An in vitro intestinal epithelial wound/repair model in which epithelium is stripped from villus tips and the wound is resealed during the following 60 minutes has previously been described. The process, termed epithelial restitution, results in part from the rapid migration of epithelial cells shouldering the wound over the denuded basement membrane. The present report examines the requirements for epithelial cell-basement membrane interactions during restitution in this model. Addition of heparin, soluble matrix components, or a variety of antibodies to matrix components (laminin; fibronectin; collagen I, III, IV) does not impair restitution. Although inhibition of protein synthesis alone also does not retard restitution, in the simultaneous presence of antibody to type III and IV collagen restitution is impeded as judged functionally and structurally. Preincubation of tissues with 20 mmol/L cis-OH-proline (a condition known to inhibit cellular secretion of newly synthesized collagen) similarly inhibited structurally and functionally defined restitution only if antibodies to type III and IV collagen were simultaneously present. These results suggest that collagen-epithelial cell interactions are important in restitution after injury, and if necessary, collagen can be produced locally and rapidly at the site of injury to allow restitution to normally proceed.

Clostridium difficile toxin A, a 308-kilodalton protein exotoxin, is the principal causative agent of antibiotic-associated, C. difficile-induced colitis. In the current study, the prevalence of specific human serum and secretory antibody to toxin A and the possible protective effect of secretory, intestinal anti-toxin A antibody are examined. Serum (n = 35), colonic aspirates (n = 35), and duodenal aspirates (n = 20) were collected from adults at diagnostic endoscopy. Patients with evidence of colitis or a history of recent antibiotic use were excluded from the study. Specific serum immunoglobulin (Ig) A and IgG antitoxin A antibodies were detected in 60% and 57% of subjects, respectively, by enzyme-linked immunosorbent assay. Fifty-seven percent of colonic aspirates contained IgA antitoxin, whereas only 10% of duodenal aspirates were positive (P = 0.002). Binding of toxin A to its intestinal receptor was studied using [3H]toxin A and purified rabbit ileal brush border membranes. Toxin A binding was significantly inhibited by colonic aspirates with high IgA anti-toxin A antibody levels (0.503 +/- 0.055 pmol toxin A bound per milligram of brush border membrane protein, mean +/- SE) in comparison with antitoxin A-negative aspirates (0.778 +/- 0.089 pmol; P = 0.02) and control (0.766 +/- 0.004 pmol; P = 0.03). In the current study, a specific intestinal secretory IgA antibody response to C. difficile toxin A in humans is reported. This antibody response is more evident in the colon, the site of C. difficile infection, than in the upper intestinal tract. Our data suggest that human colonic IgA antitoxin may protect against C. difficile colitis by inhibiting the binding of toxin A to its intestinal epithelial cell receptor.

The presence of gas in a gallstone can profoundly affect the ability of shock waves to fragment the stone by various mechanisms. In the present study, the aim was to determine the gas content of gallstones and determine if increasing the gas content of the stone affected the outcome of lithotripsy. Thirty human gallstones, transferred directly from gallbladder bile into sterile saline, were studied. The initial gas content of all stones was determined by differential weighing under saline before and after degassing. Eighteen gallstones were pairs; each pair was from a single patient and of similar size and composition. One gallstone of each of the pairs was exposed to air and the other was kept under saline. Then each of the paired gallstones was subjected to 1000 pulses at power level 3 (highest) in a Diasonics Therasonic lithotripter (Diasonics, Milpitas, CA). The volume of gas present at the beginning of the experiment in all groups of stones was 2.4 +/- 2.1 mm3, and 27% of all gallstones tested contained measurable amounts of gas initially. There was no significant difference in the volume of gas present in the paired stones at the beginning of the experiment (group A, 1 +/- 0.4 mm3; group B, 2 +/- 1 mm3). After group A stones were exposed to air, the gas content was significantly higher (36 +/- 18 mm3) than in the paired group B stones stored under saline (3 +/- 2 mm3; P less than 0.05). Stones exposed to air fragmented more easily than stones stored under saline. The mean number of pulses required to cause initial fragmentation was significantly lower in the group of stones exposed to air (22 +/- 7) compared with those stored under saline (610 +/- 139; P less than 0.05). The fragments were smaller and more numerous in the group of stones exposed to air than in those stored under saline. It is concluded that gas is present in some gallstones and that the efficacy of lithotripsy increases with increasing stone gas content. Our data suggest that alterations in the physical characteristics of gallstones can have profound effects on the outcome of lithotripsy.

