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Biomedical subjects

U Karsten

Publications and source records attributed to U Karsten.

At least 19 recordsLinked to original sources
PubMed1998

Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation.

The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines.

Antibodies, Monoclonal↗
PubMed1998

Immunohistochemical characterization of a panel of 56 antibodies with normal human small intestine, colon, and breast tissues.

The epithelial mucin MUC1 is heavily but differently glycosylated depending on the origin and developmental status of the tissue, which greatly influences the reactivity of monoclonal antibodies (MAbs). A partial characterization of their epitopes is possible by mild, carbohydrate-specific periodate oxidation of tissue sections prior to immunostaining. Using this strategy, we have evaluated 56 MAbs submitted to the ISOBM TD-4 (MUC1) Workshop. Paraffin sections from normal human small intestine, colon and breast were immunostained at different defined antibody concentrations either directly or after oxidation with 20 mM periodate at pH 5 for 30 min (PO). In addition, monolayers of T-47D breast cancer cells without PO treatment were examined in immunofluorescence. The array of observed reactivities allowed us to classify the MAbs as follows. Fourteen antibodies were found to detect MUC1 largely independent of the degree of glycosylation, and are therefore classified as pan-MUC1 MAbs (Group A). Twenty-four MAbs were nonreactive with one or more types of the examined epithelia, but became reactive after PO of the tissue sections. We have called these differentiation-dependent MUC1 MAbs (Group B). They might be especially valuable in histological tumour diagnosis. According to their differential staining behaviour towards untreated small intestine, colon, and breast tissue sections, we divided these MAbs into 4 subtypes (Group B1 through Group B4). A further group of six MAbs detected PO-sensitive carbohydrate epitopes (Group C). A seventh antibody apparently also belongs to Group C by immunohistological criteria, although its corresponding epitope was not PO-sensitive. Three further MAbs are still unclear in their specificity, and another 2 are not MUC1-specific (Group D). Six preparations were found nonreactive with the examined tissues; 4 of these were also negative with T-47D cells. Generally, a broad spectrum of different immunohistological patterns has emerged which appears to be widely independent of the type of epitope (sequence versus conformational, length of sequence) and the relative affinities determined in vitro.

Antibodies, Monoclonal↗
PubMed1997

Structure analysis of acetylated and non-acetylated O-linked MUC1-glycopeptides by post-source decay matrix-assisted laser desorption/ionization mass spectrometry.

We have investigated the potential of structural elucidation of O-linked glycopeptides by post-source decay matrix-assisted laser desorption ionization mass spectrometry (PSD-MALDI-MS). In order to establish detailed fragmentation patterns and to dissect fragment ions with and without carbohydrate content, the same O-linked MUC1-derived glycopeptides with acetylated and non-acetylated sugars were analysed and compared. Furthermore, we were interested to examine possible differences in the fragmentation between glycopeptides with acetylated and non-acetylated sugars. The obtained PSD-MALDI-MS spectra showed a rather complete set of fragmentation data which allows to localize the glycan on the peptide, in parallel with sequencing a short glycan and the backbone peptide. Fragmentations of the sugars were dominated by inter-ring cleavages at the glycosidic bond. Intra-ring cleavage did also occur from the non-reducing end, but to a much lower extent. The fragmentation of the peptide backbone was not changed either by acetylated or non-acetylated sugars. Glycosylated peptide fragments occurred as fully glycosylated fragment ions, partially deglycosylated ions and completely deglycosylated ions, and was not influenced by the acetylation of sugars. However, differences occurred in the quality and quantity of fragment ions from the non-reducing end of the glycan part when comparing acetylated with non-acetylated glycopeptides.

Acetylation↗
PubMed1997

Immunodetection of epithelial mucin (MUC1, MUC3) and mucin-associated glycotopes (TF, Tn, and sialosyl-Tn) in benign and malignant lesions of colonic epithelium: apolar localization corresponds to malignant transformation.

