Expression of MUC1 and MUC2 mucins in epithelial ovarian tumours

This is the first study to describe the association between expression of MUC1 and MUC2 mucins and prognosis in ovarian cancer. Paraffin sections of epithelial ovarian tumours (n=182: 29 benign, 21 low malignant potential, and 132 invasive tumours) were analysed immunohistochemically for expression of MUC1 and MUC2 mucin core proteins. Most benign, low malignant potential, and invasive tumours showed high MUC1 expression in the cytoplasm. Low cytoplasmic expression of MUC1 was a predictor for good prognosis, particularly within stage III tumours. A minority of benign epithelial tumours, but most low malignant potential and invasive non-mucinous tumours, showed high MUC1 expression on the cell membrane. High apical MUC1 reactivity was associated with non-mucinous tumours. Low expression of MUC1 in the apical membrane was associated with early stage and good outcome for invasive tumours. Most benign and low malignant potential tumours, but only a minority of invasive tumours, showed MUC2 expression. MUC2 was found in non-mucinous as well as in mucinous tumours. The presence of MUC2 was inversely associated with high tumour grade but was not associated with altered survival. These results support experimental evidence that MUC1 influences the metastatic ability of ovarian cancer.

Die Synthese von Glycopeptiden und Glycopeptid-Protein-Konjugaten mit einer Partialstruktur des tumorassoziierten Mucins MUC1 zur Entwicklung von Tumorvakzinen

„Synthese von Glycopeptiden und Glycopeptid-Protein-Konjugaten mit einer Partialstruktur des tumorassoziierten Mucins MUC1 zur Entwicklung von Tumorvakzinen“ Das Glycoprotein MUC1 ist in Tumorepithelzellen sonderlich stark überexprimiert und wegen der vorzeitig einsetzenden Sialylierung sind die Saccharid-Epitope der O-Glycanketten stark verkürzt (sog. tumorassoziierte Antigene). Dadurch werden auch bisher verborgene Peptidepitope des Glycoprotein-Rückgrates auf der Zelloberfläche der Epithelzellen zugänglich, die als fremd von den Zellen des Immunsystems erkannt werden können. Dies macht das MUC1-Zelloberfächenmolekül zu einem Zielmolekül in der Entwicklung von Tumorvakzinen. Diese beiden strukturellen Besonderheiten wurden in der Synthese von Glycohexadecapeptiden verbunden, indem die veränderten tumorassoziierten Saccharidstrukturen TN-, STN- und T-Antigen als Glycosylaminosäure-Festphasenbausteine synthetisiert wurden und in das Peptidepitop der Wiederholungseinheit des MUC1 durch Glycopeptid-Festphasensynthese eingebaut wurden. Wegen der inhärenten schwachen Immunogenität der kurzen Glycopeptide müssen die synthetisierten Glycopeptidstrukturen an ein Trägerprotein, welches das Immunsystem stimuliert, gebunden werden. Zur Anbindung der Glycopeptide ist ein selektives Kupplungsverfahren nötig, um definierte und strukturell einheitliche Glycopeptid-Protein-Konjugate zu erhalten. Es konnte eine neue Methode entwickelt werden, bei der die Konjugation durch eine radikalische Additionsreaktion von als Allylamide funktionalisierten Glycopeptiden an ein Thiol-modifiziertes Trägerprotein erfolgte. Dazu wurde anhand von synthetisierten, als Allylamide modifizierten Modellaminosäuren untersucht, ob diese Reaktion generell für eine Biokonjugation geeignet ist und etwaige Nebenreaktionen auftreten können. Mit dieser Methode konnten verschiedene MUC1-Glycopeptid-Trägerprotein-Konjugate hergestellt werden, deren immunologische Untersuchung noch bevorsteht. Das tumorassoziierte MUC1 nimmt in der immundominanten Region seiner Wiederholungseinheit eine knaufartige Struktur ein. Für die Entwicklung von selektiven Tumorvakzinen ist es von großer Bedeutung möglichst genau die Struktur der veränderten Zelloberflächenmoleküle nachzubilden. Durch die Synthese von cyclischen (Glyco)Peptiden wurde dieses Strukturelement fixiert. Dazu wurden olefinische Aminosäure Festphasenbausteine hergestellt, die zusammen mit den oben genannten Glycosylaminosäuren mittels einer Glycopeptid-Festphasensynthese in acyclische Glycopeptide eingebaut wurden. Diese wurden dann durch Ringschlussmetathese zyklisiert und im Anschluss reduziert und vollständig deblockiert. In einem dritten Projekt wurde der Syntheseweg zur Herstellung einer C-Glycosylaminosäure mit einer N-Acetylgalactosamin-Einheit entwickelt. Wichtige Schritte bei der von Glucosamin ausgehenden Synthese sind die Keck-Allylierung, eine Epimerisierung, die Herstellung eines Brom-Dehydroalanin-Derivates und eine B-Alkyl-Suzuki-Miyaura-Kreuzkupplung sowie Schutzgruppenoperationen. Der racemische Baustein konnte dann in der Peptid-Festphasensynthese eines komplexen MUC1-Tetanustoxin-Konjugates eingesetzt werden.

