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F G Hanisch

Publications and source records attributed to F G Hanisch.

At least 55 records · Page 3Linked to original sources
PubMed1995

Expression of alpha-3/4-monofucosylated polylactosaminoglycan epitope, as defined by monoclonal antibody FW6, is a marker of the colorectal adenoma-carcinoma sequence.

BACKGROUND: The expression of a distinct alpha-3/4-monofucosylated polylactosaminoglycan epitope, which is detected by monoclonal antibody FW6, was investigated by comparative immunohistochemical analysis of colorectal tissue specimens exhibiting different grades of premalignant and malignant transformation. The presence of this peculiar epitope was compared with different lewis type 2 blood group antigens. METHODS: Paraffin embedded specimens from 8 hyperplastic polyps, 46 adenomas, 27 colorectal carcinomas, and 10 corresponding liver metastases were studied. Staining reactions included monoclonal antibodies FW6, AM-3 (anti-sialosyl-Le(x)), LeuM1 (anti-Le(x)), and 12-4LE (anti-Le(y)) in a standard peroxidase-antiperoxidase method. RESULTS: Hyperplastic polyps were not reactive with FW6 or LeuM1, but showed a slight binding of AM-3 and 12-4LE in some cases. Approximately two-thirds of the adenomatous polyps displayed a pronounced staining activity by AM-3, and approximately half of them revealed FW6, LeuM1, and 12-4LE binding. Only the expression of the FW6 (P < 0.005) epitope correlated with the presence of severe dysplasia. All antibodies were more or less reactive with colorectal carcinomas and their liver metastases, and some showed correlating binding patterns. CONCLUSIONS: FW6 revealed a high specificity for adenomas with areas of severe epithelial dysplasia. Because this monoclonal antibody also detects the great majority of carcinomas, it is reasonable to postulate that the alpha-3/4-monofucosylated polylactosaminoglycan epitope is an important marker for malignant transformation in the colorectal adenoma-carcinoma sequence.

Adenoma↗
PubMed1995

Monoclonal antibody BW835 defines a site-specific Thomsen-Friedenreich disaccharide linked to threonine within the VTSA motif of MUC1 tandem repeats.

mAb BW835 (IgG1) has been generated to breast cancer cell lines by alternating injections of MCF-7 or SW-613 cells and has been demonstrated to be of value in the serodiagnosis of mammary carcinoma. BW835 defines a carbohydrate epitope on integrated or secreted MUC1 glycoforms from carcinoma cells and human milk. To identify BW835-reactive glycopeptides on MUC1, proteolytic fragments of the mucin obtained by digestion with the Gly-C-specific endopeptidase IV from papaya corresponding to low molecular mass fragments (< 10 kilodaltons) of the tandem repeat domain were screened. A glycosylated fragment (glycopeptide 17) containing the mAb HMFG-2-defined epitope was highly reactive to BW835 antibody, while nonglycosylated tandem repeat peptide TAP25 or its in vitro-glycosylated N-acetylgalactosamine (GalNAc) derivatives were unreactive. Glycopeptide 17 bound to peanut agglutinin and to a Thomsen-Friedenreich antigen (TF alpha)-specific mAb (A78-G/A7). Binding of BW835 to glycopeptide 17 or to MUC1 was competitively inhibited by peanut agglutinin and by the synthetic glycopeptides TF alpha Ser or TF alpha Thr but not by their beta-anomers. Evidence for site specificity of binding by BW835 to glycopeptide 17 was revealed by demonstrating nonreactivity of the antibody to other TF alpha-expressing glycoproteins with peptide moieties lacking MUC1-specific motifs at putative glycosylation sites. The epitope of BW835 was localized to threonine within the VTSA-peptide motif by site-specific enzymatic beta-galactosylation of the synthetic tandem repeat peptide TAP25-GalNAc1 TAPPAHGVT(-O-alpha GalNAc)SAPDTRPAPGSTAPPA. This is the first report on a TF alpha-specific mAb that shows a strict peptide sequence dependency of binding.

Amino Acid Sequence↗
PubMed1995

Studies on the order and site specificity of GalNAc transfer to MUC1 tandem repeats by UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase from milk or mammary carcinoma cells.

