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Background: Short chain fatty acids derived from dietary fiber may protect against invasive colon cancer by modulating degradative matrix metalloproteinases (MMPs) and protective tissue inhibitor matrix metalloproteinases (TIMPs). Since invasion depends on the MMP/TIMP ratio, we hypothesized that short chain fatty acids inhibit colon cancer invasion by inhibiting MMPs and stimulating TIMPs., Materials and Methods: SW1116 colon cancer cells were seeded onto Matrigel-coated Boyden chambers and treated with unsupplemented media or media containing 10 mM acetate, propionate, or butyrate. SW1116 invasion was quantitated by light microscopy and conditioned media were assayed by ELISA for MMP-1,2,3,9; TIMP-1,2; MMP/TIMP complex; and urokinase plasminogen activator (uPA). All data are expressed as mean percentage of control +/- SE (n > 6)., Results: Although all three short chain fatty acids inhibited invasion, butyrate was more potent than either acetate or propionate, inhibiting SW1116 invasion by 35 +/- 1% of control (n = 18, P < .0001) vs. 18 +/- 9% (n = 7, P < .05) for acetate and 10 +/- 6% (n = 7, P < .05) for propionate. MMP-2 was not modulated by any of the short chain fatty acids while MMP-1 was modulated only by butyrate and MMP-3 by propionate. Acetate did not modulate MMPs, TIMP-1, or uPA, but stimulated TIMP-2. In contrast, propionate and butyrate stimulated MMP-9 and TIMP-2 by 119-233% and both inhibited uPA by 8-16%. TIMP-1 was stimulated only by butyrate and actually inhibited by propionate. Only butyrate stimulated both TIMP-1 and TIMP-2., Conclusions: These data suggest that dietary fiber may protect against invasive colon cancer through stimulation of TIMP and inhibition of uPA activities, rather than through short chain fatty acids effects on the activities of the MMPs studied.

1. Enterocyte motility depends critically on cell-matrix interactions. Although still incompletely understood, these appear critically dependent upon integrin-mediated cell adhesion. 2. In addition to providing a mechanism for cell adhesion and traction, the integrins are likely to serve as true receptors for the matrix across which cell motility occurs, initiating signals by both mechanical and chemical means that alter cell phenotype and proliferation as well as cell motility. 3. Sound rationale now exists to postulate that soluble growth factors within the extracellular milieu regulate intestinal mucosal healing not only directly but also indirectly by modulating integrin expression and organization.

We examined whether endothelial cells (ECs) inhibit smooth muscle cell (SMC) transforming growth factor-beta 1 (TGF-beta 1) activation in bilayer coculture. Western analysis showed that SMCs cocultured with ECs as a bilayer had lower amounts of active TGF-beta 1 protein compared with SMCs cultured alone and SMCs cocultured with ECs as a monolayer. EC inhibition of TGF-beta 1 activation could be blocked with plasminogen activator inhibitor-1 (PAI-1) antibody. Similarly, SMC hill-and-valley growth, a marker for TGF-beta 1 activity, was present in SMCs cultured alone and SMCs cocultured with ECs as a monolayer but was absent in SMCs cocultured as a bilayer. SMCs cocultured with ECs as a bilayer migrated at a greater rate than SMCs cultured either alone or cocultured as a monolayer. The EC effect on SMC migration was inhibited by the addition of 5 ng/ml TGF-beta 1. ECs had no effect on SMC RNA levels of TGF-beta 1. PAI-1 levels were increased in ECs and ECs cocultured with SMCs compared with SMCs cultured alone. ECs inhibit TGF-beta 1 activation in bilayer coculture. This appears to be mediated through an increase in EC PAI-1 release. Alterations in coculture conditions, in particular the degree of EC-SMC cell contact, have profound effects on this process.

Short-chain fatty acids produced by bacterial fermentation of dietary fiber may provide a tonic stimulus to colonocyte differentiation that contributes to the protective effect of fiber against colorectal malignancy. Since brush-border enzymes are common markers of colonocytic differentiation, we compared the effects of equimolar (10 mmol/L) concentrations of the three most common short-chain fatty acids, acetate, butyrate, and propionate, on the alkaline phosphatase and dipeptidyl dipeptidase specific activity of human colonic mucosal biopsies obtained from normal volunteers. Only butyrate significantly stimulated alkaline phosphatase specific activity (50.4% +/- 18.6%, P < .05). Short-chain fatty acid stimulation of dipeptidyl dipeptidase did not achieve statistical significance. Fibers yielding high colonic butyrate levels could have different effects on human colonic mucosal differentiation.

Short chain fatty acids such as sodium butyrate are concentrated in the colonic lumen and may protect against colon carcinogenesis by maintaining colonocytic differentiation, but the mechanisms by which they act are not fully understood. It has recently been suggested that short chain fatty acids modulate cellular tyrosine kinase activity in addition to altering chromatin structure via regulation of histone acetylation and DNA methylation. Therefore, the authors evaluated the influence of tyrosine kinase inhibition on the effects of 10 mM butyrate on human Caco-2 intestinal epithelial differentiation, using alkaline phosphatase and dipeptidyl dipeptidase specific activity as markers of differentiation, and two tyrosine kinase inhibitors, of different mechanisms of action and different effects on Caco-2 brush border enzyme specific activity, to block tyrosine kinase activity. As expected, butyrate stimulated both alkaline phosphatase and dipeptidyl dipeptidase specific activity. The tyrosine kinase inhibitors prevented, and indeed one inhibitor reversed the effects of butyrate on alkaline phosphatase specific activity. However, tyrosine kinase inhibition did not prevent butyrate stimulation of dipeptidyl dipeptidase specific activity. Different pathways are likely to regulate the effects of butyrate on expression of these two brush border enzymes. Butyrate stimulation of alkaline phosphatase, but not dipeptidyl dipeptidase, may involve tyrosine phosphorylation signaling., (Copyright 1998 Academic Press.)