The significance of antibodies to hepatitis C virus (HCV) found by screening enzyme-linked immunoassay testing in a low-risk blood donor population is unclear. The rate of false positivity in this group as well as the usefulness of supplemental testing were examined by correlating the results of two screening enzyme-linked immunoassays (Ortho Diagnostics, Raritan, NJ, and Abbott Laboratories, North Chicago, IL) with supplemental antibody testing by the recombinant immunoblot assays (1 and 2) (Ortho) and neutralization assay (Abbott). Polymerase chain reaction was used to detect HCV genomic RNA to confirm viremia. Among 11.243 volunteer donors who were screened for the presence of antibodies to HCV by enzyme-linked immunoassay, 60 (0.53%) sera were repeatedly reactive. Twenty-five of these 60 sera were available for further testing. Seven sera were reactive by both screening enzyme-linked immunoassays, as well as by both recombinant immunoblot and neutralization assays. Six of these seven sera had detectable HCV genomic RNA by polymerase chain reaction. Among the remaining 18 sera, none were reactive by either recombinant immunoblot assays, whereas two sera were reactive by the neutralization assay only. None of the 18 samples had detectable HCV genomic RNA. Five of the six sera with elevated aminotransferase levels were among the seven sera reactive by all immunoassays. It is concluded that there is a significant false positivity rate associated with screening enzyme-linked immunoassay testing in a low-risk blood donor population. Supplemental testing correlates well with detection of hepatitis C genomic material by polymerase chain reaction and identifies donors who are truly infected.

Anorectic and bulimic patients frequently report symptoms of constipation, bloating, and abdominal pain suggestive of abnormal gastrointestinal motility or transit. However, except for studies of gastric emptying, gastrointestinal motility and transit in these eating disorders have not been investigated. Ten anorectic and 18 bulimic inpatients were compared with 10 healthy controls. Whole-gut transit was tested by the radiopaque marker technique, and mouth-to-cecum transit time was assessed by the lactulose breath test. All anorectics and 67% of bulimics complained of constipation. Whole-gut transit time was significantly delayed in both anorectics (66.6 +/- 29.6 hours) and bulimics (70.2 +/- 32.4 hours) compared with controls (38.0 +/- 19.6 hours). Mouth-to-cecum transit time also tended to be longer in anorectics (109.0 +/- 33.5 minutes) and bulimics (106.2 +/- 24.5 minutes) than in controls (84.0 +/- 27.7 minutes), but these differences were not statistically significant. Delayed transit could contribute to or perpetuate the eating disorders by (a) causing the patient to feel bloated, thereby exacerbating fear of fatness, or (b) causing rectal distention, which may reflexly inhibit gastric emptying.

Studies were performed to define the peptidergic nature of intramural nerves in the human esophagus. Cryosections of uninvolved surgically resected tissues from 14 individuals were studied by immunofluorescence for the localization of 10 neuropeptides. Myenteric neurons showed bombesin-, calcitonin gene-related peptide-, galanin-, substance P-, vasoactive intestinal polypeptide-, leucine-enkephalin-, methionine-enkephalin-, neuropeptide Y-, and somatostatin-like immunoreactivity. Submucous neurons had all the above except neuropeptide Y, methionine-enkephalin, leucine-enkephalin, and bombesin. Both groups of neurons received nerve terminations positive for calcitonin gene-related peptide, galanin, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. Myenteric neurons additionally received terminations positive for neuropeptide Y, methionine-enkephalin, and somatostatin. All muscle layers had varicose fibers that reacted for calcitonin gene-related peptide, galanin, neuropeptide Y, and substance P. Longitudinal and circular muscle received few nerves reactive for leucine-enkephalin, whereas methionine-enkephalin was localized in a few nerve endings in the circular muscle. Somatostatin- and bombesin-reactive nerves occurred in longitudinal muscle. No cholecystokinin-reactive nerves were found. This study extends the results of previous studies and shows the previously undescribed presence of calcitonin gene-related peptide- and galanin-reactive nerves in the human esophagus and identifies neuropeptides that may serve as motor, sensory, and modulatory neurotransmitters of esophageal nerves.