Epithelial mucins are present at the apical membranes of gastrointestinal epithelial cells or in their secretions. In this study, we examined the occurrence of peptide epitopes of the mucins MUC1 and MUC3 and of three mucin-associated glycotopes (TF, Tn, and s-Tn) in a series of colorectal tissue samples (normal colon, adenomas with different grades of dysplasia, carcinoma in situ, and invasive carcinomas). A new monoclonal antibody to a conformation-dependent peptide epitope of MUC1 was employed, which does not react with the fully glycosylated mucin as found in normal gastrointestinal mucosa. We found that adenomas acquired the ability to expose Tn, s-Tn, TF and MUC1 epitopes, and this correlated with increasing malignant potential. The secretory mucin, MUC3, revealed a different pattern: it was detectable in all sections, with maximum expression in adenomas and decrease in carcinomas. Most importantly, normal mucosa and benign lesions showed supra-nuclear and/or apical distribution of these antigens, but malignant lesions and lesions with a very high risk of malignancy revealed diffuse cytoplasmic and basolateral membrane localization. The immunohistological response to a combination of MUC1-related antibodies may assist in assessing the malignant potential and status of lesions of the colon.

Adenoma↗
PubMed1997

A sequencing strategy for the localization of O-glycosylation sites of MUC1 tandem repeats by PSD-MALDI mass spectrometry.

It is demonstrated with glycopeptides of the polymorphic epithelial mucin (MUC1) that post-source decay matrix-assisted laser desorption ionization (PSD-MALDI) is a fast, highly sensitive, and reproducible method for the localization of O-glycosylation sites by reflectron time-of-flight (TOF) mass spectrometry. We have analyzed GalNAc-carrying peptides of up to 25 amino acids, and could distinguish even neighboring glycosylation sites. This method was also able to localize and characterize disaccharides (e.g., the Thomsen-Friedenreich disaccharide) on MUC1 derived peptides. PSD-MALDI-MS fragment ion patterns were recorded in the positive ion mode from the synthetic peptide TAP25 [(T1aAPPAHGVT9S10APDT14RPAPGS20) T1bAPPA], an overlapping sequence of MUC1 tandem repeats, which was glycosylated with GaINAc in vitro. The glycosylation sites found were either Thr9 or Thr1b in the monoglycosylated, Thr9 and Thr1b in the diglycosylated, and Thr9, Thr1b, and Ser20 in the triglycosylated peptide. A single PSD-MALDI-MS spectrum of the underivatized and uncleaved di- or triglycosylated TAP25 peptide was sufficient to identify the glycosylation sites, thereby distinguishing six potential, partly adjacent, glycosylation sites. The monoglycosylated fraction was found to consist of a mixture of two glycosylated species with the same molecular weight. This was shown by the analysis of proteolytic digests. PSD-MALDI-MS of the resulting peptides right out of the digestion probe was sufficient to identify the Gal-NAc-glycosylation sites as either Thr9 or Thr1b, respectively. Beyond the methodical aspects the results revealed that in vitro glycosylation of the TAP25 peptide with a transferase system from human milk differs from that obtained with a breast cancer cell transferase system.

Amino Acid Sequence↗
PubMed1997

Mucins (MUC1 and MUC3) of gastrointestinal and breast epithelia reveal different and heterogeneous tumor-associated aberrations in glycosylation.

In a comprehensive study, we examined the expression of the membrane and secretory mucins MUC1 and MUC3, respectively, in normal and neoplastic gastrointestinal and breast epithelia before and after specific alterations of their glycan structures by neuraminidase, alpha-fucosidase, or carbohydrate-specific periodate oxidation. MUC1 mRNA was also identified in normal colorectal tissues by in situ hybridization. The data revealed that normal colorectal epithelia express both MUC1 mRNA and protein, which were detectable after periodate oxidation with all tested MUC1-specific antibodies. During tumorigenesis in the colon, MUC1 became recognizable without periodate treatment concomitantly with highly dysplastic lesions and the malignant state. In the breast, in which MUC1 is detectable with most antibodies in normal epithelium as well as in carcinomas, staining could be enhanced by pretreatment with periodate and casually by enzyme treatments. MUC3 was detectable in normal and neoplastic colorectal tissues and was more intensely stained after periodate oxidation. It was absent in normal breast even after pretreatment but was expressed in seven of 20 breast carcinomas. Therefore, incomplete glycosylation, abnormal distribution, and ectopic expression of mucins are characteristics of malignancy. Periodate oxidation may be widely applicable to immunohistochemistry for examining changes in glycosylation and for detecting antigens masked by glycans.