Synthese potentieller Antitumorvakzine aus tumorassoziierten Glycopeptiden des Mucins MUC1 und Versuche zur Festphasensynthese von Oligosacchariden

Festphasenoligosaccharidsynthesen: Um den mit der Synthese der Saccharidbausteine verbundenen Zeitaufwand zu reduzieren, sollten festphasenunterstützte, leicht automatisierbare Strategien entwickelt werden. Für die Optimierungen ist es notwendig, über Analysetechniken zu verfügen, die eine quantitative Verfolgung des Reaktionsablaufes erlauben. Zur Bestimmung der Konzentration freier Hydroxylgruppen wurde hierbei eine einfache und effiziente Reaktionsabfolge gefunden, die eine UV-spektroskopische Analyse erlaubt. rnZur Verknüpfung zwischen Kohlenhydrat und polymerem Träger (Polystyrol, Tentagel) kamen der PTMSE-, ein p-Alkoxybenzyl- und zwei Alkylthioanker zum Einsatz. Die Bedingungen zur Anknüpfung der mit Carboxylgruppen ausgestatteten Anker über Amidbindungen an das Harz, zur Spaltung der Lävulinoylschutzgruppe, sowie zur reduktiven Öffnung der Benzylidenacetalschutzgruppe wurden optimiert, sodass sich diese Reaktionen nun in sehr hohen Ausbeuten am polymeren Träger durchführen lassen. rnSpeziell Glycosylierungsreaktionen zeigten jedoch erhebliche Kompatibilitätsprobleme mit der polymeren Matrix. Die Ausbeuten aller an der festen Phase durchgeführten Glycosylierungen lagen bei maximal 5 Prozent. rnGlycopeptidsynthesen: Einige Tumorzellen unterscheiden sich von gesunden Zellen durch ein verändertes Profil an Oberflächenglycoproteinen. So ist das membranständige Glycoprotein MUC1 in malignen Epithelzellen stark überexprimiert und zeigt infolge veränderter Aktivitäten mehrerer Glycosyltransferasen ein modifiziertes Glycosylierungsmuster. Diese tumorspezifischen Strukturveränderungen stellen einen interessanten Angriffspunkt zur Entwicklung von Antitumorvakzinen dar. Um zu potentiellen Impfstoffen zu gelangen, ist es jedoch nötig, die Immunogenität der selbst nur schwach immunogenen Glycopeptide durch Konjugation zu erhöhen. rnIm Rahmen dieser Arbeit sollten vollsynthetische Konjugate, auf Basis einer 16 Aminosäuren langen, tumorassoziierten Glycopeptidpartialstruktur aus dem MUC1, zur möglichen Verwendung als Antitumorvakzine synthetisiert werden. Die Darstellung erfolgte in einer automatisierten Festphasensynthese nach der Fmoc-Strategie. rnZur Steigerung der Immunogenität wurde das glycosylierte Hexadecapeptid über einen Spacer mit einem T-Zell-Epitop aus dem Tetanustoxoidprotein sowie mit einem Mitogen verknüpft. Zur Synthese des Mitogenkonjugates sind noch Optimierungen im Syntheseablauf und insbesondere bei der Reinigung notwendig. Die immunologische Evaluierung des MUC1-Tetanustoxin-Heterotopkonjugates als mögliches Antitumorvakzin ist geplant. rn

Heparin and cancer revisited: Mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis

Independent studies indicate that expression of sialylated fucosylated mucins by human carcinomas portends a poor prognosis because of enhanced metastatic spread of tumor cells, that carcinoma metastasis in mice is facilitated by formation of tumor cell complexes with blood platelets, and that metastasis can be attenuated by a background of P-selectin deficiency or by treatment with heparin. The effects of heparin are not primarily due to its anticoagulant action. Other explanations have been suggested but not proven. Here, we bring together all these unexplained and seemingly disparate observations, showing that heparin treatment attenuates tumor metastasis in mice by inhibiting P-selectin-mediated interactions of platelets with carcinoma cell-surface mucin ligands. Selective removal of tumor mucin P-selectin ligands, a single heparin dose, or a background of P-selectin deficiency each reduces tumor cell-platelet interactions in vitro and in vivo. Although each of these maneuvers reduced the in vivo interactions for only a few hours, all markedly reduce long-term organ colonization by tumor cells. Three-dimensional reconstructions by using volume-rendering software show that each situation interferes with formation of the platelet “cloak” around tumor cells while permitting an increased interaction of monocytes (macrophage precursors) with the malignant cells. Finally, we show that human P-selectin is even more sensitive to heparin than mouse P-selectin, giving significant inhibition at concentrations that are in the clinically acceptable range. We suggest that heparin therapy for metastasis prevention in humans be revisited, with these mechanistic paradigms in mind.

GAEC1 and colorectal cancer : a study of the relationships between a novel oncogene and clinicopathologic features

GAEC1 is a novel gene located at 7q22.1 that was detected in our previous work in esophageal cancer. The aims of the present study are to identify the copy number of GAEC1 in different colorectal tissues including carcinomas, adenomas, and nonneoplastic tissues and characterize any links to pathologic factors. The copy number of GAEC1 was studied by evaluating the quantitative amplification of GAEC1 DNA in 259 colorectal tissues (144 adenocarcinomas, 31 adenomas, and 84 nonneoplastic tissues) using real-time polymerase chain reaction. Copy number of GAEC1 DNA in colorectal adenocarcinomas was higher in comparison with nonneoplastic colorectum. Seventy-nine percent of the colorectal adenocarcinomas showed amplification and 15% showed deletion of GAEC1 (P < .0001). Of the adenomas, 90% showed deletion of GAEC1, with the remaining 10% showing normal copy number. The differences in GAEC1 copy number between colorectal adenocarcinoma, colorectal adenoma, and nonneoplastic colorectal tissue are significant (P < .0001). GAEC1 copy number was significantly higher in adenocarcinomas located in distal colorectum compared with proximal colon (P = .03). In conclusion, GAEC1 copy number was significantly different between colorectal adenocarcinomas, adenomas, and nonneoplastic colorectal tissues. The copy number was also related to the site of the cancer. These findings along with previous work in esophageal cancer imply that GAEC1 is commonly involved in the pathogenesis of colorectal adenocarcinoma.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.

Exploring the Transcriptome of Atlantic Salmon (Salmo salar) Skin, a Major Defense Organ

The skin of fish is the first line of defense against pathogens and parasites. The skin transcriptome of the Atlantic salmon is poorly characterized, and currently only 2,089 expressed sequence tags (ESTs) out of a total of half a million sequences are generated from skin-derived cDNA libraries. The primary aim of this study was to enhance the transcriptomic knowledge of salmon skin by using next-generation sequencing (NGS) technology, namely the Roche-454 platform. An equimolar mixture of high-quality RNA from skin and epidermal samples of salmon reared in either freshwater or seawater was used for 454-sequencing. This technique yielded over 600,000 reads, which were assembled into 34,696 isotigs using Newbler. Of these isotigs, 12 % had not been sequenced in Atlantic salmon, hence representing previously unreported salmon mRNAs that can potentially be skin-specific. Many full-length genes have been acquired, representing numerous biological processes. Mucin proteins are the main structural component of mucus and we examined in greater detail the sequences we obtained for these genes. Several isotigs exhibited homology to mammalian mucins (MUC2, MUC5AC and MUC5B). Mucin mRNAs are generally > 10 kbp and contain large repetitive units, which pose a challenge towards full-length sequence discovery. To date, we have not unearthed any full-length salmon mucin genes with this dataset, but have both N- and C-terminal regions of a mucin type 5. This highlights the fact that, while NGS is indeed a formidable tool for sequence data mining of non-model species, it must be complemented with additional experimental and bioinformatic work to characterize some mRNA sequences with complex features.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 411 71 using lactose as the substrate Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno (R) in reducing Salmonella enterica serovar Typhimurium (S Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS Here we wished to test the hypothesis that GOS, derived from Bimuno (R) may confer the direct anti-invasive and protective effects of Bimuno (R) In this study the efficacy of Bimuno (R), a basal solution of Bimuno (R) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno (R)] and purified GOS to reduce S Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of similar to 5 mg Bimuno (R) ml(-1) or similar to 2 5 mg GOS ml(-1) significantly reduced the invasion of S Typhimurium (SL1344nal(r)) (P<0 0001) Furthermore, similar to 2 5 mg GOS ml(-1) significantly reduced the adherence of S Typhimurium (SU 344nal(r)) (P<0 0001) It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions In the murine ligated deal gut loops, the presence of Bimuno (R) or GOS prevented the adherence or invasion of S Typhimurium to enterocytes, and thus reduced its associated pathology This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen In all assays, Bimuno (R) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S Typhimurium Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno (R) can be attributed to GOS