Peptide YY (PYY) is produced by colonic mucosal endocrine cells and modulates gastrointestinal endocrine activity through specific Y-receptors. The direct effects of PYY on intestinal mucosal growth and differentiation remain uncharacterized. The abundance of PYY in colonic mucosa suggests that PYY acts locally to maintain colonocytic differentiation. We tested this hypothesis in human Caco-2 intestinal epithelial cells, which express alkaline phosphatase (AP) and dipeptidyl dipeptidase (DP), brush-border enzymes differentially concentrated in large and small intestinal mucosa, respectively. The effects of PYY on enzyme specific activity were compared with those of pancreatic polypeptide, neuropeptide-Y, vasoactive intestinal peptide, pentagastrin, bombesin, and selective Y1- and Y2-receptor agonists. Brush-border enzyme activity was assessed by AP and DP specific activity in cell lysates quantitated spectrophotometrically following synthetic substrate digestion. PYY, neuropeptide-Y, pancreatic polypeptide, and vasoactive intestinal peptide (10(-7) mol/L) stimulated AP activity. PYY brought about the greatest increase (38.0%+/-11.0%, n=48). Only PYY decreased DP specific activity (7.9%+/-2.2%, n=48). The Y2-agonist but not the Y1-agonist mimicked these PYY effects (increasing AP 28.3%+/-3.5% and decreasing DP 10.4%+/-3.6%). These data suggest that PYY promotes differentiation toward a colonocytic phenotype in Caco-2 intestinal epithelial cells and that this effect may be mediated through the Y2-receptor subtype.

Sucralfate, which binds to the matrix of the ulcer bed, is theoretically advantageous for duodenal ulcer therapy, but has not fulfilled its promise clinically. We examined the effects of sucralfate and related compounds in a human intestinal epithelial (Caco-2) cell culture model of restitution on sheet migration across and adhesion to collagen I. Migration was quantitated across a collagen I matrix treated with sucralfate or related compounds and correlated with cell adhesion. Caco-2 motility was significantly and dose-responsively inhibited by sucralfate at therapeutic luminal concentrations. Sucrose octaacetate, the sucralfate backbone, and lactose octaacetate exhibited similar effects while the beta-bonded disaccharide maltose octaacetate had little effect. Sucrose itself slightly stimulated motility. Adhesion effects paralleled motility. Thus, sucralfate may inhibit intestinal epithelial motility by sterically interfering with adhesion to collagen I. A sucralfate analog with a lactose octaacetate backbone might retain growth factor binding without inhibiting enterocyte motility, perhaps improving its clinical efficacy.

Objectives: Provision of nutrients to the apical membrane of intestinal epithelial cells by the enteral route is critical for normal gut mucosal function and for the sheet migration required for mucosal healing. The present work attempts to determine whether supplemental nutrient delivery to the basal epithelial surface is important for intestinal epithelial biology. Since attempts to regulate intestinal epithelial cell biology by manipulation of parenteral nutrition solutions have met with some success, we hypothesized that basally delivered nutrients might also be important for intestinal epithelial biology., Design: To test this hypothesis, we compared the brush border enzyme activity, proliferation, and motility of human intestinal epithelial (Caco-2) cells cultured on a type I collagen substrate either on cell culture dishes with culture medium above the apical side of the cell monolayer or in culture inserts on 0.45-mu semipermeable membranes with culture medium beneath the monolayers as well as above them. Proliferation was assessed by serial hematocytometric counts over 13-day period. Doubling times were calculated by logarithmic transformation of cell counts 48 hrs apart. The specific activity of the brush border enzymes, dipeptidyl dipeptidase and alkaline phosphatase, was assayed by the digestion of synthetic chromogenic substrates in protein-matched aliquots of cell lysates. Sheet migration was quantitated by the expansion of Caco-2 monolayers across collagen. Motility was dissociated from the proliferative component of monolayer expansion by blocking proliferation with mitomycin C., Setting: Laboratory for gastrointestinal mucosal biology., Subjects: A well-differentiated subclone of cells derived from the established human Caco-2 colonic epithelial cell line., Measurements and Main Results: Basal nutrient delivery promoted Caco-2 proliferation, brush border enzyme activity, monolayer expansion, and cell motility. Proliferation was actually increased by 694 +/- 9.89% (n = 90, p < .0001) in cells nourished apically and basally compared with a 314 +/- 3.31% increase (n = 90, p < .0001) in those cells receiving only apical nutrition. The addition of basal nutrient delivery to the cell culture system augmented both alkaline phosphatase and dipeptidyl dipeptidase specific activity by 116 +/- 5.4% and 256 +/- 14.0%, respectively (p < .0001, n = 6 for each group). The effects of basal nutrient delivery were maintained after mitomycin blockade of proliferation for both alkaline phosphatase (392 +/- 89.8% of control, n = 3, p < .0005) and dipeptidyl dipeptidase (374 +/- 79.1% of control, n = 3, p < .005), suggesting that the increased digestive enzyme-specific activity reflected differentiation rather than indirect effects of slowing of proliferation. Epithelial sheet migration increased by 389 +/- 8.8% and proliferation-blocked cell motility also increased by 76.5 +/- 1.56% (p < .0005, n = 12 for each) compared with apical nutrient delivery only., Conclusions: These results suggest that although apical nutrition may be critical for intestinal epithelial cell biology, nutrient delivery to the basal surface of intestinal epithelial cell membranes may also promote intestinal epithelial differentiation, proliferation, and mucosal healing.

Colonic butyrate may maintain mucosal differentiation and oppose carcinogenesis. We characterized butyrate effects on differentiation, proliferation, and matrix interactions in Caco-2 and SW620 human colonic cells. Differentiation was assessed by brush border enzyme activity and doubling time by serial cell counts. Motility across matrix proteins was quantitated by monolayer expansion and correlated with adhesiveness to matrix. Integrin subunit surface pools were measured by immunoprecipitation. Butyrate-stimulated differentiation inhibited proliferation and was significantly more potent than acetate in this regard. Butyrate also inhibited motility across collagen I, collagen IV, and laminin, as well as decreasing adhesiveness to these matrices and beta 1, alpha 1, and alpha 2 integrin subunit surface expression. Butyrate acts in cultured cells at clinically relevant concentrations to oppose classical malignant behavior, inhibiting proliferation and motility while promoting differentiation. Since butyrate is derived from fermentation of dietary fiber, such mechanisms may contribute to the apparent protective action of fiber against colon carcinogenesis.

