic injury, are unclear. This study assessed expression of cytochrome P450 monooxygenases (CYP1A, CYP2B, CYP2C, CYP3A, CYP4A), glutathione-S-transferase (GSTA1/2, GSTA3, GSTA4, AND GSTM1), and uridine diphosphate glucuronosyl transferase (UGT) in rat intestine, between fetal life and maturity. Enzyme induction and DNA adduct formation were assessed after 3-methylcholanthrene (MC) exposure. Untreated rat intestine expressed CYP2B, GSTA1/2, GSTA4 and UGT at all stages of maturation, although CYP2B and GSTA1/2 increased in postnatal life. MC induced new expression of CYP1A, GSTA3 and enhanced expression of GSTA1/2 and UGT. Age-dependent differences of enzyme induction and DNA adduct formation between pre- and postnatal intestine and during postnatal maturation, were observed. Rat intestinal epithelium shows variable competence for MC metabolism and sustains disparate levels of DNA adducts during pre- and postnatal development.
Chemico-Biological Interactions 06/1998; 113(1):27-37. · 2.46 Impact Factor
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ABSTRACT: The carcinogenic potency of many mutagens is increased in conditions of tissue regeneration. This involves fundamental changes of cellular division and differentiation, in intestinal epithelium. However, effects on epithelial capacity for carcinogen metabolism and susceptibility to genotoxic injury are unknown. Using a novel rat model, this study assessed expression of cytochrome P450 mono-oxygenases (Cyps), glutathione S-transferases (GSTs) and uridine diphosphoglucuronosyl transferase (UGT) in intestinal epithelium during sequential stages of regeneration. Enzyme induction and DNA adduct formation were also assessed after benzo[a]pyrene (BaP) exposure. Control assays were carried out in normal intestinal epithelium. Fewer phase I and II xenobiotic metabolizing enzymes were expressed in regenerating intestinal epithelium than in normal control intestinal epithelium (GSTA3, UGT in regeneration vs Cyp2B, GSTA1/2, GSTA4, GSTP1, UGT in control). Benzo[a]pyrene induced GSTA3 and UGT in regeneration vs Cyp1A, Cyp2B, GSTA1/2, GSTA3, GSTA4, GSTP1 and UGT in control normal intestinal epithelium. Benzo[a]pyrene induced low levels of GSTA3 in early regenerating intestinal epithelium but induction increased by >2-fold at late stage regeneration. Higher levels of benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE) DNA adducts were formed at early stages of regeneration, than at later stages. Intestinal epithelium displayed reduced metabolic competence and differential susceptibility to genotoxic injury from BaP, during regeneration.
Carcinogenesis 12/1997; 18(11):2171-7. · 5.70 Impact Factor
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ABSTRACT: The relationship of small-bowel dysmotility to dietary intake in irritable bowel syndrome (IBS) is obscure.
This study evaluated postprandial jejunal motility in IBS patients classified as constipation-predominant (n = 25) or diarrhoea-predominant (n = 35) and compared results against 18 volunteers. Twenty-four-hour ambulatory jejunal manometry was carried out in all subjects, and recordings were analysed by microcomputer and visual assessment.
By means of analysis of variance (fitting factors for channels, meals, and time periods) postprandial contraction frequency was greater in both patient groups compared with normal (constipation-predominant versus normal, diarrhoea-predominant versus normal; P < 0.001). In the constipation-predominant cohort, contraction amplitudes were lower (constipation-predominant versus normal; P < 0.002). Discrete cluster contractions occurred with similar frequency and duration in both patient and volunteer groups.
Quantitative differences of postprandial jejunal contraction characteristics have been shown between patients with IBS and healthy volunteers. Contraction frequency is greater than normal in both diarrhoea- and constipation-predominant categories, whereas contraction amplitudes are lower in constipation-predominant patients.
Scandinavian Journal of Gastroenterology 02/1997; 32(1):39-47. · 2.02 Impact Factor
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ABSTRACT: Conventional models of postnatal mucosal regeneration are cumbersome and limited: a novel model is described here. In addition, the influence of cell interactions on mucosal regeneration is examined within the model.
Postnatal rat small intestinal mucosa was digested by enzymes to yield heterotypic cell aggregates (CA). CA colony forming ability, growth, and limited cytodifferentiation were assessed in vitro. CA were transplanted subcutaneously and retrieved for histological examination at staggered intervals to assess neomucosal morphogenesis and cytodifferentiation in vivo. Cell interactions in CA were disrupted by enzymes, thus producing cell suspensions (CS). Regeneration by CA and CS were compared.
CA produced proliferative colonies in vitro and showed a temporal sequence of neomucosal morphogenesis and differentiation in vivo. CA colonies were more numerous within 24 hours of primary culture and had greater cellularity by 96 hours than CS colonies. Alkaline phosphatase was expressed only by 258 of 696 CA colonies (37%). CA subcutaneous grafts (48 of 56 (87%)) regenerated small intestinal neomucosa while CS were unsuccessful.