Biomarkers, Tumor↗
PubMed1997

Novel alphaGalNAc containing glycans on cytokeratins are recognized invitro by galectins with type II carbohydrate recognition domains.

We report on a novel posttranslational modification of cytoplasmic proteins. Presented evidences suggest that cytokeratins are bound in vitro by mammalian galectin-3 and the galectins from the sponge Geodia cydonium via their type II carbohydrate recognition domains, whose highest binding affinity is directed towards terminal alpha-N-acetylgalactosamine-bearing glycans with the general sequence GalNAcalpha1-3Gal(NAc)beta. Specificity analyses and the characterization of the critical sugar residue on cytokeratins for galectin binding were done with cytochemical and biochemical methods using various plant and animal lectins. Binding of GalNAc-specific lectins was saturable, sensitive to mild periodate oxidation, inhibitable by glycoconjugates carrying terminal GalNAc, and abolished after treatment of the cytokeratins with alpha-N-acetylgalactosaminidase. Binding to bacterially expressed recombinant cytokeratins did not exceed background binding. The presence of GalNAc residues on highly purified cytokeratins from MCF-7 and HeLa SS6 cells was confirmed by sugar composition analyses using gas chromatography/mass spectrometry. This novel posttranslational modification was not restricted to cytokeratins of MCF-7 cells, but did also occur in all of 9 other examined human carcinoma cell lines and in a normal human mammary epithelial cell line. From these cytochemical and biochemical in vitro studies we hypothesize that this glycan with its terminal alpha1-3 linked GalNAc determinant might represent the first natural cytoplasmic ligand for endogenous galectins-3 detected so far.

Acetylgalactosamine↗
PubMed1996

Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study.

A broad variety of normal human tissues were examined for the expression of Thomsen-Friedenreich (TF)-related histo-blood group antigens, TF (Gal beta 1-3GalNAc alpha 1-R), Tn (TF precursor, GalNAc alpha 1-R), sialosyl-Tn (NeuAc alpha 2-6GalNAc alpha 1-R), considered to be useful in cancer diagnosis and immunotherapy, and sialosyl-TF, the cryptic form of TF. These antigens or, more correctly, glycotopes, were determined by immunohistochemistry with at least two monoclonal antibodies (mAbs) each (except sialosyl-TF) as well as by lectin histochemistry. For a better dissection of sialosyl-TF and TF glycotopes, tissue sections were pretreated with galactose oxidase or the galactose oxidase-Schiff sequence. Staining with mAbs appeared to be more restricted than with the lectins used. Distribution patterns among normal epithelia were different for all four antigens. These antigens were also detected in some non-epithelial tissues. They can be classified in the following sequence according to the frequency of their occurrence in normal tissues: sialosyl-TF > > sialosyl-Tn > Tn > TF. Most of the positively staining sites for TF, Tn, and sialosyl-Tn are located in immunologically privileged areas. The complex results obtained with anti-TF mAbs (after treatment of the tissue sections with sialidase from Vibrio cholerae) and the lectins amaranthin and jacalin revealed a differential distribution of the subtypes of sialosyl-TF [NeuAc alpha 2-3Gal beta 1-3GalNAc alpha 1-R and Gal beta 1-3 (NeuAc alpha 2-6)GalNAc alpha 1-R] in normal human tissues. From our data it can be inferred that TF, Tn, and sialosyl-Tn are promising targets for a cancer vaccine.

ABO Blood-Group System↗
PubMed1995

New monoclonal antibodies recognizing phosphorylated proteins in mitotic cells.

