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Differential diagnosis between adrenal cortical and adrenal medullary lesions may be difficult in many cases. Different immunohistochemical, histochemical tools as well as ultrastructural diagnostic techniques have been employed to aid in differentiating between these lesions. Recently, both inhibin-A and BCL-2 have been shown to stain selectively adrenal cortical tissue and its derived neoplasms but not adrenal medulla or pheochromocytomas. In this study we compared the staining reactions of inhibin-A and BCL-2 in cases of adrenal cortical adenomas and carcinomas as well as pheochromocytomas. We found that both inhibin-A and BCL-2 stained cortical derived tissues, but not medullary derived tissues. Staining intensity for inhibin-A was significantly weaker than for BCL-2. We found that fixation techniques may influence the staining reactivity, as some cases did not immunoreact with any of the antibodies. We conclude that both inhibin-A, and, preferentially, BCL-2 are useful additions to a staining protocol to help in the differential diagnosis of cortical and medullary neoplasms.

Neutrophil infiltration of the colonic mucosa is a hallmark of Clostridium difficile toxin A-mediated enterocolitis. Macrophage-inflammatory protein-2 (MIP-2) is a potent neutrophil chemoattractant secreted by rat macrophages and epithelial cells in response to inflammatory stimuli. In this work, we report that administration of toxin A into rat ileal loops increased mucosal levels of MIP-2 before the onset of fluid secretion and mucosal neutrophil infiltration. Administration of rabbit anti-MIP-2 IgG, but not control IgG, reduced toxin A-mediated secretion (by 58%), mucosal permeability (by 80%), and myeloperoxidase activity (by 85%). Immunohistochemical analysis demonstrated increased MIP-2 expression in intestinal epithelial and lamina propria cells 1 h after toxin A administration. Intestinal epithelial cells purified from toxin A-exposed ileal loops also showed increased MIP-2 mRNA expression and MIP-2 protein release that was inhibited by pretreatment of rats with the transcriptional inhibitor actinomycin D. These results indicate that C. difficile toxin A induces MIP-2 release from intestinal epithelial cells and that MIP-2 contributes to neutrophil mucosal influx during toxin A enteritis.

We recently reported that immobilization stress increased colonic motility, mucin, and prostaglandin E2 (PGE2) release and mucosal mast cell degranulation in rat colon [Proc. Natl. Acad. Sci. USA 93: 12611-12615, 1996; Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G884-G892, 1996]. To directly assess the contribution of mast cells, we compared colonic responses to stress in mast cell-deficient KitW/KitW-v and normal(+/+) mice. Mucin and PGE2 release were measured in colonic explants cultured from KitW/KitW-v and (+/+) mice 30 min after immobilization stress. We found that stress stimulated colonic mucin release (1.8-fold), goblet cell depletion (3-fold), and PGE2 (2.3-fold) release in (+/+) but not mast cell-deficient KitW/KitW-v mice. However, mast cell-deficient mice that had their mast cell population reconstituted by injection of bone marrow-derived mast cells from (+/+) mice had colonic responses to stress similar to those of normal (+/+) mice. In contrast, colonic transit changes in response to stress, estimated by fecal output, were similar between KitW/KitW-v and normal (+/+) mice. We conclude that mast cells regulate colonic mucin and PGE2 release but not colonic transit changes in response to immobilization stress.

Interleukin-11 (IL-11) is a stromal cell-derived cytokine with several biological activities against hematopoietic cells. Recent results indicated that IL-11 reduced mucosal damage in animal models of colitis. This study aimed to explore the action of recombinant human IL-11 (rhIL-11) on the intestinal effects of Clostridium difficile toxin A, an inflammatory enterotoxin, and cholera toxin, a noninflammatory enterotoxin in rat ileum. We administered rhIL-11 subcutaneously to rats before injection of toxin A into ileal loops and measured fluid secretion, epithelial permeability to mannitol, histopathological damage, and release of rat mast cell protease II (RMCP II) from intestinal mast cells and of tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) from lamina propria macrophages. rhIL-11 (50-1,000 micrograms/kg) inhibited toxin A but not cholera toxin-mediated secretion and permeability in a dose-dependent fashion and reduced toxin A-induced epithelial cell damage. Rats treated with rhIL-11 also showed reduced RMCP II, TNF-alpha, and MIP-2 release in response to toxin A. Exposure of rat peripheral monocytes in vitro to rhIL-11 (1 microgram/ml) inhibited lipopolysaccharide and toxin A-mediated increases in TNF-alpha mRNA and protein levels. We conclude that rhIL-11 blocks the intestinal effects of C. difficile toxin A, possibly by inhibiting release of inflammatory mediators from mucosal mast cells and intestinal macrophages.

Topoisomerase II alpha (TP) excises and reconnects double-stranded super-coiled DNA during the replicative cell cycle. We studied the localization of TP and Ki-67 in inflammatory and neoplastic mucosal lesions of the stomach and of TP in similar conditions of the colon. TP expression was correlated with tumor stage, grade, and survival time in the colonic tumors to evaluate its potential utility as a predictive marker for clinical outcome. Forty-three sections of chronic gastritis, lesions indefinite for dysplasia, low- and high-grade dysplasia, and gastric adenocarcinomas were immunostained with antibody against TP and Ki-67. For the colon, 71 sections of normal mucosa, chronic colitis, hyperplastic polyps, adenomas, and carcinomas were examined; fresh tissue was analyzed by flow cytometry. Expression of TP in non-neoplastic gastric mucosa was maximal in neck/foveolar cells and focal in surface and deep gland cells. Increased surface and deep gland positivity was found in low-grade dysplasia and a diffuse distribution of positive cells in high-grade dysplasia and carcinoma. The Ki-67 staining pattern was similar. TP in non-neoplastic colon was restricted to the lower crypt zone; it was greatly expanded in the surface/upper crypt region in adenomas and was diffuse in carcinomas. Flow cytometric analysis revealed TP expression mainly in the S and G2/M phase, with higher labeling index in tumors. There was no correlation of TP with stage, grade, or survival times in the colonic tumors. Staining for TP and Ki-67 might help in the distinction of inflammatory and neoplastic lesions of the stomach and colon.

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans.

We determined the effects of immobilization stress on rat colonic mucus release and mast cell degranulation and examined whether corticotropin releasing factor (CRF) was involved in these responses. After 30-min immobilization, rats were killed, colonic mucosal explants were cultured, and levels of rat mast cell protease II (RMCP II) and prostaglandin E2 (PGE2) were measured. Mucin release from explants was assayed by incorporation of [3H]glucosamine into colonic mucin and by histological evaluation of goblet cell depletion. Stress caused significant increases of colonic RMCP II, PGE2, and mucin release and fecal pellet output and caused an approximately 10-fold increase in colonic mucosal levels of cyclooxygenase-2 (COX-2) mRNA. These stress-associated changes were reproduced by intravenous or intracerebral injection of CRF in conscious, nonstressed rats. Pretreatment of rats with the CRF antagonist alpha-helical-CRF9-41, hexamethonium, atropine, or bretylium, or the mast cell stabilizer lodoxamide inhibited stress-induced release of RMCP II, PGE2, and mucin, whereas indomethacin prevented mucin release but not mast cell degranulation. Hexamethonium and CP-96,345, a substance P antagonist, inhibited fecal pellet output caused by stress. We conclude that CRF released during immobilization stress increases colonic transit via a neuronal pathway and stimulates colonic mucin secretion via activation of neurons and mast cells.