Little is known about the effects of repetitive deformation during peristaltic distension and contraction or repetitive villus shortening on the proliferation and differentiation of the intestinal epithelium. We sought to characterize the effects of repetitive deformation of a physiologically relevant magnitude and frequency on the proliferation and differentiation of human intestinal epithelial Caco-2 cells, a common cell culture model for intestinal epithelial biology. Human intestinal epithelial Caco-2 cells were cultured on collagen-coated membranes deformed by -20 kPa vacuum at 10 cycles/minute, producing an average 10% strain on the adherent cells. Proliferation was assessed by cell counting and 3H-thymidine incorporation. Alkaline phosphatase and dipeptidyl dipeptidase specific activity were measured in cell lysates. Since cells at the membrane periphery experience higher strain than cells in the center, the topography of brush border enzyme histochemical and immunohistochemical staining was analyzed for strain-dependence. Cyclic strain stimulated proliferation compared to static cells. Proliferation was highest in the membrane periphery where strain was maximal. Strain also modulated differentiation independently of its mitogenic effects, selectively stimulating dipeptidyl dipeptidase while inhibiting alkaline phosphatase. Strain-associated enzyme changes were also maximal in areas of greatest strain. The PKC inhibitors staurosporine and calphostin C ablated strain mitogenic effects while intracellular PKC activity was increased by strain. The strain-associated brush border enzyme changes were attenuated but not blocked by PKC inhibition. Thus, strain of a physiologically relevant frequency and magnitude promotes proliferation and modulates the differentiation of a well-differentiated human intestinal epithelial cell line in an amplitude-dependent fashion. PKC may be involved in coupling strain to increased proliferation.

Purpose: Smooth muscle cell (SMC) migration is a critical element in the development of intimal hyperplasia. The effect of endothelial cells (ECs) on SMC migration and the modulation of this cell-to-cell interaction by extracellular matrix is not well understood., Methods: To examine this relationship SMCs and ECs were cocultured on opposite sides of a semipermeable membrane and were compared with SMCs cultured alone. To assess migration SMCs were plated at confluent density into the center of the membrane with a steel fence. After the fence was removed, SMCs were treated for 2 hours with mitomycin C (20 micrograms/ml) to assess migration independent of proliferation. Cell migration was measured with morphometry. Experiments were performed on plastic and membranes coated with fibronectin or type I collagen (n > or = 8/group). Cell adhesiveness was quantitated by cell attachment and spreading assays., Results: ECs stimulated SMC migration by 187% when compared with SMCs cultured alone on plastic and by 160% when cultured on fibronectin (p < 0.01). Type I collagen stimulated migration of SMCs cultured alone and prevented EC stimulated migration in cocultured SMCs (p < 0.01). Cell adhesiveness was significantly increased in cocultured SMCs compared with SMCs cultured alone regardless of whether cells were cultured on plastic (EC/SMC, 13.5 +/- 0.6 SMCs/high power field vs SMC, 8.9 +/- 0.5, p < 0.01), fibronectin (16.3 +/- 0.8 vs 12.3 +/- 0.7, p < 0.01) or type I collagen (15.5 +/- 1.0 vs 12.4 +/- 0.6, p < 0.01). ECs increased SMC cell spreading on plastic and fibronectin when compared with SMCs cultured alone. No difference in SMC cell spreading was seen in the presence or absence of ECs when cells were cultured on type I collagen. EC-SMC contact was not required; EC-conditioned media alone increased SMC migration by 75% when compared with SMCs cultured alone. Our data suggest that ECs increase SMC migration by a diffusable molecule that may also alter SMC adhesion molecule expression. Extracellular matrix composition can attenuate these effects.

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.

Somatostatin modulates gastrointestinal mucosal growth and differentiation indirectly via inhibition of bioactive peptides and directly by less well understood mechanisms. We studied the direct effects of the somatostatin analog octreotide on proliferation, brush-border enzyme activity, cell-matrix interactions and intracellular cAMP in Caco-2 human intestinal epithelial cells. Proliferation was assessed by cell counting and [3H]thymidine uptake. The brush-border enzymes alkaline phosphatase (AP) and dipeptidyl dipeptidase (DP) were quantitated by synthetic substrate digestion. Adhesion and migration on purified matrix proteins were also measured. Octreotide (10(-9)-10(-5)M) shortened doubling time (46.5 +/- 6.2% at 10(-5) M, n = 20, P < 0.0001) and stimulated [3H]thymidine uptake. Octreotide decreased intracellular cAMP by 19.4 +/- 5.0% (n = 7, P < 0.0001) while dibutyryl-cAMP (10(-6) M) prolonged doubling time by 10.1 +/- 1.5% (n = 8, P < 0.0001), and blocked the octreotide effect. Octreotide decreased AP and DP with maximal effect at 10(-6) M (36.8 +/- 8.3% and 20.5 +/- 9.1%, n > 7, P < 0.0005 respectively). However, mitomycin proliferative blockade prevented octreotide inhibition of AP and DP, suggesting that the mitogenic effects of octreotide had simply decreased average maturity of the cells. Octreotide did not alter Caco-2 adhesion, EGF-or matrix-modulated motility, or integrin surface expression. Octreotide appears to directly stimulate Caco-2 proliferation by decreasing cAMP. These proliferative effects modulate Caco-2 differentiation but do not affect cell-matrix interactions.

Intestinal epithelial cells migrating across a mucosal defect are generally described as dedifferentiated, a term that suggests a loss of regulatory biology. Since cell biology may be more readily studied in established cell lines than in vivo, a model is developed using the human Caco-2 intestinal epithelial cell migrating across matrix proteins. This resembles in vivo models of mucosal healing in its sheet migration and loss of the brush border enzymes, which are conventional markers for intestinal epithelial differentiation. Immunohistochemical studies of migrating Caco-2 cells suggest, however, that the rearrangements of cytoskeletal, cell-cell and cell-matrix proteins during migration are not random but seem adapted to the migratory state. Indeed, Caco-2 migration may be substantially regulated by a variety of physiologic and pharmacologic stimuli and differentiation, measured by the specific activity of the intestinal epithelial brush border enzymes alkaline phosphatase and dipeptidyl dipeptidase, may be independently pharmacologically programmed during the stimulation or inhibition of cell motility.

