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C Hanski

Publications and source records attributed to C Hanski.

At least 55 records · Page 3Linked to original sources
PubMed1992

Sialyl Lewis(x) antigen as defined by monoclonal antibody AM-3 is a marker of dysplasia in the colonic adenoma-carcinoma sequence.

Monoclonal antibody AM-3 (MAb AM-3) raised against a sialomucin from human colorectal carcinoma has previously been shown to define a carbohydrate epitope, which is detectable by immunocytochemistry on all investigated colonic carcinomas and is expressed in correlation with the grade of dysplasia in colonic adenomas (Hanski et al., J. Clin. Pathol., 43: 379-385, 1990). Epitope analyses in solid-phase enzyme immunoassays revealed that AM-3 antibody recognizes the sialylated Lewis(x) sequence on a branched O-linked glycan and its reductively cleaved alditol from human amniotic mucins. In comparative binding and binding inhibition studies MAbs AM-3 and CSLEX1 displayed reciprocal affinities to mucins versus gangliosides. Correspondingly, the weaker binding activities of AM-3 versus CSLEX to III3-alpha Fuc-IV3-alpha NeuAc-nLcOse4-Cer or to monogangliosides from human granulocytes were measured. Gangliosides from a human colon carcinoma were recognized by MAb CSLEX1 exhibiting a broader specificity to various sialyl-Lewis(x) antigens and by MAb FH6 reactive to sialyl-dimeric Lewis(x) antigen, but not by MAb AM-3. In conclusion, MAb AM-3 is distinguished from other sialyl Lewis(x)-specific MAbs by its selective reactivity to mucin-carried epitopes on the monomeric antigen.

Adenoma↗
PubMed1992

Tumorigenicity, mucin production and AM-3 epitope expression in clones selected from the HT-29 colon carcinoma cell line.

Two mucin-producing cell clones (16.2 and 12.2) and a mucin-deficient clone (15.2) were selected from the established human adenocarcinoma cell line HT-29 by limiting dilution and Alcian blue staining. The amounts of the mucin antigen detectable on the cell surface with the monoclonal antibody (MAb) AM-3 decreased in the order HT-29 greater than 16.2 greater than 12.2 greater than 15.2 = 0. The binding avidity of AM-3 antibody to cells as well as to mucin extracts from each cell line decreased in the same order, indicating that the epitope density on the cell-bound mucins was highest in HT-29 and lowest in 12.2 cells. The parental line and the mucin-producing cell clones 16.2 and 12.2 showed no contact inhibition and grew as aggregates, while the 15.2 cells were well spread and formed a regular monolayer. The mucin-producing cell lines injected into nude mice yielded solid tumors with different growth rates (HT-29 greater than 16.2 greater than 12.2), while the 15.2 cell clone was not tumorigenic at all. The relative amounts of total mucin-bound hexoses and of the mucin epitope AM-3 decreased in the xenografts in the order HT-29 greater than 16.2 greater than 12.2. The present system is suitable for investigating the role of mucins in growth of colon carcinoma cells and indicates that increased tumorigenicity in nude mice coincides with the increase in total mucin expression and the expression of the AM-3 mucin epitope in tumor tissue.

Adenocarcinoma↗
PubMed1991

Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the rat.

