David R. Mack

Elevated extrahepatic expression and secretion of mammary-associated serum amyloid A 3 (M-SAA3) into colostrum.

Mammary-associated serum amyloid A 3 (M-SAA3) was secreted at highly elevated levels in bovine, equine and ovine colostrum and found at lower levels in milk 4 days postparturition. N-terminal sequencing of the mature M-SAA3 protein from all the three species revealed a conserved four amino acid motif (TFLK) within the first eight residues. This motif has not been reported to be present in any of the hepatically-produced acute phase SAA (A-SAA) isoforms. Cloning of the bovine M-Saa3 cDNA from mammary gland epithelial cells revealed an open reading frame that encoded a precursor protein of 131 amino acids which included an 18 amino acid signal peptide. The predicted 113 residue mature M-SAA3 protein had a theoretical molecular mass of 12,826Da that corresponded with the observed 12.8kDa molecular mass obtained for M-SAA3 in immunoblot analysis. The high abundance of this extrahepatically produced SAA3 isoform in the colostrum of healthy animals suggests that M-SAA3 may play an important functional role associated with newborn adaptation to extrauterine life and possibly mammary tissue remodeling.

Introduction of 6-mercaptopurine in Crohn's disease patients during the perioperative period: a preliminary evaluation of recurrence of disease.

BACKGROUND Recurrence of Crohns disease after surgery is a common occurrence, pointing to the need for a strategy to prevent recurrent disease. We report the postoperative course of 10 patients who required intestinal resections for complications related to Crohns disease. METHODS All patients had a Pediatric Crohns Disease Activity Index score of 10 or greater. Among these patients, 5 began treatment with 6-mercaptopurine in the perioperative period. All 10 had received various combinations of prednisone and salicylate compounds. Patients who were given 6-mercaptopurine did not discontinue the medication until 2 years after the surgery. RESULTS To date, none of the five patients who were placed on 6-mercaptopurine have had recurrence of their Crohns disease (mean disease-free period 32.6 +/- 18.4 months). Among those five patients not receiving 6-mercaptopurine there have been three relapses (mean time to relapse 3.7 +/- 1.2 months). Log-rank sum analyses of Kaplan-Meier survival curves show benefit to patients receiving 6-mercaptopurine in preventing relapses after intestinal resection (p < 0.05). CONCLUSIONS Although the underlying pathophysiologic reasons leading to the high relapse rate after intestinal surgery in Crohns disease are unknown, we conclude that treatment with 6-mercaptopurine in the perioperative period may be warranted to help prevent the recurrence of Crohns disease after surgery.

Functional heterogeneity of colonic adenocarcinoma mucins for inhibition of Entamoeba histolytica adherence to target cells.

Mucins secreted from the gastrointestinal epithelium form the basis of the adherent mucus layer which is the hosts first line of defense against invasion by Entamoeba histolytica. Galactose and N‐acetyl‐D‐galactosamine residues of mucins specifically inhibit binding of the amebic 170 kDa heavy subunit Gal‐lectin to target cells, an absolute prerequisite for pathogenesis. Herein we characterized the secretory mucins isolated from the human colon and from three human colonic adenocarcinoma cell lines: two with goblet cell‐like (LS174T and T84) and one with absorptive cell‐like morphology (Caco‐2). By Northern blot analysis the intestinal mucin genes MUC2 and MUC3 were constitutively expressed by confluent LS174T and Caco‐2 cells, whereas T84 cells only transcribed MUC2 and not MUC3 mRNA. 3H‐glucosamine and 3H‐threonine metabolically labeled proteins separated as high Mr mucins in the void (Vo > 106 Da) of Sepharose‐4B column chromatography and remained in the stacking gel of SDS‐PAGE as depicted by fluorography. All mucin preparations contained high amounts of N‐acetyl‐glucosamine, galactose, N‐acetyl‐galactosamine, fucose and sialic acid, saccharides typical of the O‐linked carbohydrate side chains. Mucin samples from the human colon and from LS174T and Caco‐2 cells inhibited E. histolytica adherence to Chinese hamster ovary cells, whereas mucins from T84 cells did not. These results suggest that genetic heterogeneity and/or posttranslational modification in glycosylation of colonic mucins can affect specific epithelial barrier function against intestinal pathogens.

ALTERED EXPRESSION OF SIALYLATED CARBOHYDRATE ANTIGENS IN HT29 COLONIC CARCINOMA CELLS

To determine whether cell growth conditions impacted carbohydrate expression, HT29 cells were gradually transferred from a conventional glucose-containing media to a glucose-free galactose containing media. Indirect immunofluorescence on acetone fixed cells showed increased expression of sialyl Lewis A antigen (CA19-9), sialyl Lewis C (DUPAN2) and Tn/sialyl-Tn on the surface of HT29 cells grown in the glucose-free galactose containing media compared to those grown in the glucose containing media. Sialyltransferases responsible for the synthesis for these sialylated epitopes were increased in the galactose-fed HT29 cells. Media overlying the cells was subjected to isopycnic ultracentrifugation in cesium chloride and the fractions derived from both glucose and galactose media with equivalent buoyant densities of 1.56 g/L, which are predicted to contain mucin glycoforms, were further separated by HPLC using a Mono-Q anion exchange column. The chromatograph of eluent from the sample derived from the cells growing in the galactose containing media showed an increased peak that reacted with the anti-sialyl Lewis A antibody, CA19-9. These results show that alteration of in vitro culture conditions may cause HT29 colonic carcinoma cells to alter the expression of sialylated carbohydrates.

Hydrophobicity and the gastrointestinal tract: methods of determination, its source and implications for bacterial adherence

Abstract Increasing research efforts are now focused on characterizing the physicochemical properties of microbial pathogens and the biologic surfaces to which the organisms adhere. Interest is intense because there is the potential to develop novel strategies to disrupt the infectious process and thereby employ therapeutic agents other than antimicrobial compounds. This review will focus on the gut as a model system in order to highlight advances in the field.