A synthetic peptide [TAP25, (T1aAPPAHGVT9S10APDT14RPAPGS20)T1bAPPA5b] corresponding to one repeat (T1a-S20) and five overlapping amino acids (T1b-A5b) of the MUC1 core protein served as an acceptor substrate for in vitro glycosylation. TAP25 was glycosylated using the detergent-solubilized UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases from the breast carcinoma cell line T47D, the colon carcinoma cell line HT29 and from human premature skim milk. The glycosylated peptides were isolated by ultrafiltration, purified by reverse-phase HPLC and further analysed by liquid secondary ion mass spectrometry (LSIMS). Three different glycosylation species, mono-, di- and triglycosylated peptides were identified. Automated Edman sequencing and LSIMS of proteolytic fragments independently revealed the sites of GalNAc incorporation and confirmed that the threonine residues Thr9 and Thr1b are the preferred sites of glycosylation independent of the enzyme source, while Thr14 remained non-glycosylated even with the enzyme preparation from milk. In addition, evidence was obtained that at least 20% of the glycosylated peptides exhibited GalNAc incorporation at Ser20. On the basis of kinetic studies a preferred sequence of GalNAc addition to the three acceptor sites has been concluded (Thr9-->Thr1b-->Ser20). Although Thr14 within the PDTRP motif of the tandem repeats remained non-glycosylated, the introduction of GalNAc into adjacent positions significantly decreased the immunoreactivity of antibodies SM-3, HMFG-1 and HMFG-2 defining overlapping epitopes of this motif. It is assumed that glycosylation at Thr9, Thr1b and Ser20 distorts the peptide conformation of the binding epitope.

Amino Acid Sequence↗
PubMed1995

[Mucin-associated antigens as markers of gastrointestinal differentiation and carcinogenesis].

Mucins are heavily glycosylated glycoproteins which exhibit a variety of antigenic determinants consisting of carbohydrates and/or peptide sequences. The application of monoclonal antibodies and lectins in immunohistochemistry resulted in a considerable extension of knowledge regarding their topography during histogenesis. Additionally, typical alterations of antigenic profiles during neoplastic transformation of cells and tissues were described. A number of new results are in keeping with the assumption that mucin-associated antigens play an important role in tumor biology, for example metastasis, and as markers of prognosis. The purpose of the present paper is to give a review, including the authors' own results, of knowledge on the gastro-intestinal mucin antigens in experimental and clinical pathology.

Animals↗
PubMed1995

A new monoclonal antibody (A78-G/A7) to the Thomsen-Friedenreich pan-tumor antigen.

A new monoclonal antibody to the Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope; Gal beta 1-3GalNAc-) has been developed that is specific for both anomeric forms of this disaccharide (TF alpha and TF beta, including related structures on glycolipids), and not assay restricted. We demonstrate that this avid antibody (A78-G/A7) is well suited for immunohistochemistry on paraffin-embedded and cryosectioned tissues, immunoblotting, ELISA techniques, and hemagglutination. Immunohistochemistry on paraffin sections does not require proteolytic or microwave pretreatment. The binding characteristics of this antibody are largely independent of variations in pH (6.0-8.2) and temperature (4-37 degrees C). Immunoblotting with KG-1 (human acute myelogenous leukemia) cells revealed a series of TF-active glycoproteins with a main band at about 155 kDa. Immunoprecipitation was performed using a new technique applicable to IgM-type antibodies.

Animals↗
PubMed1995

Carbohydrate and peptide antigens in macrophage populations derived from human bone marrow and milk: an immunomorphological and immunochemical analysis.

An immunomorphological and immunochemical study was performed to elucidate the pattern of carbohydrate antigens and their relationships to the cluster differentiation (CD) 68 epitopes on macrophages derived from human bone marrow and milk. Core and backbone antigens recognized by lectins from Bauhinia purpurea (BPA), Helix pomatia (HPA), Arachis hypogaea (PNA), Glycine max. (SBA), Griffonia simplicifolia (GSA-I-B4), Lycopersicon esculentum (LEA) and Erythrina cristagalli (ECA) were expressed by both macrophage populations. Additionally, they exhibited various peripheral type 1 and type 2 carbohydrate antigens. In bone marrow trephine biopsies, the number of macrophages stained by the CD68-specific monoclonal antibody PG-M1 exceeded significantly (range 30-40%) the subpopulation expressing SBA, GSA-I-B4, and ECA binding sites as well as the Lewisa antigen. This result is very interesting since, from in vitro studies GSA-I-B4 and SBA are known to react especially with activated macrophages. Western blotting experiments on milk macrophage lysates revealed that ECA, GSA-I-B4, BPA, PNA and MAA visualize a 110 kDa band isographic with the CD68 antigen detected by PG-M1, KP1 and Ki-M1P monoclonal antibodies. These antibodies recognize peptide epitopes as shown by enzyme-linked immunosorbent assays after biochemical modification of milk macrophage lysates. This result is in keeping with the assumption that the CD68 antigen consists of a highly glycosylated mucin-type glycoprotein comprising various differentiation-dependent epitopes.

