Biomedical subjects

D Bitter-Suermann

Publications and source records attributed to D Bitter-Suermann.

At least 37 records · Page 2Linked to original sources

PubMed1991

Primary structure of the murine monoclonal IgG2a antibody mAb735 against alpha(2-8) polysialic acid. 1) Amino-acid sequence of the light (L-) chain, kappa-isotype.

Here we report the complete amino-acid sequence of the anti-alpha(2-8)polysialic acid antibody mAb735 light chain. The sequence was determined after digestion of the reduced and carboxymethylated L-chain with trypsin, SV-8 proteinase, and Asp-N proteinase, isolation of the generated peptides by RP-HPLC and characterization of these fragments by sequence analysis, amino-acid analysis and/or plasma desorption mass spectrometry. According to Kabat et al. the variable region belongs to the V kappa-II subgroup, whilst according to Hum et al. it belongs to the V kappa-1B subgroup. With the exception of proline at position 46, the sequence from position 1 to 95 is identical to the translated DNA sequence of a V kappa-germline gene segment previously reported.

Amino Acid Sequence↗

PubMed1991

Polysialic acid of the neural cell adhesion molecule distinguishes small cell lung carcinoma from carcinoids.

The neural cell adhesion molecule (NCAM) exists in various types of neuroendocrine cells and their tumors. A typical feature of NCAM is polysialic acid, of which the chain length is developmentally regulated. The authors have performed a comparative immunohistochemical study on small cell lung carcinomas and bronchial as well as gastrointestinal carcinoids with the monoclonal antibody (MAb) 735 reactive with the long-chain form of polysialic acid. The small cell lung carcinomas, irrespective of their histological type, were positive for polysialic acid. Metastatic tumor cell complexes also exhibited immunostaining. The tumor cell-surface-associated immunostaining for polysialic acid was sensitive to endoneuraminidase. The mature and atypical bronchial and gastrointestinal carcinoids were not immunoreactive for polysialic acid. Cytoplasmic staining in groups of cells of carcinoids (2 of 28 cases) was due to nonspecific antibody binding, which could be prevented by increased ion strength. These data indicate that neuroendocrine tumors of the lung can be distinguished by their content of highly sialylated NCAM.

Antibodies, Monoclonal↗

PubMed1991

Complement and monocytes are essential for provoking glomerular injury in passive Heymann nephritis in rats. Terminal complement components are not the sole mediators of proteinuria.

Complement but not polymorphonuclear granulocytes (PMN) causes glomerular injury in passive Heymann nephritis in rats. We have now identified monocytes as another important mediator in this model. Passive Heymann nephritis was induced in Wistar rats by intravenous injection of sheep anti-rat Fx1A antiserum. Four groups (all receiving anti-rat Fx1A antiserum) were studied: (a) rats given normal sheep globulin (nephritic controls), (b) rats given sheep anti-rat PMN globulin (PMN-depleted), (c) rats given sheep anti-rat monocyte globulin (monocyte-depleted), (d) rats injected with cobra venom factor (complement-depleted). In vitro specificity controls for anti-cell antisera were made by cytotoxicity tests and inhibition of phagocytosis. In vivo specificity controls were performed in heterologous Masugi nephritis (PMN-dependent) and accelerated Masugi nephritis (monocyte-dependent). Complement and monocyte depletion significantly delayed the onset of proteinuria (p less than 0.001 versus nephritic controls on day 5), PMN depletion had no significant effect. Monocyte infiltration was seen in control nephritic rats, but monocyte depletion prevented this influx. In the monocyte-depleted group, no differences in glomerular deposition of C3, C9, and C5b-9 were seen in comparison to the nephritic control rats. Serum C3 levels were comparable in groups a, b, and c, the complement system was biologically active in the monocyte depleted-group (c), and the amount of anti-Fx1A antibody bound was the same in all groups. This shows that, besides complement, monocytes are required for induction of renal damage in passive Heymann nephritis. The concept of a sole role for complement in glomerular immune injury involving subepithelial immune deposits should be reconsidered.

Animals↗

PubMed1990

Giant liposomes as model membranes for immunological studies: spontaneous insertion of purified K1-antigen (poly-alpha-2,8-NeuAc) of Escherichia coli.