Glycosyltransferase activities of highly purified fractions of Golgi apparatus, plasma membrane and endoplasmic reticulum, all from the same homogenates, were analyzed and compared. Additionally, Golgi apparatus were unstacked and the individual cisternae separated into fractions enriched in cis, median and trans elements using the technique of preparative free-flow electrophoresis. Golgi apparatus from both liver and hepatomas were enriched in all glycosyltransferases compared to endoplasmic reticulum and plasma membranes. However, Golgi apparatus from hepatomas showed both elevated fucosyltransferase and galactosyltransferase activities but reduced sialyltransferase and dipeptidyl peptidase IV (DPP IV) activities compared to liver. Activity of N-acetylglucosaminyltransferase was approximately the same in both liver and hepatoma Golgi apparatus. With normal liver, sialyl- and galactosyltransferase activities and DPP IV showed a marked cis-to-trans gradient of activity. Fucosyltransferase was concentrated in two regions of the electrophoretic separations, one corresponding to cis cisternae and one corresponding to trans cisternae. N-Acetylglucosaminyltransferase activity was more widely distributed but the endogenous acceptor activity was predominantly cis. With hepatoma Golgi apparatus, the pattern for DPP IV was similar to that for liver but those of sialyl- and galactosyltransferases differed markedly from liver. Instead of activity increasing cis to trans, the activities for sialyl- and galactosyltransferases decreased. For fucosyltransferases, activity dependent on exogenous acceptor was medial whereas with endogenous acceptor, two activity peaks, cis and trans, still were observed. For N-acetylglucosaminyltransferase the pattern for hepatoma was similar to that for liver. The results indicate alterations in the distribution of glycosyltransferase activities within the Golgi apparatus in hepatotumorigenesis that may reflect altered cell surface glycosylation patterns.

Animals↗
PubMed1991

Increased number of accessible sugar epitopes defined with monoclonal antibody AM-3 on colonic mucins is associated with malignant transformation of colonic mucosa.

Monoclonal antibody AM-3 detects a mucin sugar epitope (AM-3 epitope) the expression of which increases in the course of human colon carcinogenesis parallel to the gradual morphological alterations (so called adenoma-carcinoma sequence). In the present report the AM-3-positive mucin has been purified from human normal and carcinomatous colonic tissue. About 300-fold enrichment of the epitope per protein from both sources was achieved after ultracentrifugation, gel filtration on Sepharose CL-6B and isopyknic gradient centrifugation. Slot-blot and enzyme-linked immunosorbent assays of the purified preparation indicated not only different amounts of the mucins but also a consistent qualitative difference between the molecules from both sources. The qualitative difference could be obliterated by a partial removal of the AM-3 epitope from the tumor-derived mucin with neuraminidase. The visualization of the molecules by rotary shadowing indicated that the mucins from both sources have similar length distribution, 80% of the molecules being 100-600 nm long. The reaction with AM-3 antibody followed by rotary shadowing showed that in the purified preparations more than 95% of the tumor-derived molecules and 74% of the normal colon tissue-derived molecules carried the epitope. The tumor-derived mucins bound, on the average, 34 +/- 15 (SD) antibodies/1000 nm of the protein core while the mucin from normal colon tissue carried 12 +/- 11 antibodies/1000 nm of the protein core. These data indicate that the increased expression of AM-3 epitopes during malignant transformation of the human colon is due to accumulation of AM-3-positive mucin as well as a higher number of accessible AM-3 epitopes on this mucin.

Antibodies, Monoclonal↗
PubMed1991

Expression in vivo of additional plasmid-mediated proteins during intestinal infection with Yersinia enterocolitica serotype O8.

The decisive aspect of Yersinia enterocolitica virulence in vivo is the ability of the plasmid-bearing bacteria to resist the immune response within the host tissue. The expression of plasmid-mediated virulence proteins was investigated in the intestinal lumen and in the Peyer's patches of infected mice. Three novel plasmid-mediated outer-membrane proteins have been identified with antisera raised against bacteria grown in vivo. When the bacteria were grown in the intestinal lumen, all plasmid-coded proteins known to be expressed in vitro, except the 26-Kda protein were expressed. Additionally, a novel outer-membrane protein of 23 Kda was synthesised. After penetration into the Peyer's patches, two further proteins of 240 and 210 Kda were expressed. None of these three proteins was detected in the outer membrane of bacteria grown in vitro. By contrast, plasmid-coded released proteins, which are abundantly synthesised in Ca(2+)-deficient media in vitro, were not detectable in the ileal lumen nor in the tissue of infected Peyer's patches, which suggests that they are not required for Y. enterocolitica pathogenesis.