Antibodies, Monoclonal↗
PubMed1994

Carbohydrate antigens of human megakaryocytes and platelet glycoproteins: a comparative study.

Until now, carbohydrate antigens of human megakaryocytes have not been studied very extensively. For this reason, we investigated the staining pattern of 25 lectins and carbohydrate-specific monoclonal antibodies on paraffin-embedded trephine biopsies and acetone-fixed smears from patients with reactive and neoplastic bone marrow lesions. A biotin-streptavidin-alkaline phosphatase assay was used to visualize the binding of lectins or antibodies. Ulex europaeus agglutinin I (UEA-I) stained megakaryocytes in all cases tested. Monoclonal antibodies detecting fucosylated Lewis type 2 chain antigens (19-OLE, 12-4LE and LeuM1) were also reactive. Several lectins detecting backbone and core oligosaccharides [Helix pomatia agglutinin (HPA), peanut agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), soybean agglutinin (SBA)] bound to megakaryocytes only after neuraminidase digestion. Moreover, we investigated human platelet lysates to gain some information about the carbohydrate residues of platelet glycoproteins which are synthesized by megakaryocytes. The carbohydrate expression of platelets showed striking similarities to that of megakaryocytes. Immunoblotting experiments revealed a strong binding of UEA-I, 19-OLE and 12-4LE to a band isographic to glycoprotein (gp) Ib. After desialylation of glycoproteins transblotted to nitrocellulose, ECA and PNA also reacted with a band of this molecular weight. Gp Ib is known to contain a mucin-like peptide core with a great number of potential O-glycosylation sites. Therefore, it is tempting to speculate that carbohydrate residues characterized in this study are involved in the complex biological interactions of gp Ib.

Antibodies, Monoclonal↗
PubMed1994

The ganglioside GD1 alpha' IV3Neu5Ac, III6Neu5Ac-GgOse4Cer, is a major disialoganglioside in the highly metastatic murine lymphoreticular tumour cell line MDAY-D2.

The aim of the present study was to investigate the ganglioside expression of the highly metastatic murine lymphoreticular tumour cell line MDAY-D2. Cells were propagated under controlled pH conditions and oxygen supply in bioreactors of 1 and 7.5 l volumes by repeated batch fermentation. Gangliosides were isolated from 2.7 x 10(11) cells, purified by silica gel chromatography and separated into mono- and disialoganglioside fractions by preparative DEAE anion exchange high performance liquid chromatography. Individual gangliosides were obtained by preparative thin layer chromatography. Their structural features were established by immunostaining, fast atom bombardment and gas chromatography mass spectrometry. In addition to gangliosides of the GM1a-pathway (GM2, GM1a and GD1a) and GM1b (IV3Neu5Ac-GgOse4Cer) and GalNAc-GM1b of the Gm1b-pathway, the disialoganglioside GD1 alpha (IV3Neu5Ac, III6Neu5Ac-GgOse4Cer) was found in equal amounts compared to GD1a (IV3Neu5Ac, II3Neu5Ac-GgOse4Cer). All gangliosides were substituted with C24:0, 24:1 and C16:0 fatty acids, sphingosine and N-acetylneuraminic acid as the sole sialic acid.

Animals↗
PubMed1993

Core-typing of O-linked glycans from human gastric mucins. Lack of evidence for the occurrence of the core sequence Gal1-6GalNAc.

Mucins from the pooled gastric juice of Lewis-positive secretors were investigated to establish their glycosylation patterns with particular reference to the type and abundance of the glycan-core structures. Following reductive beta-elimination, the neutral glycan alditols from these mucins were fractionated by ion exchange and size-exclusion chromatographies and subjected to structural analyses. It was possible to gain insights into the core sequences of the neutral O-linked glycan alditols by matching (a) composition data from liquid secondary-ion mass spectrometry of the native alditol fractions, (b) specific structural information on the core sequences by thin-layer-chromatography mass spectrometry of alditol-derived neoglycolipids and (c) data from electron-impact mass spectrometry of permethylated glycan alditols or their partially methylated alditol acetates. The predominant core structures detected among the neutral glycans representing about 77% (by mass) of the total carbohydrates released from gastric mucins were core 1, Gal beta 1-3GalNAc (Ac, acetyl) and core 2, Gal beta 1-3(GlcNAc beta 1-6)GalNAc in the approximate ratio 1:2. Core 3, GlcNAc beta 1-3GalNAc, and core 4, GlcNAc beta 1-3(GlcNAc beta 1-6)GalNAc, were much less abundant (< 10%), while core 5, GalNAc alpha 1-3GalNAc, core 6, GlcNAc beta 1-6GalNAc, and a recently described sequence GalNAc alpha 1-6GalNAc (core 7) were not detected. This investigation also addressed the question of the presence of the sequence Gal beta 1-6GalNAc which has been reported previously to occur as a core-structure element in gastric mucins. This was greatly assisted by the availability of the authentic chemically synthetized disaccharide alditol which, when converted into a neoglycolipid after mild periodate oxidation, gives diagnostic ions in mass spectrometry and can be detected with high sensitivity. No evidence was found for the presence of this unusual sequence among the oligosaccharides in gastric mucins.