A flow chamber has been constructed to use giant liposomes (diameter 5-50 microns) as model membranes for immunological studies and other experiments involving the interaction with water-soluble compounds. As an example of immunological importance, the insertion of purified K-antigen from Escherichia coli K1 has been studied. Despite its large hydrophilic part (poly-alpha-2,8-NeuAc), which is capped at its potential reducing end with phosphatidic acid acting as a lipid anchor group, this water-soluble material is readily incorporated into liposomal membranes of dimyristoylphosphatidylcholine (DMPC). The incorporation has been proven by immunofluorescence using a FITC-labeled monoclonal anti-K1-IgG. Without the lipid residue, however, no binding of poly-alpha-2,8-NeuAc to the liposomes has been observed. This could be shown by using colominic acid, an oligomeric form of alpha-2,8-NeuAc with free reducing ends instead of purified K1-antigen. The possibility for further manipulation of this model system has been shown by using a poly-alpha-2,8-NeuAc cleaving enzyme (endoneuraminidase). The function of the endoneuraminidase has been proven by showing no binding of the antibody after enzyme treatment of K1-bearing liposomes as well as by rapid loss of fluorescence of a previously bound FITC-antibody.

Antibodies, Monoclonal↗

PubMed1990

Expression of neural cell adhesion molecule-related sialoglycoprotein in small cell lung cancer and neuroblastoma cell lines H69 and CHP-212.

Monoclonal antibodies (MAbs) 123C3 and 123A8 generated against a membrane preparation of a small cell lung carcinoma (SCLC) specimen recognize not only SCLC and bronchial carcinoids but also a significant portion of non-small cell lung carcinomas (non-SCLC) of various histological types. Together with 13 other monoclonal antibodies, which show preference for SCLC, they have been ranked as SCLC cluster 1 (SC-1) Mabs. In this study we show that SC-1 MAbs are directed against a restricted number of epitopes, and that SC-1 MAbs and a polyclonal antiserum directed against the neural cell adhesion molecule (NCAM) recognize identical glycoproteins, indicating that SC-1 antigens are closely related to or identical with NCAM. Long polysialic acid units composed of alpha-(2,8)-linked N-acetylneuraminic acid units, which in mammals are found exclusively on NCAM, were present on SC-1 antigens in SCLC. This provides further evidence that SC-1 MAbs recognize NCAM. The SC-1 antigens in the SCLC cell line H69 were present in two forms, NCAM-containing alpha-(2,8)-polysialic acid units identified by antiserum 735, the NCAM-H form, and the less sialylated NCAM-L form. The NCAM-H form consisted of diffusely migrating sialoglycoproteins with a molecular weight of 200,000-250,000, which resolved after neuraminidase treatment into two proteins with molecular weights of 140,000 and 180,000. Since the NCAM-H form is expressed in the lung tumor type with a poor prognosis, our results suggest that NCAM might be implicated in the invasive behavior of these NCAM-positive lung tumors.

Antibodies, Monoclonal↗

PubMed1990

Immunocytological localization of the highly polysialylated form of the neural cell adhesion molecule during development of the murine cerebellar cortex.

The expression of the highly polysialylated form of the neural cell adhesion molecule (N-CAM)--the so-called embryonic N-CAM (E-N-CAM)--was investigated in the developing and adult mouse cerebellar cortex by immunohistology and immunocytology at the light and electron microscopic levels. E-N-CAM was never (from embryonic day 14 to postnatal day 15) detectable in the germinal zone of neuroblasts destined to form or forming the external granular layer and was only observed once small cerebellar interneurons had become postmitotic before the beginning of granule cell migration. Granule cells expressed E-N-CAM on cell bodies, axons, and leading and trailing processes also during migration but ceased to reveal detectable levels of E-N-CAM at the end of migration after having reached their final position in the internal granular layer. Other cerebellar cell types, such as Purkinje cells, Bergmann glia, astrocytes, oligodendrocytes, and most prominently, stellate and basket cells, also expressed E-N-CAM, but became E-N-CAM-negative during the third and fourth postnatal weeks, coinciding with overt cessation of cerebellar histogenesis. Thus, except for neuroblasts, E-N-CAM appeared characteristic of growing and moving cellular structures, in agreement with the notion that the highly polysialylated form of N-CAM is less adhesive than the adult form.