Regulation of intestinal epithelial differentiation is critical to normal function, malignant transformation, and healing. However, the intracellular regulation of intestinal epithelial differentiation is incompletely understood. We studied the effects of intracellular cyclic AMP and cyclic GMP on brush border enzyme activity in the human Caco-2 intestinal epithelial cell using pharmacologic agonists and antagonists of cAMP and cGMP mediated pathways as probes. The stable cyclic nucleotide analogs dibutyryl cAMP and dibutyryl cGMP selectively decreased Caco-2 dipeptidyl dipeptidase specific activity while increasing alkaline phosphatase. The inhibitors of adenylate and guanylate cyclase KT5720 and KT5823 each exerted the opposite effects. Combinations of dibutyryl cAMP and dibutyryl cGMP demonstrated synergistic effects on each brush border enzyme but KT5720 and KT5823 were less than additive. Thus, cAMP and CGMP may regulate human intestinal epithelial differentiation by interacting pathways.

Caco-2 intestinal epithelial cells differentiate spontaneously after confluence when contact inhibition slows proliferation. We hypothesized that such reversible differentiation might be dependent on DNA synthesis and repair. We studied the effects of the topoisomerase II inhibitor etoposide on Caco-2 proliferation and on the differentiation markers alkaline phosphatase and dipeptidyl dipeptidase specific activity, as well as cell motility. Etoposide (0.3-10 microM) dose-dependently inhibited proliferation and alkaline phosphatase activity. However, etoposide (0.7-3 microM dose-dependently stimulated dipeptidyl dipeptidase activity. Above this concentration, dipeptidyl dipeptidase was also inhibited. Similar effects on enzyme activity were observed when proliferation was blocked with mitomycin C. Etoposide (1-10 microM) also dose-dependently inhibited cell motility. The selective stimulation of dipeptidyl dipeptidase activity by etoposide may offer a clue to the regulation of intestinal brush border enzyme expression at the molecular level.

Background: Although screening for colorectal cancer facilitates earlier detection and improves survival, cost-effective screening requires identification of patients at high risk for colorectal cancer. Rectal prolapse has not been clearly linked to colorectal carcinoma. Whether patients with rectal prolapse should be screened for colorectal cancer is therefore unclear., Methods: We retrospectively identified 70 consecutive patients treated for rectal prolapse at a community hospital during a period of 16 years and monitored for an average of 4.4 +/- 2.7 years and 350 patients of similar age treated for chronic unrelated conditions in the same institution during a similar period. We determined their incidence of colorectal cancer and sought demographic correlates., Results: The prevalence of rectosigmoid carcinoma among patients with prolapse was 5.7% during the study. Only 1.4% of the comparative group had colorectal cancer. Thus patients with rectal prolapse exhibited a 4.2-fold (95% confidence interval, 1.1 to 16.0) relative risk for colorectal cancer over the comparative group (p < 0.02)., Conclusions: To the extent to which these patients represent the population of patients with rectal prolapse, routine initial screening of patients with symptomatic rectal prolapse by use of flexible sigmoidoscopy may be appropriate.

Protein kinase C (PKC) isoforms are altered in colon tumors and upon exposure of intestinal mucosa to nutrients. We evaluated the effects of the PKC inhibitors staurosporine and calphostin C on human Caco-2 intestinal epithelial proliferation, motility, and differentiation. Motility was quantitated by monolayer expansion and the brush border enzymes dipeptidyl dipeptidase (DPDD) and alkaline phosphatase (AP) by synthetic substrate digestion. Staurosporine (0.03-1.0 ng/ml) and calphostin C (10(-12) M-10(-4) M) dose-dependently inhibited monolayer expansion and AP but stimulated DPDD. Proliferation was also inhibited but the effects of each inhibitor on motility, AP, and DPDD were preserved after mitomycin C proliferative blockade, suggesting that these effects were proliferation-independent. PKC inhibitors independently inhibit motility, AP and proliferation in human intestinal Caco-2 epithelial cells, but selectively stimulate the small intestinal differentiation marker DPDD. PKC may regulate small intestinal epithelial differentiation.

Much is known about intestinal epithelial regulation by growth factors and nutrients but the intracellular signals governing cell phenotype are less well understood. In an initial attempt to evaluate the role of protein kinase C in these events, we studied the effects of protein kinase C modulation by the phorbol ester TPA upon the differentiation, motility, and doubling time of the human intestinal epithelial Caco-2 cell line, a common model for enterocytic brush border enzyme expression. We also compared the effects of TPA to those of 4 alpha-phorbol 12,13-didecanoate, which does not modulate protein kinase C activity. Differentiation was studied by quantitating brush order dipeptidyl peptidase (DPDD)-specific activity in protein-matched Caco-2 lysates via synthetic substrate digestion. Alkaline phosphatase (AP) was studied for comparison. Doubling time was assessed by log transformation of serial cell counts and motility by monolayer expansion across type I collagen. TPA (0.03-0.7 micrograms/ml) dose-dependently stimulated DPDD, with a maximal 455 +/- 26% increase at 0.7 micrograms/ml (P < 0.01, n = 5). However, TPA dose-dependently inhibited AP to a maximal 91.6 +/- 0.3% decrease (P < 0.01, n = 5). TPA also dose-dependently prolonged the cell doubling time from 26.5 +/- 0.4 to 64.5 +/- 8.8 hr (n = 20, P < 0.01) with a maximal effect at 1.0 micrograms/ml and inhibited migration with essentially complete ablation of cell motility at 0.1 micrograms/ml (n = 10, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Primary intestinal lymphoma is uncommon but not rare. Its pathology and potential therapy are complex. Its epidemiology has changed radically over the past century, reflecting a sharp surge in the incidence of intestinal lymphoma, particularly that associated with immunosuppression. Recent advances in diagnosis, a new approach to pathologic classification by immunotyping, and a new staging system have influenced therapy. New multimodal therapeutic protocols have improved the prognosis of this once deadly disease.