Animals↗
PubMed1991

Humoral and cellular defense against intestinal murine infection with Yersinia enterocolitica.

The role of phagocytes and the complement system as potential host defense mechanisms against bacterial infection were studied in mice with two isogenic strains of Yersinia enterocolitica serotype O8 differing in pathogenicity because of differences in plasmid content. Complement depletion in mice by intraperitoneal injection of cobra venom factor did not affect the course of colonization of the intestinal tissue by each strain, indicating that in mice complement is not essential for the elimination of these bacteria. This conclusion is supported by the fact that fresh murine serum had no bactericidal effect in vitro either on the pathogenic or on the nonpathogenic strain. However, in the intestinal tissue as well as in the peritoneal cavity, only the pathogenic, plasmid-bearing Y. enterocolitica strain survived, while the nonpathogenic, plasmidless strain was rapidly eliminated. Since elimination from the peritoneal cavity is due to phagocytosis by polymorphonuclear leukocytes and macrophages, resistance to phagocytosis in vivo seems to be the decisive factor determining the virulence of pathogenic Y. enterocolitica strains.

Animals↗
PubMed1990

Identification of laminin binding proteins in cell membranes of a human colon adenocarcinoma cell line.

The invasion of malignant cells through the basement membrane is a critical step in local infiltration and metastasis. Adhesion and invasion of malignant cells may be modulated by their receptor mediated binding to the basement membrane glycoprotein laminin. We studied the specific adhesion of human colon adenocarcinoma derived HT 29 cells to laminin and its proteolytic fragments. The major cell adhesion domain of laminin was localised in the central part of the cross shaped molecule. Immunoblotting experiments on separated HT 29 cell membranes using specific antibodies or radiolabelled laminin fragments revealed two major laminin-binding cell surface components with Mr of 67,000 and 69,000 D similar to the putative laminin receptor described for other tissues. Using a nitrocellulose filter disk assay, the specific interaction between cell surface binding proteins and proteolytic fragments originating from the central core of the laminin molecule could be further corroborated. In contrast, interaction of HT 29 cell membranes with the pentapeptide YIGSR (tyr-ile-gly-ser-arg), a sequence domain of the B1-chain of the laminin molecule, thought to be responsible for cell adhesion, was significantly weaker.

Adenocarcinoma↗
PubMed1990

Involvement of M cells in the bacterial invasion of Peyer's patches: a common mechanism shared by Yersinia enterocolitica and other enteroinvasive bacteria.

Recent evidence indicates that ileal Peyer's patches represent the main infection route for Yersinia enterocolitica. In this study transmission and scanning electron microscopy have shown that only a small fraction of bacteria present in the lumen adhere to the follicle-associated murine epithelium with no discernible preference for either M or absorptive cells. Yersiniae attached to M cells are phagocytosed and transported from the lumen into the lamina propria. No invasion of columnar absorptive cells was observed. These data, in combination with recently published reports, indicate that the involvement of M cells is a common step in bacterial invasion of Peyer's patches.

Animals↗
PubMed1990

Detection of a mucin marker for the adenoma-carcinoma sequence inhuman colonic mucosa by monoclonal antibody AM-3.

The monoclonal antibody AM-3 was raised against mucins extracted from human colorectal carcinomas. It reacted strongly with sections of paraffin wax embedded colorectal carcinoma. In colonic adenoma tissue the percentage of cells expressing the epitope detected by AM-3 correlated with the degree of dysplasia. In contrast to immunohistochemical staining, which did not show the presence of the antigen in histologically normal mucosa, the more sensitive enzyme linked immunosorbent assay (ELISA) and immunoblot assays showed that it was weakly expressed in this tissue. AM-3 reacted with variable frequency with several normal and malignant human tissues, indicating that the detected epitope is not restricted to colonic tissue. In colonic carcinomas it is present on a sialomucin of apparent relative molecular mass of more than 440,000. These data suggest that the antigen detectable with AM-3 may be useful in the assessment of premalignant changes in colonic adenomas.