The effects of clonidine, an alpha 2-adrenergic agonist, and verapamil, a Ca2+ channel blocker, on Na+ and Cl- absorption were studied in stripped jejunal mucosa from control and transmissible-gastroenteritis-virus-infected piglets. All infected piglets developed severe diarrhea 18-24 hours after oral inoculation. Jejunum from infected animals, as compared with control jejunum, had decreased mucosal-to-serosal, serosal-to-mucosal, and net Na+ and Cl- fluxes. Clonidine and verapamil caused a decrease in short-circuit current and stimulation of Na+ and Cl- absorption in control jejunum. In infected piglets, although the jejunum exhibited severe villus atrophy, both drugs stimulated Na+ and Cl- absorption and the magnitude of Na+ and Cl- absorption was similar in control and transmissible-gastroenteritis-infected jejunum. In contrast, D-glucose stimulated Na+ absorption, and the decrease in short-circuit current caused by verapamil and clonidine, were decreased in transmissible-gastroenteritis-infected jejunum. Such pharmacological stimulation of Na+ and Cl- absorption might be useful in the management and treatment of certain viral diarrheal diseases.

Normal human lamina propria lymphocytes are in a heightened state of activation compared with peripheral blood with regard to cell-surface activation antigen expression (transferrin receptor, interleukin-2 receptor, 4F2) and the increased spontaneous secretion of immunoglobulins in vitro. This study evaluates the cell-surface expression of activation-associated antigens in different subpopulations of isolated colonic lamina propria mononuclear cells in inflammatory bowel disease. In pilot studies using three-color flow cytometry, autofluorescence was observed that was emitted by unstained lamina propria mononuclear cells, which interfered with both the sensitivity and the specificity of the analyses. Because a major portion of the intestinal lymphocyte populations of interest were autofluorescent, a method to remove autofluorescence signals was developed by designing a computer program for the subtraction of autofluorescence from the emissions of each individual cell. This technique increases both the sensitivity and specificity of flow-cytometric analyses of intestinal lamina propria mononuclear cells. Using fluorescence-activated cell-sorter analyses with subtraction of autofluorescence on a single-cell basis, increased expression of lymphocyte activation antigens (interleukin-2 receptor, transferrin receptor, 4F2) was found on the cell surface of isolated intestinal B cells, T cells, CD4+ T cells, and CD8+ T cells in both Crohn's disease and ulcerative colitis. Therefore, markedly increased intestinal lymphocyte activation is a major immunological alteration in inflammatory bowel disease and includes all lymphocyte subpopulations investigated in this study. In addition, 5-aminosalicylic acid, which is used for the treatment of intestinal inflammation in inflammatory bowel disease, inhibits the expression of cell-surface activation antigens on mitogen-activated peripheral blood lymphocytes in a dose-dependent manner. These observations suggest that lymphocyte activation may play an important role in underlying immune processes that lead to chronicity and perpetuation of inflammatory bowel disease and may implicate an additional mechanism for the therapeutic action of 5-aminosalicylic acid.

A 54-year-old man with chronic B hepatitis was treated with interferon alfa. Despite resolution of the hepatitis B viral infection, he experienced severe jaundice, ascites, and encephalopathy. Further work-up showed hyperglobulinemia, chiefly immunoglobulin G, and positive smooth muscle and anti-nuclear antibodies. Because of these "autoimmune" features, the patient was treated with prednisone. One month later, a significant clinical and biochemical improvement was observed. A possible autoimmune mechanism induced by interferon alfa is proposed as the cause for the perpetuation of the necroinflammatory activity.