Vasoactive intestinal polypeptide (VIP) is the pathophysiologic mediator of several small intestinal hypersecretion states. VIP exerts its effect by binding mucosal receptors and ultimately increasing intracellular levels of cAMP. Peptide YY (PYY), a GI hormone concentrated in the distal ileum and colon, has been demonstrated to decrease VIP-mediated secretion in the colon through a specific Y4 mucosal receptor. Characterization of PYY's effect on VIP-stimulated small intestinal secretion may provide a basis for future therapeutic interventions. We hypothesized that ion transport in the small intestine is mediated through a novel Y receptor subtype. We performed Ussing chamber ion transport studies on rabbit ileum using VIP, PYY, and other pancreatic polypeptide (PP)-fold peptides in order to specifically examine: (1) the effects of VIP and PYY on basal and VIP-stimulated short circuit current (Isc), and (2) the changes in VIP-stimulated Isc in response to NPY, PP, leucine31,proline31 neuropeptide Y fragment, ([Leu31,Pro34]NPY) and the carboxy-terminal fragment of NPY (NPY13-36). VIP increased basal Isc in a concentration-dependent manner, while PYY decreased basal Isc. Graded concentrations of PYY decreased VIP-stimulated increases in Isc. PYY added prior to VIP had no effect on VIP-stimulated increases in ISC. Inhibition of VIP-stimulated Isc increases was seen with NPY, but not with [Leu31,Pro34]NPY, PP, or NPY13-36. This distinct pattern of binding affinity characterizes a novel Y receptor subtype. Additionally, increases in Isc by VIP despite pretreatment with PYY suggests that VIP-stimulated ion transport is mediated through mechanisms other than increases in cAMP.

Glutamine supplementation has been advocated for patients requiring parenteral nutritional support. However, the direct effect of glutamine on neoplastic cells is poorly understood. We therefore investigated the effects of glutamine on the proliferation, differentiation, and cell-matrix interactions of two human colon carcinoma cell lines (Caco-2 and SW620) adapted to glutamine-free media. Doubling times were calculated by logarithmic transformation of serial cell counts. Alkaline phosphatase, cathepsin C (dipeptidyl peptidase), lactase, and isomaltase expression (markers of differentiation) were assayed by digestion of synthetic substrates. Adhesion to matrix proteins was assessed by colorimetric quantitation of toluidine blue staining of adherent cells. Surface expression of Caco-2 receptors for matrix proteins (integrins) was studied by biotinylation and immunoprecipitation with specific antibodies. Glutamine (1-10 mM) dose-dependently stimulated Caco-2 proliferation on all matrices studied with maximal effect at 7 mM. For instance, Caco-2 doubling time on collagen IV decreased by 57 +/- 0.2% (SE) (P < 0.001). Glutamine inhibited the expression of all four digestive enzymes with maximal inhibition ranging from 10 to 40% (P < 0.05 for all). Adhesion to matrix proteins was markedly diminished (51 +/- 1%, P < 0.01) by glutamine (5 mM) treatment, correlating with decreased alpha 2 and beta 1 integrin subunit surface expression. Glutamine had similar effects on SW620 cells, stimulating proliferation, inhibiting digestive enzyme expression, and diminishing both adhesion and integrin surface expression. Glutamine supplementation modulates the phenotype of at least two human colon carcinoma cell lines, increasing proliferation, decreasing differentiation, and decreasing adhesion to matrix proteins in association with decreased integrin expression. Although the mechanisms of these effects await elucidation, such characteristics would appear to predict more aggressive tumor behavior and raise the possibility that nutritional supplementation with glutamine may be deleterious in patients with cancer.

Mucosal healing requires enterocyte migration (restitution) supplemented by proliferation. Proliferation and migration may be studied independently by thymidine uptake and proliferation-blocked cell migration using human Caco-2 enterocyte monolayers in culture. Since epidermal growth factor (EGF) promotes mucosal healing and the EGF receptor is a tyrosine kinase, we hypothesized that tyrosine kinases might therefore modulate enterocyte migration and proliferation. The tyrosine kinase inhibitors genistein and 2,5-dihydroxymethylcinnamate, which block kinase ATP-binding and substrate-binding sites, respectively, were studied alone and with EGF. Proliferation was blocked with mitomycin. Although each inhibitor decreased basal and EGF-stimulated monolayer expansion when cell proliferation occurred, neither genistein nor 2,5-dihydroxymethylcinnamate decreased migration when proliferation was blocked. However, each inhibitor prevented EGF stimulation of proliferation-blocked migration and thymidine uptake. More substantial inhibition of basal proliferation by genistein correlated with increased protein-linked DNA breaks, which may reflect nonspecific inhibition of DNA topoisomerase activity by genistein. The more specific 2,5-dihydroxymethylcinnamate blocked changes in the alpha 2 integrin subunit organization which may modulate EGF-stimulated migration. Antiproliferative effects of tyrosine kinase inhibitors decrease basal monolayer expansion but true basal enterocyte migration appears independent of tyrosine kinase regulation. However, a specific tyrosine kinase-dependent modulation of cell-matrix interaction inhibits EGF-stimulated migration.

The closely related processes of healing and adaptation in the intestinal mucosa are of substantial clinical significance in patients after injury, intestinal surgery, and inflammatory or infectious ulceration, as well as all patients who are fasting. Recent investigations demonstrate that mucosal healing and adaptation are complex processes that seem likely to be regulated by a variety of autocrine and juxtacrine growth factors as well as conventional gut peptides. Further studies of the biology of mucosal healing may permit pharmacological intervention to promote healing and adaptation in the future.

Over 80 peptides and amines secreted by more than 20 different types of neuroendocrine cells scattered throughout the gut have been identified. The physiologic function and clinical relevance of many of these hormones await elucidation. Nevertheless, the clinical use of these agents in either diagnostic or therapeutic modalities has greatly expanded the appreciation of the relevance of many of these peptides to malignant and nonmalignant pathobiology.