Adenoma↗
PubMed1990

Further characterization of the mucin antigen associated with malignant transformation of human colonic mucosa, detectable with the monoclonal antibody AM-3.

The recently obtained monoclonal antibody AM-3 reacts with an antigenic determinant present in several normal and neoplastic human tissues. In the large intestine the expression of the antigen is very low, but it increases dramatically after malignant transformation. In colonic adenomas the antigen expression correlates with the grade of dysplasia. The detected epitope is membrane-bound in the epithelial membranes of the liver, kidney and esophagus, while the membrane-bound and secretory forms are detectable in the salivary gland and in transformed colonic mucosa. However, the epitope is located on different molecules in all the investigated tissues. Its resistance to denaturing agents and its sensitivity to neuraminidase treatment in preparations from every investigated tissue suggest that it is a common carbohydrate-containing epitope.

Antibodies, Monoclonal↗
PubMed1989

Determinants of invasion and survival of Yersinia enterocolitica in intestinal tissue. An in vivo study.

The invasion of intestinal tissue by Yersinia enterocolitica was studied after infection of mice with plasmid-bearing or with plasmidless strains of serotype 08 and 03. Within the first 15 h of infection each strain colonized the mouse ileum to the same extent, indicating that the initial binding and invasion are not plasmid dependent. This conclusion is supported by the finding that the plasmidless and the plasmid-bearing Yersiniae (serotype 08) colonize the ileum in a quantitatively similar, dose-dependent manner. The data show that the virulence of Yersiniae depends primarily on their ability to survive and to proliferate within the tissue. This character was expressed by the plasmid-bearing 08 strain, but not by the plasmidless 08 and 03 strain nor by the plasmid-bearing 03 strain. An orally induced rabbit antiserum was used to identify plasmid-coded antigens (relative molecular masses = 47, 45 and 42 kDa), which are expressed in vivo by the plasmid-bearing 08 strain, but not by the three other strains investigated. These antigens may be crucial for virulence of the 08 strain.

Animals↗
PubMed1989

Biochemical properties of dipeptidyl peptidase IV in liver and hepatoma plasma membranes.

In the present investigation we compared the glycoprotein DPP IV from rat liver and Morris hepatoma 7777 by means of biochemical and immunological methods. For that purpose nine monoclonal anti-DPP IV-antibodies recognizing four different epitopes and a monospecific anti-DPP IV-antiserum were applied. In the homogenates of both tissues a plasma membrane-bound and a soluble form were detected. The immunological cross-reactivity of both forms was demonstrated with the antiserum and the monoclonal antibodies against the epitopes A, B and C while epitope D was restricted to liver plasma membrane. Differences of the distinct DPP IV forms were exhibited in the molecular weights, isoelectric points and peptide maps. In the hepatoma homogenate only 10% of DPP IV activity was found compared to normal liver but the ratio of soluble to membrane-bound form is higher in the hepatoma than in the liver. The fractionation of the homogenates into different cell components revealed for the liver a continuous increase of DPP IV activity from the endoplasmic reticulum fractions to the Golgi apparatus and finally to the plasma membranes. By contrast, in hepatoma the flow from the Golgi apparatus to plasma membrane was greatly reduced. The loss of DPP IV from the surface of cultured hepatoma cells was concomitant with a decrease of cell-substratum adhesion. DPP IV was found to be inserted into the liver plasma membrane by two different mechanisms, a phospholipase C-sensitive and a papain-sensitive one. In the hepatoma the phospholipase C-sensitive anchorage was not expressed. Besides liver and hepatoma the distribution of DPP IV was characterized in various rat organs by enzyme activity, histochemistry and immunohistochemistry with the anti-DPP IV-antibodies.