Three monoclonal antibodies which showed strong staining of mitotic cells by screening on the human cell line MCF-7 were isolated. The antigens detected by the DH7 and BF6 monoclonal antibodies were located predominantly in multiple extranucleolar patches in interphase cell nuclei. In mitotic cells a strong increase in the fluorescence intensity was accompanied by its redistribution into a fine speckled form. Metaphase chromosomes were unstained. Centrosomes, spindle poles or midbodies were not stained either before or after extraction of the cells with Triton X-100 under conditions which preserve microtubular structures. In immunoblots of interphase cell extracts only very few bands reacted with DH7 whereas in mitotic cell extracts approximately 30 bands were stained. BF6 also showed an increase in the intensity and number of bands detected in mitotic compared to interphase cell extracts, and the pattern was clearly different from that obtained with DH7. The BF6 antigen were extracted by 0.5% Triton X-100, whereas the DH7 antigen was not. Dephosphorylation of the antigens strongly reduced the binding of both antibodies as measured by immunoblotting and ELISA assays. The results suggested that BF6 and DH7 detect two different phosphorylated epitopes, each of which is shared by a different subset of proteins from mitotic cells. The third antibody, BD 12, bound to several polypeptides, including one of high molecular weight that appeared to correspond to the NuMA antigen. The epitope recognized by BD 12 was not sensitive to phosphatases.

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↗
PubMed1994

Mast cells detected in cultures of neonatal rat heart cells.

Although the presence of mast cells in heart muscle is well documented, their possible presence in heart cell cultures has not yet been considered. In this paper we show for the first time that cultures from neonatal rat hearts contain mast cells in proportions which may exert physiological influences on heart muscle cells. Evidence is based on cytochemistry, immunocytochemistry with a monoclonal antibody (F2) specific for connective tissue mast cells of the rat, and direct estimations of both cellular histamine and histamine released into the culture medium. Since the number of nonmuscle cells immunoreactive with antibody F2 exceeded the number of cells reacting with conventional cytochemical stains for mast cells, a substantial proportion of the former may represent less differentiated (precursor) cells.

Animals↗
PubMed1993

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells.

Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected' cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le(y), Le(x), sialosyl-Le(a), precursor of Thomsen Friedenreich antigen (Tn), but not Thomsen-Friedenreich antigen and sialosyl-Tn]. Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2+ cells develop from the basal cell layer of the mammary gland.

Adult↗
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↗
PubMed1991

[Expression of intermediate filaments in cutaneous cylindroma].

Using different monoclonal anti-cytokeratin antibodies (broad spectrum, cytokeratin 7, 8, 18, 19) and the antibody A55-A/A9 until now characterized only by immunohistology the staining of ductal structures and some cells within the lobulae of dermal cylindromata was demonstrated. In regard to the cytokeratin polypeptide patterns of the adnexes in uninvolved skin an exact histogenetic correlation by our findings is not possible, but the pattern is consistent with the conception of the glandular origin of the tumours.

Antibodies, Monoclonal↗
PubMed1991

Distribution of (fucogalactosyl-)epitope expressing glycoconjugates in rat brain.

Glycoconjugates are known to be concentrated in plasma membranes, especially in synaptic junctions, where they subserve various functions in neural connectivity. Here we report the cellular distribution of a new monoclonal antibody recognizing (fucogalactosyl) sequences in carbohydrate structures. The most pronounced immunoreactivity was found in fibrous astrocytes, in many parts of the brain and with lower density in various neuronal elements. This points to the expression of identical carbohydrate sequences on molecules within certain glial and neuronal elements. Previous intracerebral injections of the antibody interfered with long term memory formation. Therefore, functions mediated by corresponding glycoproteins in neurons and glia cells or even neuron-glial interactions, might be relevant for information-processing.

Animals↗
PubMed1991

Detection of cancer metastases in regional lymph nodes: comparative histological and immunohistological investigations with the broad-range anticytokeratin monoclonal antibody A45-B/B3.

A total of 113 patients with carcinomas of breast, testis, stomach and colon were examined for lymph node metastases by means of an exact case-by-case comparison by conventional histology and by immunohistochemistry using the anticytokeratin monoclonal antibody A45-B/B3. Among 891 examined lymph nodes, 90% of metastases were recognized by both methods, about 2% by histology alone, and more than 10% by immunohistochemistry alone. The method can be applied for intraoperative frozen section diagnosis.

Breast Neoplasms↗