These methods provide a model of mucosal regeneration which includes constituent processes of colony formation, growth, neomucosal morphogenesis, and cytodifferentiation. Preservation of cell interactions within CA seems advantageous to regeneration within the model.
Gut 06/1996; 38(5):679-86. · 10.11 Impact Factor
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ABSTRACT: The relation between symptom severity in gastro-oesophageal reflux disease (GORD) and quantitated oesophageal acid reflux is variable. Furthermore, when oesophageal acid exposure lies within the conventional normal range, the cause of the symptoms is unknown. This prospective study evaluated 24 hour ambulatory oesophageal pH profiles in relation to objective symptom scores in 100 dyspeptic patients who were free from ulcer and gall stones. Twenty patients had raised oesophageal acid exposure and reflux symptoms consistent with GORD, and 80 had oesophageal pH profiles within the conventional normal range. Forty four of the 80 had severe or moderate reflux symptoms and were classified as having reflux like functional dyspepsia (RFD); 36 had minimal or absent reflux symptoms, and were categorised as having non-reflux dyspepsia (NFD). While oesophageal pH profiles lay within the conventional normal range in both functional dyspepsia subgroups, patients with RFD had consistently greater acid exposure values as follows: mean (SEM) total oesophageal acid exposure time, RFD 16.2 (2.56) min v NFD 9.05 (2.0) min (p < 0.03); percentage of time with pH < 4, RFD 1.4 (0.2) v NFD 0.8 (0.2) (p < 0.03); DeMeester scores, RFD 12.8 (0.5) v NFD 11.4 (0.4) (p < 0.03). The RFD group had a pain/reflux event correlation of 23.8 (5.3)% v 8.1 (3.7)% for the NFD group (p < 0.01). This study shows that patients with RFD have oesophageal acid exposure that lies below the diagnostic threshold for GORD, but exceeds that of patients with NFD. The high pain/reflux event correlation in RFD, suggests that subthreshold oesophageal acid exposure may be associated with troublesome reflux symptoms.
Gut 02/1995; 36(2):189-92. · 10.11 Impact Factor
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ABSTRACT: Small intestinal epithelium digests and absorbs nutrients. Crypt stem cell transplantation can generate neomucosa with normal morphology, but the digestive and absorptive capacities of this neomucosa are unknown. This study evaluates stem cell induced neomucosal brush border digestive enzyme activity and nutrient transport function. Rodent small intestinal epithelial stem cells were isolated by enzymatic digestion, then grafted to inbred recipients. Grafts were retrieved at 25 days, and apical brush border membrane vesicles prepared for quantitative assays. Neomucosal lactase, sucrase, aminopeptidase N, and alkaline phosphatase activity was determined by incubation with enzyme specific substrate. Neomucosal sodium dependent D-glucose transport was evaluated by incubation with D-[U-14C] glucose. Comparative assays were performed in age-matched control intestine. Neomucosal digestive enzyme activities and D-glucose transport were all similar in neomucosa and control small intestine.
The American Journal of Surgery 02/1995; 169(1):120-5. · 2.78 Impact Factor
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ABSTRACT: Transport of the carcinogen benzo[a]pyrene in apical membrane vesicles (AMV) from normal human intestine, was investigated. Benzo[a]pyrene transport was found in AMV throughout the small intestine, but was greatest in colon. Evidence suggesting involvement of P-Glycoprotein (P-Gp), included (1) comparable transport of P-Gp substrate doxorubicin, (2) transport stimulation by ATP and (3) transport suppression by the P-Gp inhibitor, verapamil.
Biochimica et Biophysica Acta 06/1994; 1226(2):232-6. · 4.66 Impact Factor
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ABSTRACT: A novel method to study the generation of rat small intestinal mucosa, by transplantation of disaggregated postnatal rat small intestinal epithelium is described. Cellular aggregates, comprised of epithelium with attached proliferative cells and closely associated stromal tissue, were isolated from postnatal rat small intestine by enzymatic digestion, then grafted immediately to the subcutaneous plane of adult recipients. On graft retrieval after 14 days, 39% of cellular transplants to nude mice, and 84% of cellular transplants to inbred rats had developed into small intestine-like structures. These structures were comprised of a circumferential layer of epithelium surrounding a central mucin filled lumen. This neomucosal layer exhibited well formed crypts and villi, and contained all epithelial stem cell lineages i.e. absorptive enterocytes, goblet cells, Paneth's cells and entero-endocrine cells. Proliferative activity within this neomucosa was confined to crypt regions as in normal postnatal small intestine. Developmental maturation within the regenerated neomucosa was demonstrated by organotypic morphogenesis, i.e. formation of mature crypts and villi, and progressive cytodifferentiation with increased numbers of goblet cells, entero-endocrine cells and Paneth's cells. Altered patterns of brush border enzyme expression further confirmed a temporal progression of development within neomucosal enterocytes. It is concluded that after "extensive" mucosal disaggregation, postnatal small intestinal epithelial progenitor cells retain the capacity for organotypic regeneration of neomucosa when transplanted to ectopic sites in adult recipients. These small aggregates of epithelium and stroma are capable of generating the topographical signals necessary for the three dimensional regeneration of this tissue. Furthermore, the multipotent generative potential of the stem cells within these cellular aggregates is maintained with production of all progeny.