Animals↗
PubMed1989

Immunohistochemical and electron microscopic study of interaction of Yersinia enterocolitica serotype O8 with intestinal mucosa during experimental enteritis.

The experimental infection of mice with Yersinia enterocolitica serotype O8 was investigated in a quantitative and histological study. The course of bacterial penetration and spreading was precisely determined by immunohistochemical staining. After oral administration, the bacteria passed the epithelial barrier of the ileum and spread into the lamina propria. By preference they entered Peyer's patches, which were about 1,000 times more heavily colonized than the surrounding epithelium of a comparable surface area. The bacteria proliferated in the follicles, from which they spread into the lamina propria of the villi. At either site most of the bacteria multiplied extracellularly, with only a small percentage observed to be present within the phagocytes. The bacteria did not appear to be able to pass the intact basement membrane; hence, the integrity of the basement membrane is likely to play a role in determining the route of entry and limit of spread of Y. enterocolitica infection.

Animals↗
PubMed1988

Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV.

The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.

Animals↗
PubMed1988

Characterization of different forms of dipeptidyl peptidase IV from rat liver and hepatoma by monoclonal antibodies.

Nine monoclonal antibodies directed against DPP IV from rat liver plasma membranes were obtained. They recognized four different epitopes (A, B, C and D) of the enzyme. The epitopes A, B and C were located on the outside of the hepatocyte plasma membrane and were shared by DPP IV from hepatoma plasma membrane and the soluble form. Epitope D appeared to be partly inserted in the membrane and was found exclusively in the liver. Epitopes A, B and C and DPP IV revealed by histochemical means showed similar distribution patterns on frozen sections of various rat tissues, while epitope D did not show such a correlation. DPP IV is inserted in liver plasma membrane by two different mechanisms, one being phospholipase C-sensitive, while in hepatoma the enzyme is anchored in this membrane by a phospholipase C-resistant mechanism only.

Animals↗
PubMed1988

Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry.

Immunohistochemistry and activity histochemistry were used to study the localization of dipeptidyl peptidase (DPP) IV in rats. For immunohistochemistry, polyclonal as well as monoclonal anti-DPP IV antibodies were employed. The pattern of DPP IV immunoreactivity, determined with polyclonal anti-DPP IV antibody, corresponds to the histochemical pattern found for the enzymic activity of DPP IV. Immunoreactivity was present, in addition, in nerve cells, lateral membranes of certain surface epithelia, e.g., Fallopian tube, uterus and vesicular gland, in the luminal cytoplasm of e.g., vesicular gland epithelium, and in mucous cells of Brunner's gland. The monoclonal antibodies against DPP IV recognized four different epitopes (A-D) of the DPP IV molecule, and revealed that certain epitopes were not detectable by immunohistochemistry in some organs. Generally, the staining intensities for epitopes A, B, C and D decreased in that order. Usually, the monoclonal antibodies against epitopes A and B showed similar reaction patterns to those as obtained with the polyclonal antibody. Epitope D was recognized in the lumen of the duct system of exocrine glands and the intestine. Furthermore, high reactivity of this epitope was detected in goblet cells of the intestine, where no DPP IV activity was present.

Animals↗
PubMed1988

Direct evidence for the binding of rat liver DPP IV to collagen in vitro.

Previous studies have shown that the tripeptide Gly-Pro-Ala, a substrate for dipeptidyl peptidase IV (DPP IV, EC 3.3.14.5), interferes with initial spreading of hepatocytes on a matrix consisting of fibronectin and denatured collagen. In the present investigation we report that the tripeptide as well as the anti-DPP IV antibody inhibits the initial spreading of hepatocytes also on native collagen. This effect appears to be due to the interaction of DPP IV from hepatocyte plasma membrane with native collagen. It is shown in vitro by immunohistochemistry, catalytic histochemistry, and by affinity chromatography of solubilized plasma membrane on collagen-Sepharose that DPP IV has a binding affinity to collagen. This binding does not affect the activity of DPP IV.

Animals↗