Carbohydrate Sequence↗
PubMed1993

Monoclonal antibody 2B5 defines a truncated O-glycan, GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6 (GalNAc), on mucins from deep gastric and duodenal glands as well as metaplasia and neoplasia of gastric differentiation.

Monoclonal antibody (MAb) 2B5 previously generated in BALB/c mice using gastric mucosa of the corpus as immunogen was characterized with regard to its binding epitope. Binding assays on structurally defined neoglycolipids from mucin glycans revealed that MAb 2B5 recognizes a carbohydrate epitope on a neutral O-glycan sequence from gastric mucins. This oligosaccharide chain has been characterized by mass spectrometry and methylation analysis and sequential exoglycosidase treatment of the derived neoglycolipid as GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6OY (where 6OY corresponds to the GalNAc-ol fragment--O--(CH2)2 conjugated to dipalmitoylphosphatidylethanolamine). The selective binding of MAb 2B5 to mucus from deep gastric and duodenal glands, to gastric metaplasia or neoplasia with gastric differentiation may imply that mucin glycosylation in the respective cells is incomplete resulting in the accumulation of truncated blood group precursor chains. MAb 2B5 is the first monoclonal antibody which defines an epitope on M2-type antigens and may substitute, accordingly, for the inconvenient "paradoxical concanavalin A-horseradish peroxidase method" in histochemistry.

Animals↗
PubMed1993

Monoclonal antibody FW6 defines an epitope on alpha 3/4-monofucosylated polylactosaminoglycans expressed by fetal and colon carcinoma-associated mucins.

A mouse IgM monoclonal antibody FW6 was established after immunization of mice with mucins from human amniotic fluid and was characterized with regard to its binding epitope. According to a series of biochemical criteria the epitope is located on O-linked neutral carbohydrates of M(r) 700,000 and M(r) 570,000 mucins in human amniotic fluid. The epitope is presumed to contain alpha 3/4-linked fucose and terminal beta 3/4-linked galactose that are labile to the fucosidase I from almond emulsion or to the galactosidase from bovine testes, respectively. Immunoreactive fractions of glycan alditols from human amniotic fluid mucins were partially characterized by fast atom bombardment-mass spectrometry and methylation analysis to be composed of monofucosylated polylactosamine-type deca- or nonasaccharides. According to antibody competition studies, inhibition assays with defined carbohydrates and binding assays on neoglycolipids monoclonal antibody FW6 are presumed to recognize a novel epitope that is distinct from known carbohydrate markers of the Lex/Ley family associated with colonic carcinomas. The selective reactivity of this monoclonal antibody to the majority of human colonic carcinomas and its nonreactivity to normal colonic mucosa may render this antibody as a valuable tool in cancer diagnosis or cancer treatment.

Amino Sugars↗
PubMed1993

Altered glycosylation of the MUC-1 protein core contributes to the colon carcinoma-associated increase of mucin-bound sialyl-Lewis(x) expression.

The mucin carbohydrate epitope sialyl-Le(x), detected with the monoclonal antibody AM-3, is strongly overexpressed in > 90% of human colon carcinomas. We show here that in colon carcinoma one of the mucin cores bearing the sialyl-Le(x) group is MUC-1, whereas sialyl-Le(x) present in normal colon is not detectable on MUC-1. The amounts of MUC-1 core detectable with the monoclonal antibody BC3 in extracts of tumor tissue are 60-180% of those in normal tissue. Two other carbohydrate epitopes located on MUC-1 in mucins from normal and tumor tissue have also been characterized. In contrast to sialyl-Le(x), their expression on MUC-1 is variable and does not correlate with the malignant transformation of colonic mucosa. The transfer of the sialyl-Le(x) group onto the MUC-1 core contributes to the colon carcinoma-associated overexpression of the sialyl-Le(x) epitope.

Antigens↗
PubMed1993

Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes.