Peptide YY (PYY) and enteroglucagon are hormonal peptides found in endocrine cells of the distal intestinal mucosa. Although it is known that plasma concentrations of both peptides increase in response to feeding, the mechanism by which ingested food causes release of colonic hormones is not understood. The release of PYY and enteroglucagon was measured in response to intraluminal stimuli in 176 patients having investigative colonoscopy. Introduction of air, saline (isotonic and hypertonic), glucose (isotonic and hypertonic), oleic acid (without bile salts), and casein hydrolysate all failed to release PYY but glucose caused a small but significant increase in enteroglucagon concentrations. In contrast with the lack of effect of nutrients, infusion of deoxycholic acid produced a rapid and marked dose responsive increase in plasma PYY concentrations when introduced into the sigmoid colon. PYY release was statistically significant at doses between 3.3 mM to 30 mM; for example 10 mM deoxycholate caused a sixfold increase in plasma PYY concentrations. Infusion of 10 mM deoxycholate into the transverse colon or caecum produced an increase of PYY that was similar to the responses in the sigmoid colon. There was also a significant release of enteroglucagon in response to infusion of this bile salt into the sigmoid colon at doses between 3.3 mM and 30 mM. The enteroglucagon response to 10 mM deoxycholate was similar in all three colonic regions. When oleic acid was added to deoxycholate as an emulsion, the release of PYY and enteroglucagon was similar to that seen with the bile salt alone. These findings suggest that bile salts may play an important part in the control of colonic endocrine function and may explain the increased circulating concentrations of colonic regulatory peptides that are seen in malabsorption states and after small bowel resection in humans.

Neuroendocrine tumors of the gastrointestinal tract pose substantial diagnostic and therapeutic challenges. The numerous biologically active peptides and amines produced by these lesions generate complex clinical syndromes. Unfortunately, little is known regarding the genesis of these lesions. Recent advances in understanding of the pathobiology of these lesions has facilitated characterization of the biochemistry of tumor secretions. Similarly, pharmacological blockade of tumor bioactivity using somatostatin analogs, pharmacotherapeutic probes, or specific antagonists has enhanced treatment options. Effective biochemical palliation of tumor symptoms has in turn motivated a more aggressive approach to cytoreduction of these tumors by surgery or hepatic embolization. The combination of chemical blockade and surgical therapy may permit more extended and symptom-free survival for a significant proportion of patients. We evaluate the basic principles of diagnosis and management of gastrointestinal neuroendocrine tumors.

Midgut carcinoid tumors derive from gut entoderm. These tumors may cause a complex of symptoms comprising the carcinoid syndrome by secreting a wide variety of bioactive agents in addition to serotonin. Such symptoms generally follow metastases to the liver but may also occur in primary ovarian or retroperitoneal tumors. After localization and biochemical characterization, the bioactivity of these tumors should be blocked by octreotide, sometimes in combination with other pharmacologic antagonists, so that primary resection may be performed safely. If curative resection is impossible, then a cytoreductive management scheme should be employed that includes surgical debulking and hepatic arterial embolization, followed by palliation with octreotide.

Small bowel tumors include adenocarcinomas, carcinoids, lymphomas, and sarcomas, as well as a variety of benign polyps, which should be excised to rule out malignancy. Although small bowel tumors are traditionally difficult to diagnose, a heightened index of suspicion should be combined with improved diagnostic techniques to increase yield and decrease delays in identification. The management of bioactive carcinoid tumors has rapidly advanced over the past year. Increasing experience with the stable somatostatin analogue octreotide has enabled regulation of hormonal secretion by these tumors both chronically and during acute "carcinoid crisis." In addition, early identification may be facilitated by the use of isotopically labeled variants of the compound. Although the development of sophisticated pharmacotherapeutic probes has improved the management of secretory small intestinal tumors, the prognosis for adenocarcinomas, lymphomas, and sarcomas still primarily reflects on the time of delay in diagnosis and awaits an improved understanding of the pathobiology of these malignant lesions.

Gastric stromal tumors are heterogeneous and poorly understood. Investigators have studied these tumors by histologic and immunocytochemical characteristics, but interlaboratory variations have impeded clinical application of these techniques. Although many gastric stromal tumors can be categorized as benign or malignant with some certainty, occasional instances may present great difficulty in this distinction. Uncertainty about malignant potential and the biologic factors of the lesion will therefore generally suggest excision with a 1 to 2 centimeter margin for diagnosis and definitive therapy of these tumors. Subsequent prognosis will be dictated primarily by tumor size, histologic factors and mitotic index. Gastric stromal tumors in young women should suggest the possibility of Carney's triad. Functional extra-adrenal paragangliomas and pulmonary chondromas should be sought preoperatively. The paraganglioma should be managed first and the gastric lesion then addressed. Distal gastrectomy of at least 50 percent would seem to represent a reasonable resection limit. Identification of the specific cellular biologic factors of these lesions will be necessary to accurately classify particular tumors, gauge prognosis and individualize their management.

In vitro, BAEC and BASMC migratory phenotypes are known to be reciprocally modulated by both soluble factors and extracellular matrix proteins. In addition, integrin matrix receptors mediate endothelial and smooth muscle cell attachment and migration. To further elucidate these phenomena, we studied the effects of TGF-beta 1 on integrin expression by vascular BASMC and BAEC in tissue culture. TGF-beta 1 upregulated mRNA levels and surface pools of BASMC beta 3 integrin classes without modulating beta 1 integrin mRNA levels or expression of beta 1 integrin organization. In contrast to its effects on BASMC, TGF-beta 1 increased BAEC mRNA levels and surface expression of beta 1 and beta 3 integrins without altering their organization. Conversely, extracellular matrix components (fibronectin, laminin, and fibrinogen) organized cell surface integrins in both BASMC and BAEC without affecting the size of their cell surface pools. These data are consistent with the hypothesis that SMC and EC behavior in neointimal lesions may be modulated, in part, through a coordination of soluble factor and extracellular matrix protein regulation of integrin surface expression and organization.