Animals↗

PubMed1990

Reevaluation of the C3a active site using short synthetic C3a analogues.

In 1980 the C-terminal pentapeptide LGLAR (C3a 73-77) was described (Caporale, L. H. et al. J. Biol. Chem. 1980, 255: 10758) as the minimal sequence inducing a C3a-specific activity. We have synthesized C3a-analogue peptides connected to non-peptidic acyl residues known to potentiate biological activity. Starting from the acylated hexapeptide fluorenylmethoxycarbonyl(Fmoc)-aminohexanoyl(Ahx)-ALGLAR+ ++, a related series of shorter peptides was synthesized. C3a-specific activity was measured as ATP release from guinea pig platelets. Even the tripeptide LAR, acylated with Fmoc-Ahx, exhibited C3a-specific activity. With 0.34% C3a activity, it was even more potent than the native LGLAR sequence which has 0.01% activity. N-terminal extension of the acylated tripeptide LAR by adding one to three alanines increased activity tenfold up to 3.26% (Fmoc-Ahx-AAALAR), while N-terminal addition of three glycine residues (Fmoc-Ahx-GGGLAR) only increased activity to 0.83% of native C3a. Furthermore, a stimulus-specific desensitization could be observed. Fmoc-Ahx-R and Fmoc-Ahx-AR exhibited neither activity nor desensitizing capacity, but the addition of four alanines to the dipeptide AR led to a sequence (Fmoc-Ahx-AAAAAR) with a C3a-specific activity of 0.14%. Even arginine prolonged N-terminally with five glycines (Fmoc-Ahx-GGGGGR) exhibited some C3a-specific activity so that for biological activity only the appropriate presentation of arginine seems to be essential.

Adenosine Triphosphate↗

PubMed1990

C3 deficiencies.

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Animals↗

PubMed1990

Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping.

Sequences of antigenic determinants were identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonal antibody-bound peptide fragments by 252Cf plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453 [Burger, R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141, 553-558] were isolated from immobilized antigen-antibody complexes by partial trypsin digestion. A synthetic eicosapeptide comprised of the C-terminal sequence of the human complement component polypeptide des-Arg77-C3a as well as guinea pig des-Arg78-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded the antigens without inactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from the antibody with 4 M MgCl2. The antigenic peptides were purified by HPLC and identified by 252Cf plasma desorption mass spectrometry. The epitope recognized by h453 resides on the C-terminal tryptic peptides of human (residues 70-76) and guinea pig (residues 70-77) C3a. As an estimation of accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of the antigen within a distance of two amino acid residues upstream from the epitope.

Amino Acid Sequence↗

PubMed1990

Embryonic neural cell adhesion molecule in cerebrospinal fluid of younger children: age-dependent decrease during the first year.

Poly-alpha-2,8-N-acetylneuraminic acid (poly-alpha-2,8-NeuAc) is developmentally expressed in neural tissue of higher animals, where it is covalently attached to the neural cell adhesion molecule (NCAM), a large integral membrane glycoprotein mediating cell-cell adhesion during neuronal development. NCAM exists in several molecular forms, of which only embryonic NCAM carries lengthy chains (n greater than 5) of poly-alpha-2,8-NeuAc. Chemically identical poly-alpha-2,8-NeuAc of bacterial origin is an important virulence factor in infections caused by Neisseria meningitidis group B and Escherichia coli K1, the predominant pathogens of bacterial meningitis. A quantitative enzyme-linked immunoassay was developed using monoclonal antibody (MAb) 735, an MAb specifically recognizing poly-alpha-2,8-NeuAc, and applied to CSF specimens from younger children. Poly-alpha-2,8-NeuAc contents were within the range of 20-0.2 micrograms/ml, decreasing from day 1 to day 300. Immunoprecipitation, immunoblot with a rabbit anti-mouse NCAM serum recognizing the protein part of human NCAM by cross-reactivity, affinity enrichment using immobilized MAb 735, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that poly-alpha-2,8-NeuAc in CSF is bound to human NCAM, probably NCAM-120.