Ocular melanoma and mammary mucinous carcinoma in an African lion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ovarian mucinous cystadenoma in a gray brocket deer (Mazama gouazoupira)

An ovarian mucinous cystadenoma is described in a gray brocket deer (Mazama gouazoupira). The tumor was histologically characterized by the presence of cysts and proliferation of papillae, both lined by single- or multi-layered pleomorphic epithelial cells that contained alcian blue-positive mucins.

Staining methods applied to glycol methacrylate embedded tissue sections

The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. on the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues.Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections.The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA. (C) 2003 Elsevier Ltd. All rights reserved.

REVERSIBILITY OF ADENOMATOUS HYPERPLASIA IN THE GASTRIC STUMP AFTER DIVERSION OF BILE REFLUX IN RATS

Morphological and phenotypical patterns of proliferative epithelial lesions induced in the gastric stump mucosa by duodenal content reflux after Billroth II partial gastrectomy (BII) were evaluated in rats. Control animals were either sham-operated or submitted at different times after BII to Roux-en-Y (RY) surgical procedure which prevents duodenal reflux. The lesions were analysed using routine haematoxylin and eosin staining, immunohistochemical staining for pepsinogen isoenzyme 1 and histochemical procedures for mucins (paradoxical Concanavalin A, galactose oxidase Schiff and sialidase galactose oxidase Schiff reactions). Mucosal hyperplasia (H) was observed in the group submitted to BII procedure 6 weeks after surgery. Adenomatous hyperplasia (AH) also appeared 6 weeks after induction of the reflux and its incidence and size increased until the 54th week of the experiment. RY procedure performed in the normal animals at the beginning of the experiment or at the 24th week after BII gastrectomy led to a significantly lower incidence of AH which was related to the moment of surgery. Most of H was due to pyloric mucosal hyperplasia. AH consisted mainly of gastric type glands but in some animals glands of the intestinal type Were present probably originating from the intestinal mucosa. Six mucinous adenocarcinomas were observed, all of them of intestinal type. This study demonstrates that AH induced by BII procedure is a reversible lesion and that the anomalous epithelial proliferation in the stoma may lead to adenocarcinomas.

REDUCTION OF CELL PROLIFERATIVE ACTIVITIES OF GASTRIC STUMP ADENOMATOUS HYPERPLASIAS AFTER BILE REFLUX DIVERSION IN RATS

Previously we reported the majority of lesions induced hy bile reflux, in the absence of chemical carcinogens, in the rat remnant stomach to consist primarily of gastric type and secondarily of intestinal type cells, and that they are reversible after diversion of bile reflux. The present study was designed to evaluate changes in proliferative activities in cells of each type under these conditions. The frequency of adenomatous hyperplasia (AH) induced in the gastric stump mucosa by duodenal content reflux after Billroth II partial gastrectomy (BII) increased until the 54th week of the experiment. Roux-en-Y (RY) surgical procedure which prevents duodenal reflux performed at the 24th or 36th week after BII led to a decrease in AH. Cell content of the lesions was analyzed using routine H&E staining, immunohistochemical staining for pepsinogen isoenzyme 1 and histochemical procedures for mucins (paradoxical concanavalin A, galactose oxidase Schiff and sialidase galactose oxidase Schiff reactions) and proliferation in each compartment evaluated by an immunohistochemical method using bromodeoxyuridine (BrdU) and a monoclonal antibody against BrdU. At the 54th week the number of BrdU-labeled cells per normal pyloric column was significantly (P < 0.05) increased to 10.63/pit after the BII operation, while it diminished to 5.23/pit after RY diversion, this being the same level as with the RY procedure alone. AH maintained a high rate of BrdU incorporation at 12.7% after BII operation, which was also significantly reduced (P < 0.01) to 7.0% by the RY surgery. The intestinal type cell showed highest (22.2%), the surface mucous type cell showed the next (16.5%) and the pyloric gland type cell showed lowest (5.2%) BrdU labeling indices after BII operation. All the cell types in AH showed similar proportional decreases in BrdU incorporation after RY diversion. Thus surgical intervention reverses the cell proliferation caused by bile reflux in the gastric stump.