Gallstone formation in the cholesterol-fed prairie dog is preceded by an increase in mucin secretion by the gallbladder epithelium, and mucin hypersecretion is believed to promote cholesterol gallstone formation by accelerating the nucleation of cholesterol monohydrate crystals. Some studies have suggested that gallbladder mucin hypersecretion is mediated by increases in gallbladder prostaglandin synthesis, but other observations are difficult to reconcile with this view. An organ culture technique was used to measure mucin secretion in normal prairie dog gallbladder in response to exogenous prostaglandins and agents that increased or decreased endogenous prostaglandin production. Incubation with indomethacin produced a concentration-dependent inhibition of endogenous prostaglandin synthesis with virtually complete inhibition at 10(-5) mol/L indomethacin. However, indomethacin had no effect on gallbladder mucin secretion at concentrations as high as 10(-5) mol/L, and significant inhibition of mucin secretion was only found at 10(-4) mol/L indomethacin, a concentration that also produced a significant increase in lactate dehydrogenase release from cultured explants. Incubation of gallbladder explants with the calcium ionophore A23187 significantly stimulated endogenous prostaglandin synthesis in a concentration-dependent manner, increasing synthesis of prostaglandins E and F to as much as 278% +/- 20% and 335% +/- 21% of basal values, respectively; however, the same concentrations of A23187 did not stimulate mucin secretion. Incubation of gallbladder explants in the presence of exogenous prostaglandin E2 or prostaglandin F2a in concentrations as high as 10(-6) mol/L also did not stimulate mucin secretion. Prairie dogs fed a lithogenic 1.2% cholesterol diet showed a significant increase in gallbladder mucin secretion after 1 week (117.5 +/- 10.2% of control, P less than 0.05), and 4 of 5 had formed cholesterol monohydrate crystals after 3 weeks. Long-term treatment with indomethacin, 1.2 mg.kg-1.day-1, failed to inhibit gallbladder mucin hypersecretion (129.2 +/- 10.7% of control after 1 week) or cholesterol monohydrate crystal formation (3/5) in cholesterol-fed prairie dogs. Furthermore, incubation of explants with 10(-5) mol/L indomethacin failed to prevent in vitro mucin hypersecretion in cholesterol-fed animals. These findings suggest that prostaglandins do not regulate gallbladder mucin secretion in the prairie dog, and it is unlikely that increases in gallbladder prostaglandin synthesis are responsible for mediating gallbladder mucin hypersecretion during cholelithiasis in the prairie dog.

The genetic disease abetalipoproteinemia is characterized by a total absence of apolipoprotein B-containing lipoproteins from plasma. A presumed synthetic defect in apolipoprotein B synthesis was thought to be responsible for this disorder. The present study quantitates apoprotein B synthesis and apolipoprotein B messenger RNA levels in duodenal mucosa from normal patients and four patients with abetalipoproteinemia. After in vitro [3H]leucine incorporation, small intestinal biopsy specimens from three of four patients with abetalipoproteinemia synthesized immunoprecipitable apolipoprotein B of identical mobility (on sodium dodecyl sulfate gel electrophoresis) to normal apolipoprotein B. In abetalipoproteinemia, the apolipoprotein B content of intestinal mucosa by radioimmunoassay was 15% of normal mucosal values, whereas apolipoprotein B messenger RNA quantitation showed 3-20-fold increased levels compared with normal mucosa. In one patient, smaller-molecular-weight fragments of apolipoprotein B were immunoprecipitated from duodenal biopsy specimens. The synthesis rates and messenger RNA levels of two other chylomicron apoproteins (apolipoprotein A-I and apolipoprotein A-IV) were found to be reduced by 50%. These results show the synthesis of immunologically recognizable apolipoprotein B48 in abetalipoproteinemia. The significance of mucosal apolipoprotein B content in abetalipoproteinemia is discussed in terms of factors controlling apolipoprotein B synthesis in normal mucosa and in abetalipoproteinemia.