Antigens, Surface↗

PubMed1990

Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.

Antibodies, Monoclonal↗

PubMed1990

Immunohistochemical localization of polysialic acid in tissue sections: differential binding to polynucleotides and DNA of a murine IgG and a human IgM monoclonal antibody.

For immunolocalization of alpha(2-8)-linked polysialic acid, which forms part of the neural cell adhesion molecule (N-CAM), two monoclonal antibodies, MAb735 and IgMNOV, were employed. Both antibodies have previously been shown to bind the extremely low immunogenic capsular polysaccharide of group B meningococci, which also consists of alpha(2-8) polysialic acid, but not to other, even closely related forms of polysialic acid. Despite the identical polysaccharide specificity of these two MAb, we observed marked differences of the staining pattern in tissue sections. We showed that these differences in immunostaining were due to the crossreactivity of IgMNOV with polynucleotides and DNA. MAb735, however, was shown to react exclusively with alpha(2-8) polysialic acid. Moreover, the specificity of MAb735 proved to be unique among eleven other MAb directed against various bacterial polysaccharides, as it was the only one unreactive with polynucleotides. Thus, MAb735, the only IgG type mouse monoclonal antibody to polysialic acid thus far reported, can be considered a specific probe for the unambiguous detection of alpha(2-8) polysialic acid in tissue sections, and should therefore help to further elucidate the role of polysialic acid in developmental processes.

Animals↗

PubMed1989

Sandwich enzyme-linked immunosorbent assays for the quantification of the C4 isotypes (C4A and C4B) in human plasma.

Sandwich enzyme-linked immunosorbent assays were developed to determine the concentration of the isotypes of the fourth component of human complement (C4A and C4B) in human plasma. In the case of C4A a monoclonal antibody against a common determinant of the alpha chain was used to capture the protein. The bound antigen was then detected with a biotinylated monoclonal antibody reacting exclusively with the C4A isotype, followed by peroxidase labeled avidin. For the quantification of C4B, C4B-specific monoclonal antibodies were coated onto a microtitration plate in order to capture the protein. Bound antigen was then detected with a biotinylated monoclonal antibody directed against C4 followed by peroxidase labeled avidin. The assays, which were rapid, selective and specific for C4A and C4B, respectively, provide an alternative to gel electrophoresis and blot procedures for the study of unexpressed alleles ('null alleles') at each of the C4 loci.

Antibodies, Monoclonal↗

PubMed1989

Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes.

The expression of specific membrane receptors for C3a was determined on guinea pig C3a-sensitive (gp R+) platelets and human polymorphonuclear leukocytes (hu PMNL). Binding studies with 125I-labeled C3a from gp or hu sources and Scatchard analysis applied to the binding data revealed the existence of two receptor classes on gp R+ platelets; a high-affinity class with about 200 binding sites/cell and Kd = 1.7 x 10(-9) M, and a relatively low-affinity class with Kd = 10(-8) M and about 500 sites/cell. Hu PMNL express a homogeneous receptor class with Kd = 3 x 10(-8) M and 40,000 sites/cell. Molecular characterization of the C3a receptor on gp R+ platelets was achieved by (a) cross-linking photoaffinity-labeled receptors to bound 125I-labeled C3a; (b) photoaffinity labeling receptors with a 13-amino acid residue C3a analogue 125I-Nap-Ahx-13; and (c) use of chemical cross-linkers like disuccinimidylsuberate to cross-link receptors with 125I-C3a. All three techniques gave rise to very similar labeling patterns. With the photoaffinity labeling methods, a diffuse band pattern was observed with an apparent molecular mass of 95-123 kDa with 125I-C3a as label, and 85-105 kDa with 125I-Nap-Ahx-13 as label. Chemical cross-linking of 125I-C3a revealed three distinct bands with molecular masses of approximately 123, 108 and 95 kDa. Subtracting the contribution of the cross-linked ligands, the C3a receptor on gp R+ platelets appears to be a protein complex, consisting of one to three components with estimated molecular masses between 83-114 kDa.