Background: Gastrointestinal mucosa heals by restitution and proliferation. These are difficult to distinguish in vivo., Methods: Human Caco-2 enterocytes were cultured on matrix proteins (collagen I, laminin, fibronectin) with growth factors (epidermal growth factor [EGF] and transforming growth factor-beta 1 [TGF-beta 1]) and the tyrosine kinase and prostaglandin inhibitors genistein and indomethacin. Healing was modeled by means of monolayer expansion, proliferation by means of 3H-thymidine uptake, and restitution by means of mitomycin-blocked migration., Results: Changing matrix composition failed to alter proliferation, but collagen I stimulated migration more than laminin or fibronectin (laminin/collagen, 68% +/- 2%; p less than 0.05). EGF (30 ng/ml) increased proliferation on both collagen (225% +/- 11% of basal) and laminin (206% +/- 26%) but increased migration only over laminin (210% +/- 17%) (all, p less than 0.05). TGF-beta 1 (200 pg/ml) stimulated migration over laminin (187% +/- 18%, p less than 0.005) but inhibited migration over collagen (89% +/- 3%, p less than 0.01) and did not affect 3H-thymidine uptake. When cultured on laminin, EGF but not TGF-beta 1 altered organization of the alpha 2 integrin subunit. Genistein (100 mumol/L) inhibited basal and EGF-stimulated 3H-thymidine uptake. In addition, it prevented EGF stimulation of replication-blocked migration (81% +/- 10% vs 190% +/- 20% of basal, p less than 0.0001) without altering basal replication-blocked migration. Indomethacin (10(-5) mol/L) did not alter migration but inhibited basal and EGF-stimulated proliferation by 7% +/- 1% (each, p less than 0.005)., Conclusions: Restitution and proliferation appear independently regulated by matrix and growth factors. It may be possible to individually target specific phases of mucosal healing by means of pharmacologic agents.

The modulation of enterocyte sheet migration was studied using Caco-2 cells, a well-differentiated human colonic cell line. Although Caco-2 cells attached and spread equivalently over collagen types I, III, IV, and V and laminin, migration over laminin was significantly slower than migration over the collagen types. Fibronectin was a poor substrate for attachment, spreading, and migration. Epidermal growth factor (EGF) stimulated migration over laminin but did not alter Caco-2 migration over collagen or fibronectin. This effect was independent of cell proliferation, which was stimulated equivalently on both laminin and collagen I. Expression and organization of cell surface receptors for matrix (integrins) were studied using antibodies specific for beta and alpha integrin subunits. Integrin surface expression was assessed by immunoprecipitation of surface 125iodinated control and EGF-treated cells. Beta 1 surface pools did not change substantially in any condition studied. Alpha 1 subunit pools were decreased after EGF treatment on collagen I but alpha 1 pools increased after EGF treatment on laminin. Surface pools of alpha 2 subunits were increased following EGF treatment whether cells were cultured on laminin or collagen I. However, traditional immunofluorescent and laser confocal imaging demonstrated substantial differences in the character of alpha 2 subunit organization between collagen and laminin in the migrating cell front. Furthermore, a functional antibody to the alpha 2 subunit inhibited EGF stimulation of migration over laminin without substantial effects on basal migration over laminin or collagen I. Thus, EGF appears to exert a matrix-specific effect on enterocyte migration by modulation of integrin expression and organization.

Options for the management of patients with acute variceal hemorrhage must be compared for their ability to stop bleeding and prevent rebleeding, for their safety, and for their impact on hepatic reserve. Pharmacologic treatment may stop bleeding in up to 50% of patients, but cannot prevent rebleeding. Balloon tamponade controls bleeding in 90% of patients but is only transiently effective and may cause complications. Immediate sclerotherapy may stop acute bleeding and may provide chronic control with repeated treatments. However, patients who fail to respond to such measures must be submitted for surgical intervention. Emergency portosystemic shunt or esophageal transection represent the current procedures of choice for these patients. Surgery may also prove more cost effective than repeated sclerotherapy as primary therapy in a select group of patients with a high risk of rebleeding. Ultimately, however, primary pharmacologic therapy or hepatic transplantation to correct the pathophysiology underlying the esophageal variceal hemorrhage must be developed and refined if the natural history of this disease is to be substantially altered.

The psychological effects of nonneurologic trauma on children are poorly recognized. We hypothesized that physical trauma in children, with or without head injury, would result in substantial and persistent psychological and behavioral abnormalities. Using a short telephone survey followed by a detailed behavioral checklist, we studied psychobehavioral dysfunction in children who had experienced trauma either with or without minor head injury (n = 40 each) as well as in a comparative group of children after emergency appendectomy (n = 80). Substantial behavioral disability was identified by the detailed checklist in 35% and 28% of children without and with head injury, respectively, but in none after appendectomy. Dysfunctions included phobias, major scholastic difficulties, rage attacks, and episodic depression that continued for a long period. Even in the 67% of children who eventually fully recovered, the duration of symptoms after the time of injury was an average of 19 months. Demographics, socioeconomic status, severity of injury, and length of hospitalization did not correlate with dysfunction, and these traumatized children's siblings had no reported history of trauma or psychological difficulties. Thus, parental opinion about behavioral dysfunction appears sensitive and specific and is therefore a useful screening index. These results suggest that injured children, even after minor trauma, may suffer substantial and long-lasting behavioral changes to a degree hitherto unrecognized.

Small GTP-binding proteins are important regulators of intracellular traffic. The presence of several small GTP-binding proteins was documented in subfractions of rabbit parietal cells. Upon maximal stimulation of the cells with a combination of histamine and forskolin, one 23 kDa GTP-binding band was observed to decrease in a 50,000 g membrane fraction while increasing in 4000 g membranes. The 23 kDa band resolved into one major and two minor species on two-dimensional gels. GTP-binding species of 23 kDa, 24 kDa and 25 kDa were present in purified preparations of tubulovesicles. The three isoelectric species of the 23 kDa proteins observed in parietal cell 50,000 g microsomes were enriched in tubulovesicle preparations. None of the tubulovesicle-associated GTP-binding proteins were substrates for ADP-ribosylation by a preparation of botulinum D toxin. These results indicate that tubulovesicles contain discrete small GTP-binding proteins which redistribute during parietal cell stimulation.