Compared with classic achalasia, vigorous achalasia has been defined as achalasia with relatively high esophageal contraction amplitudes, often with minimal esophageal dilation and prominent tertiary contractions on radiographs, and with the presence of chest pain. However, no study using current manometric techniques has compared manometric, radiographic, and clinical findings in vigorous and classic achalasia or questioned the usefulness of making this distinction. Fifty-four cases involving patients with achalasia whose radiographic and manometric studies were performed within 6 months of each other were available for review. Patients with vigorous achalasia (n = 17), defined by amplitude greater than or equal to 37 mm Hg, and patients with classic achalasia (n = 37), defined as amplitude less than 37 mm Hg, had substantial overlap in radiographic parameters of esophageal dilation, tortuosity, and tertiary contractions. Manometric properties of repetitive waves and lower esophageal sphincter pressure and clinical aspects of chest pain, dysphagia, heartburn, and satisfactory responses to pneumatic dilation were similar in both forms of achalasia. A separate analysis of patients with mean contraction amplitude greater than 60 mm Hg revealed similar findings. It is concluded that use of amplitude as a criterion for classifying achalasia is arbitrary and of dubious value.

Two cases of Clonorchis-associated cholangiocarcinoma are described along with their cholangiographic features to illustrate the spectrum of pathology ascribed to the injurious effects of the flukes on the bile duct epithelium. This includes adenomatous hyperplasia, extensive fibrosis, and carcinoma. The first case was also complicated by hepatic abscesses, left hepatic lobar atrophy, gastrobiliary and biliarocutaneous fistulae. The second case features an unusually dilated pancreatic duct containing pancreaticoliths that was found later to consist of hyperplastic bile duct epithelium, presumably carried by worm migration in the biliary tree. Liver sections from both patients showed typical features of hepatic clonorchiasis with the cancer. A knowledge of the wide spectrum of clinical presentation of clonorchiasis, particularly cholangiocarcinoma, might aid Western physicians in averting this serious sequela through prompt eradication of the helminthic infection and early recognition and treatment of its complications.

In this study, the presence of a bicarbonate gradient-dependent, carrier-mediated anion exchange process for butyrate (a representative short-chain fatty acid) uptake in apical membrane vesicles isolated from rat distal colon is described. An outward gradient of both butyrate- and bicarbonate-stimulated [14C]butyrate uptake and resulted in transient accumulation (an "overshoot" phenomenon). Butyrate gradient-stimulated [14C]butyrate uptake was not altered either by an imposed pH gradient or at different pH values. In contrast, bicarbonate gradient-stimulated [14C]butyrate uptake was stimulated severalfold by an additional imposition of an outward pH gradient (pHi = 7.5; pH0 = 6.0). This bicarbonate- and pH gradient-stimulated butyrate uptake was not inhibited by either voltage clamping, with equimolar intravesicular and extravesicular K+ and valinomycin, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an anion-exchange inhibitor. Increasing butyrate concentrations saturated the bicarbonate- and pH gradient-stimulated butyrate uptake with a half-maximal concentration (Km) of 26.9 +/- 1.6 mmol/L. Butyrate uptake was substantially inhibited by 20 mmol/L propionate (45%) and acetate (60%) but was not inhibited by oxalate, inorganic anions (SO4(2-) and NO3-), and transport inhibitors (amiloride, acetazolamide, furosemide, and ouabain). It is concluded from these results that bicarbonate gradient-stimulated butyrate uptake in apical membrane vesicles of rat distal colon occurs via a carrier-mediated anion-exchange process that differs from other DIDS-sensitive anion exchanges [e.g., the Cl- -OH- (HCO3-) process].