Adenosine Triphosphate↗

PubMed1989

Embryonic neural cell adhesion molecules on human natural killer cells.

The neural cell adhesion molecules (NCAM) are surface glycoproteins that were first described in brain tissue. NCAM mediate adhesion in a variety of cell-cell interactions. In the present study we show that the so-called "embryonic" NCAM, i.e., the highly polysialylated forms of these proteins, are expressed on natural killer cells and some CD3+ cells in man. Homotypic binding of NCAM, believed to be of importance for cell-cell adhesion in neural tissues, appears not to be essential for NK cell-mediated killing. Yet, NCAM might be involved in NK cell migration, homing or related functions.

Antibodies, Monoclonal↗

PubMed1989

Virus-transformed macrophage-like cell line as tool for eicosanoid research.

The ability of a virus-transformed murine macrophage like cell line HA 38 to produce different eicosanoid metabolites was examined. HA 38 cells release similar amounts of prostaglandins and leukotrienes as did murine peritoneal macrophages in response to both physiological and non-physiological stimuli. Enzyme systems known to be involved in the regulation of eicosanoid synthesis are expressed. HA 38 cells thus are a well defined macrophage model system and are well suited to study eicosanoid synthesis in macrophages and effects of drugs on the prostaglandin and leukotriene synthesis pathways.

Animals↗

PubMed1989

Differentiation-regulated loss of the polysialylated embryonic form and expression of the different polypeptides of the neural cell adhesion molecule by cultured oligodendrocytes and myelin.

The expression of the neural cell adhesion molecule (N-CAM) on cultured murine oligodendrocytes, their precursors, and myelin was examined by indirect immunofluorescence, biosynthetic radiolabeling followed by immunoprecipitation and Western blot analysis, using antibodies specific for various forms of the molecule. In all culture systems studied, whether the oligodendrocytes were cultured as an enriched fraction containing precursor cells or in the presence of astrocytes and neurons, a similar differentiation-stage-related expression of N-CAM was seen. At early developmental stages many tetanus toxin receptor- and A2B5 antigen-positive putative oligodendrocyte precursors with bipolar morphology were seen and found to express N-CAM in its embryonic form. Of the 04 antigen-positive immature oligodendrocytes with few slender processes most expressed N-CAM, but few the embryonic form of N-CAM. The more mature 01 or 010 antigen-positive oligodendrocytes were found to express exclusively the adult form of N-CAM. Oligodendrocytes synthesized the 120 and 140 kD forms of N-CAM (N-CAM 120 and N-CAM 140), but not N-CAM 180, although with differentiation, N-CAM 120 predominated in oligodendrocytes and also in pure myelin. N-CAM 120 could be released from oligodendrocytes and myelin by phosphatidylinositol-specific phospholipase C, suggesting that in both oligodendrocytes and myelin N-CAM 120 is inserted into the membrane by covalent linkage to phosphatidylinositol.

Animals↗

PubMed1989

Expression of the embryonal neural cell adhesion molecule N-CAM in lung carcinoma. Diagnostic usefulness of monoclonal antibody 735 for the distinction between small cell lung cancer and non-small cell lung cancer.

Paraffin sections of 19 surgically resected small cell lung carcinomas (SCLC), 33 non-small cell lung carcinomas (NSCLC) of various types, and four bronchial carcinoids were immunostained with monoclonal antibodies (MoAbs) 735 and anti-Leu 7, both recognizing some sugar epitopes present on the neural cell adhesion molecule N-CAM. With MoAb 735, all SCLC were stained focally or diffusely, and one carcinoid was stained focally. Only three of the 33 NSCLC were faintly and focally positive with MoAb 735; these three tumours showed relatively small tumour cells and small, oval nuclei. Anti-Leu 7 stained all the carcinoids, only eight SCLC, sometimes focally, and eight NSCLC. MoAb 735 was thus superior to anti-Leu 7 in distinguishing between SCLC and NSCLE. Since MoAb 735 stained all SCLC strongly and is applicable on paraffin sections, it provides a well-needed addition to the immunomarkers used in the diagnostic distinction of SCLC and NSCLC.

Antibodies, Monoclonal↗