Differentiation 05/1994; 56(1-2):91-100. · 2.81 Impact Factor
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ABSTRACT: A novel method of colonic mucosal replacement by transplantation of disaggregated small intestinal epithelium is described. Thirty-one inbred rats had the ascending colon isolated, and surgical mucosectomy was performed on the "free" loop. Epithelial cell aggregates were isolated from postnatal small intestine using collagenase and dispase digestion, then 20 microL of the cell suspension was "seeded" over the denuded colonic muscle of 25 recipient rats. Six control rats had surgical mucosectomy only. All loops were retrieved after 14 days for histologic examination. Stem cell lineage studies were used with selective staining protocols to identify enterocytes, goblet cells, entero-endocrine cells, and Paneth cells. A neomucosa with typical small bowel morphology including crypts and villi and all four stem cell lineages was regenerated by transplanted cells on the colonic muscle in 19 of 25 (76%) recipients. Control loops showed no epithelial regrowth confirming total mucosectomy. With appropriate stromal support, transplanted small intestinal stem cells have the capacity to re-epithelialize denuded colonic muscle with small bowel neomucosa.
The American Journal of Surgery 02/1994; 167(1):67-72. · 2.78 Impact Factor
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Gut 10/1993; 34(9):1153-5. · 10.11 Impact Factor
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ABSTRACT: The characteristics of the phases of the migrating motor complex (MMC) were studied in the antrum, duodenum, and jejunum after alteration of intraluminal gas and acaloric fluid in 17 healthy volunteers. Aspiration of gas and fluid from the upper gastrointestinal tract reduced motor activity. In the antrum and duodenum, phase II contraction amplitude decreased, while in the duodenum and jejunum, the duration of phase II decreased and phase I increased. Phase III contraction frequency decreased in the duodenum only. Intragastric instillation of gas caused an increase of phase II duration and contraction amplitude in all regions. Similar effects were observed after intragastric instillation of fluid. Fasting periodic motor activity is responsive to volume changes of intraluminal gas and acaloric liquid content.
The American journal of physiology 11/1992; 263(4 Pt 1):G533-7.
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Transplantation Proceedings 07/1992; 24(3):1061-4. · 1.00 Impact Factor
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ABSTRACT: A method of small bowel mucosal augmentation called ileal mucosal fenestration and colonic autotransplantation (IMFCA) was devised and tested in pigs. In this technique, a vascularized mucosal graft was harvested from a 12-cm ileal loop, fenestrated by serial incision and then expanded to 20 cm. A 20-cm colonic loop was isolated and surgical mucosectomy was carried out. The fenestrated ileal mucosal graft was then autotransplanted into the prepared colon and the resulting composite structure was exteriorized as a Thiry-Vella loop. With this technique, ileal mucosal fenestrations healed by lateral epithelial in-growth, giving a new mucosal continuum within the recipient colon. At 60 days after surgery, the surface area of transplanted mucosa exceeded that within the original ileal loop by approximately 85 per cent. At this time, the transplanted mucosa had morphology and capacity for Na(+)-dependent glucose transport which were indistinguishable from those of control ileal mucosa.
British Journal of Surgery 12/1991; 78(11):1309-12. · 4.61 Impact Factor
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ABSTRACT: A computerised system is developed for the acquisition and display of gastrointestinal motility data which utilises a purpose developed software program called 'PC-motil', running on an IBM compatible microcomputer. 'PC-motil' displays data during collection, writes data to disk file and compresses all data at the end of a study on to a single monitor screen for convenient overview. Any area of interest, in single or multiple channels, may be selected and expanded for detailed examination. This system is tested by the recording of gastric and jejunal motility patterns of 11 healthy volunteers in fasting and fed states. All antral and jejunal migrating motor complexes (MMCs) in fasting studies, as well as all fed motility patterns were recognisable in both 'compressed' and 'expanded' form. The reproduction of motility patterns by the computer based system was indistinguishable from that of a conventional analogue chart recorder. This computerised system provides a convenient and cost-effective means of acquisition, storage and display of motility data in digital form.
Medical & Biological Engineering & Computing 06/1991; 29(3):304-8. · 1.88 Impact Factor