The intestinal sucrase-isomaltase precursor is cleaved at the brush border membrane by luminal proteases. Whether the lactase precursor also is cleaved by luminal proteases is uncertain. Lactase synthesis and processing was studied in 0- and 15-day-old rats after IP administration of [35S]methionine, and changes in precociously cortisone-induced sucrase-isomaltase were used as an internal control. Mucosal lactase and sucrase-isomaltase were separately immunoprecipitated and analyzed by autoradiography after electrophoresis. In both 0- and 15-day-old rats, mucosal lactase appeared as a 200K lactase precursor band at 30 minutes and as 200K and 225K lactase precursor bands at 60 minutes and was cleaved to form a 130K lactase band 120-240 minutes after labeling; sucrase-isomaltase similarly appeared as 210K and 220K bands at 30-60 minutes and was cleaved to form 140K I and 120K S subunits by 240 minutes in day 15 rats. To determine the role of luminal proteases, intestinal segments were isolated in situ and the luminal contents were flushed 30 minutes after labeling. Unflushed segments were used as controls. Only lactase precursor and sucrase-isomaltase precursor were present 240 minutes after labeling in flushed intestinal segments, but lactase precursor and sucrase-isomaltase precursor were cleaved in unflushed segments. Addition of trypsin or elastase into the lumen of flushed segments resulted in partial cleavage of lactase precursor but not of sucrase-isomaltase precursor. Luminal contents collected from the small intestine of day 15 rats 120 and 240 minutes after labeling showed 35S-labeled 130K and 80K polypeptides in lactase immunoprecipitates. It is concluded that intestinal lactase is synthesized as lactase precursor and transported to brush border membrane and cleaved by luminal proteases, and the amino end polypeptide cleaved from lactase precursor is released into the lumen.

Atrial natriuretic factor is a hormone intimately involved in water and salt homeostasis. The heart constitutes the major but not exclusive site of synthesis of this hormone. Among other functions, the gastrointestinal tract has endocrine functions, plays an important role in volume regulation of the body, and seems to be a target organ for atrial natriuretic factor. Therefore, the presence of atrial natriuretic factor was investigated in the human gut. Immunoreactive atrial natriuretic factor was found in intraoperatively obtained samples of normal human colon. Acidic extracts of human large intestine contained about 0.4 pmol/g wet wt of atrial natriuretic factor. Analysis of atrial natriuretic factor immunoreactivity by gel-filtration and reverse-phase high-performance liquid chromatography showed that about 65% of the immunoreactivity corresponded to the atrial natriuretic factor phohormone and about 35% corresponded to the C-terminal ANF99-126. Immunohistochemistry showed atrial natriuretic factor prohormone location in enterochromaffin cells of the colon mucosa. Altogether, these findings show the presence of atrial natriuretic factor prohormone in enterochromaffin cells of the human large intestine and may suggest this organ as a site of atrial natriuretic factor synthesis in humans.

The efficacy and safety of therapy with azathioprine/6-mercaptopurine was studied in 78 patients with Crohn's disease. Mean duration of therapy was 1.6 years; 52 patients were treated greater than or equal to 6 months. All patients were also on other antiinflammatory medications. Evaluations included self-assessment and physician's assessment of well-being, functional capacity, general clinical response, clinical activities indices (National Foundation for Ileitis and Colitis/International Organization for the Study of Inflammatory Bowel Disease and Harvey-Bradshaw), and achievement of specific therapeutic goals. General clinical condition improved in 70% of the patients. Median response time was 3 months. The average Harvey-Bradshaw score decreased 37% with therapy, and a decrease of greater than or equal to 30% occurred in 66% of the subjects. An overall 72% achievement rate for specified therapeutic goals included controlling refractory disease, 73%; corticosteroid "sparing," 76%; and lessening fistulization, 63%. Nine patients got worse despite therapy. Adverse effects requiring discontinuation of therapy occurred in 10%, whereas dosages were briefly lowered for mild side effects in another 10%. This study demonstrates the effectiveness and safety of azathioprine/6-mercaptopurine in the majority of selected patients with chronic, unremitting, or steroid-requiring Crohn's disease.