The structures of gangliosides from human granulocytes were elucidated by fast atom bombardment mass spectrometry and by gas chromatography/mass spectrometry as their partially methylated alditol acetates. In human granulocytes besides GM3 (II3Neu5Ac-LacCer), neolacto-series gangliosides (IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer) containing C24:1, and to some extent C22:0; and C16:0 fatty acid in their respective ceramide portions, were identified as major components. In this study we demonstrate that gangliosides from human granulocytes, the second most abundant cells in peripheral blood, can serve as receptors for influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and a parainfluenza virus Sendai virus (HNF1, Z-strain). Viruses were found to exhibit specific adhesion to terminal Neu5Ac alpha 2-3Gal and/or Neu5Ac alpha 2-6Gal sequences as well as depending on the chain length of ganglioside carbohydrate backbones from human granulocytes, these important effector cells which represent the first line of defence in immunologically mediated reactions.

Carbohydrate Sequence↗
PubMed1993

Inhibition of adhesion of S-fimbriated E. coli to buccal epithelial cells by human skim milk is predominantly mediated by mucins and depends on the period of lactation.

Expression of S-fimbriae is frequent in Escherichia coli strains causing sepsis and meningitis in the newborn period. We analysed the ability of human skim milk to inhibit adhesion of S-fimbriated E. coli to human buccal epithelia. Adhesion was inhibited by up to 90% using colostrum (5%) and up to 50% with mature milk (5%), indicating that this anti-infective mechanism depends on the period of lactation. Elimination of up to 99% of immunoglobulins and 91% of lactoferrin by affinity chromatography had no effect on the inhibition of adhesion. After separation of high- (> 10 kD) and low-molecular-weight fractions of skim milk, only the fraction > 10 kD was found to be able to inhibit bacterial adhesion. In order to further characterize receptor molecules for bacteria, we investigated binding of isolated S-fimbriae to glycoprotein bands on Western blot strips. Fimbriae mainly bound to a high-molecular-weight band (> 200 kD). According to molecular weight and staining behaviour, this band most likely represents mucins. We conclude that carbohydrate residues on secreted mucins of human skim milk are able to inhibit bacterial adhesion to mucosal surfaces. This could provide protection against neonatal sepsis and meningitis caused by E. coli.

Age Factors↗
PubMed1993

Specificity of S fimbriae on recombinant Escherichia coli: preferential binding to gangliosides expressing NeuGc alpha (2-3)Gal and NeuAc alpha (2-8)NeuAc.

The adhesins of Escherichia coli strains HB101(pANN801-13) and HB101(pAZZ50), which express S fimbriae encoded by a recombinant plasmid containing the sfaI and sfaII gene clusters, respectively, were characterized with regard to the detailed structural requirements of their binding to sialyloligosaccharides on (neo)glycoproteins and (neo)glycolipids. From binding and binding inhibition studies in solid-phase enzyme immunoassays with isolated S fimbriae, several major conclusions can be drawn. S fimbriae bind specifically to sialic acid on gangliosides. The most active structural variant of sialic acid on GM3 ganglioside is N-glycolylneuraminic acid (NeuGc). In contrast to previous reports, high binding activities were measured also for b-series gangliosides expressing NeuAc alpha (2-8)NeuAc. In agreement with earlier studies, the site of sialic acid substitution to subterminal sugars strongly influences the binding to sialyloligosaccharides, i.e., alpha-6-linked sialic acid is only poorly recognized by the adhesin compared with alpha-3-linked sialic acid. C-8 and C-9 hydroxyl groups form essential structural elements of sialic acid in the binding event.

Animals↗
PubMed1993

Monoclonal antibody BW494 defines a blood group Lewisa/type 1 chain-related antigen on carcinoma-associated mucins.

The murine monoclonal antibody (MAb) BW494 defines a carbohydrate epitope on a mucin-type glycoprotein which is expressed on the majority of well-differentiated adenocarcinomas of the pancreas. In this contribution we present evidence for the detailed structural requirements of the BW494 interaction with the glycan portions on (neo)glycoproteins or (neo)glycolipids as revealed by solid-phase binding assays and inhibition assays of BW494 binding to synthetic antigens or to the affinity-isolated cancer mucin CA494. The observed cross-reactivity of spacer-linked Gal beta 1-3GalNAc beta (TF-beta) and Gal beta 1-3GlcNAc beta (type 1 chain) is indicative of an epitope that comprises a terminal Gal beta 1-3HexNAc unit. The active conformation of this epitope is critically influenced by the chemical environment of the terminal disaccharide, i.e. by adjacent aglycon or spacer moieties or by substitution with further sugar residues at the reducing end. Although a strong enhancement of antibody binding is observed for the type 1 chain derived Lea antigen, MAb BW494 is distinct in its reactivity from Lea-specific antibodies.

Animals↗