Despite blockade and neutralization of gastric acid, acute gastric lesions cause substantial morbidity and mortality in critically ill patients. Pepsinogen release in response to noxious stimuli such as hypoxia and endotoxin might contribute to mucosal damage. Guinea pig fundic mucosa was mounted in Ussing chambers. Acid secretion, pepsinogen release, potential difference (PD), and resistance were monitored. Gassing with room air or nitrogen diminished acid secretion and PD but increased pepsinogen release 9.7- and 15.5-fold, respectively (both p less than 0.001). Similarly, endotoxin (0.01 and 0.1 units/ml) dose-dependently inhibited acid secretion and PD but increased pepsinogen release 3.3- and 6.1-fold (both p less than 0.05). Endotoxic and air-gassed tissues were edematous with scattered cellular damage by light and transmission electron microscopy; nitrogen-exposed membranes appeared necrotic. Pepsin release may therefore have resulted from cell damage rather than exocytosis. Intragastric peptic activity in critically ill H2-receptor-blocked patients (n = 20) was 5490 +/- 1701 U/ml. The gastric juice of H2-blocked convalescing surgical patients (n = 20) contained 315 +/- 101 U/ml (p less than 0.0001). Occult blood correlated with intragastric peptic activity (r = 0.59, p less than 0.0001) but not with gastric pH (r = 0.04, p = 0.6). These data suggest that the complex of pathophysiologic abnormalities common in critical illness causes substantial pepsin release. Efflux of this potent mucolytic barrier breaker may damage gastric mucosa in severely stressed patients.

Although cholecystokinin (CCK) has been reported to stimulate pepsinogen secretion, this action has been poorly characterized. To assess the ability of CCK to regulate mammalian pepsinogen secretion, guinea pig fundic mucosa was incubated in Ussing chambers with CCK-8, carbamylcholine, and pentagastrin, and with cholinergic and CCK antagonists. CCK-8 stimulated pepsinogen secretion at 10(-10) M, with an ED50 of 10(-9) M and maximally (26-fold over basal) at 10(-8) M. Carbachol stimulated pepsinogen and acid secretion with an ED50 of 3 x 10(-7) M and maximally at 10(-6) M. Pentagastrin (10(-9) M-10(-6) M) did not affect acid or pepsinogen secretion, whereas gastrin-I (10(-6) M) stimulated acid secretion slightly but did not alter pepsinogen secretion. L364, 718 (10(-5) M), a specific CCK peripheral receptor antagonist, abolished all pepsigogic effects of 3 x 10(-9) M CCK-8 without altering basal acid or pepsinogen secretion or mucosal electric characteristics. L364,718-treated tissues unresponsive to CCK-8 nevertheless secreted pepsinogen and acid in response to 3 x 10(-7) M carbachol identically to control carbachol-treated preparations. Atropine (10(-5) M) blocked the response to 3 x 10(-7) M carbachol without inhibiting 10(-9) M CCK stimulation. These results support a specific receptor-mediated role for cholecystokinin in the physiologic regulation of guinea pig pepsinogen secretion.

Pepsin is a potent proteolytic enzyme stored and secreted by chief cells in an inactive precursor form, pepsinogen. Its secretion is modulated by both cAMP and calcium-dependent mechanisms. Abnormalities in levels of pepsinogen and its various isozymogens have been linked clinically, epidemiologically, and experimentally to peptic ulcer disease and gastric carcinoma. The ulcerogenesis of pepsin stems from its ability to breach gastroduodenal mucosal barriers. Furthermore, certain isozymogens seems abundant and hyperactive in patients with peptic ulcer disease. The etiology and significance of low pepsinogen levels with disproportionate elevations of pepsinogen II and pepsin 5 in gastric cancer and its precursors is less clear. Further exploration of the patho-physiologic role of pepsin is likely to be of considerable importance in initiating further advances in the understanding and treatment of upper gastrointestinal disease.

Patterns of activity of six dehydrogenase enzymes were studied histochemically in 42 free muscle grafts in the rat. Within hours, the surviving peripheral muscle fibers can be distinguished from the central ischemic muscle fibers. The surviving muscle fibers retain their characteristic pattern of staining throughout the post-transplantation period. The central ischemic muscle fibers stain abnormally and by five or six days they lose their enzymatic activity. The zone of regeneration, between the surviving and the ischemic muscle fibers, initially shows little dehydrogenase activity, but as the regenerating muscle fibers mature, they develop first a homogeneous staining pattern and, later, differences in staining intensity among different types of muscle fibers.

In vivo observations have suggested that acid secretion may potentiate pepsinogen release. We measured pepsinogen and acid secretion by guinea pig fundic mucosal sheets stimulated by 10(-4) M histamine, 10(-8) and 10(-9) M cholecystokinin, and 3 x 10(-7) M carbamylcholine and then investigated the effects of 10(-4) M omeprazole on basal, carbachol-stimulated, and cholecystokinin-stimulated secretion. Histamine increased basal acid secretion fivefold (p less than 0.01) without altering pepsinogen secretion. Cholecystokinin did not stimulate acid secretion but increased pepsinogen secretion by factors of 23.1 at 10(-8) M and 9.1 at 10(-9) M (both p less than 0.01). The combination of 10(-4) M histamine and 10(-9) M cholecystokinin increased acid secretion 3.5-fold and pepsinogen secretion 6.4-fold, statistically equivalent to the sum of the effects of histamine and cholecystokinin alone. Carbachol increased acid secretion and pepsinogen secretion by factors of 4.0 and 10.9, respectively (both p less than 0.01). Pretreatment with 10(-4) M omeprazole abolished basal and carbachol-stimulated acid secretion. However, pepsinogen secretion was unaffected (p greater than 0.05). Furthermore, omeprazole-treated tissues increased pepsinogen secretion by factors of 10.0 with 3 x 10(-7) M carbachol and 9.1 with 10(-9) M cholecystokinin (both p less than 0.01). Thus, basal and secretagogue-stimulated pepsinogen secretion appear independent of acid secretion in intact guinea pig mucosa.