Dieter Bitter-Suermann

Biliary adenocarcinoma: Characterisation of three new human tumor cell lines

Three human cell lines from adenocarcinomas of the extrahepatic biliary tract were established in permanent tissue culture. Mz-ChA-1 and Mz-ChA-2 were cultured from mechanically dissociated gallbladder adenocarcinoma metastases and SK-ChA-1 was grown from malignant ascites of a patient with primary adenocarcinoma of the extrahepatic biliary tree. Cell doubling times in tissue culture are 3-4 days for Mz-ChA-1 and approximately 2 days for Mz-ChA-2 and SK-ChA-1. All three tumour cell lines were successfully transplanted to nude mice, inducing progressive tumour growth. Histologically, nude mouse tumours resembled the original adenocarcinomas. In vitro formation of gland-like structures were regularly seen in Mz-ChA-1 and Mz-ChA-2 but only occasionally in SK-ChA-1. All three cell lines formed contacts through interdigitating processes with desmosomes and junctional complexes. On scanning electron microscopy, an abundance of microvilli was seen at the cell surfaces. Chromosome analyses of all three tumour cell lines showed a wide range of numerical abnormalities and presence of marker chromosomes. Mz-ChA-1 appears to be highly differentiated with cells producing mucus. Mz-ChA-2 synthesizes components of complement C2, C3 and C5, while Mz-ChA-1 and SK-ChA-1 produce only C3 in detectable quantities. In addition, Mz-ChA-2 supernatants are positive for ferritin and alpha 1-fetoprotein, but not CEA; while Mz-ChA-1 and SK-ChA-1 produce only CEA. Supernatants of all three cell lines are positive for N-acetyl neuraminic acid (NANA), phosphohexoisomerase (PHI) and LDH, and negative for alpha 2-macroglobulin, alpha 1-anti-trypsin, gamma-GT, AP, coeruloplasmin, haptoglobin and albumin. A high cloning efficiency renders these new tumour cell lines suitable for continued studies on clonal heterogeneity in malignant tumours. The establishment of these cell lines in tissue culture facilitates further studies on the biology of upper gastrointestinal tract cancer in man.

Neural cell adhesion molecule expression, neuroendocrine differentiation and prognosis in lung carcinoma

We investigated the expression of the neural cell adhesion molecule (NCAM) in a series of surgically resected lung carcinomas of various histological subtypes by means of a panel of monoclonal antibodies recognising different N-CAM epitopes. In a subgroup of 56 tumours, the results of immunostaining with MAb 123C3--the antibody studied most extensively in our material--were compared to the ultrastructure, and in 231 radically resected non-small cell carcinomas, with histological tumour type and with clinical follow-up data. N-CAM expression was not limited to neuroendocrine tumours, as assessed ultrastructurally. Non-small cell lung carcinomas positive for MAb 123C3 showed post-operative overall and disease-free survival times significantly shorter than 123C3-negative non-small cell carcinomas.

Cloning and characterisation of an immunodominant major surface antigen of Echinococcus multilocularis.

A lambda gt11 cDNA expression library from mRNA of Echinococcus multilocularis protoscolices has been constructed in Escherichia coli Y1090. Immunoscreening with pooled sera obtained from patients suffering from E. multilocularis disease revealed 5 reactive clones. By partial DNA sequence comparison all clones proved to encode the same gene. The complete cDNA sequence of the clone pEM10 with the largest insert of 2.2 kb was determined and an open reading frame of 1.7 kb could be described. The derived amino acid sequence shares 42.6% identity with human microvillar cytovillin found in the membranes of placenta and carcinoma tissues. The coding region of the cDNA of pEM10 was amplified by polymerase chain reaction (PCR) and cloned in frame into expression vector pGEX-3X. Immunoblot analysis revealed the expression of a recombinant antigen of 65 kDa and a protein with the same molecular weight was also found in the lysate of E. multilocularis protoscolices. In contrast, the protein was absent from hydatid fluid or larvae of Echinococcus granulosus. By means of immunofluorescence studies this immunodominant antigen could be located in the germinal layer of brood capsules and in the tegument of E. multilocularis protoscolices. The fusion protein was purified and used for diagnostic purposes in immunoblot. The diagnostic value of this antigen is discussed.

Guillain‐barré syndrome Activated complement components C3a and C5a in CSF

Plasma and spinal fluid levels of complement activation products C3a and C5a were quantitated by radioimmunoassay in a group of 16 patients suffering from acute monophasic Guillain-Barré syndrome (GBS). Median CSF levels of C3a (118 ng/ml) and of C5a (9.6 ng/ml) were significantly elevated when compared with samples from a control group of patients with noninflammatory neurologic diseases. Plasma concentrations of these anaphylatoxic peptides were not significantly different between the two populations. Our findings indicate that the complement system is activated in the CSF of patients with acute GBS. Complement activation products may contribute to the inflammatory changes observed in this disorder.

Detection of native human complement components C3 and C5 and their primary activation peptides C3a and C5a (anaphylatoxic peptides) by ELISAs with monoclonal antibodies.

Monoclonal antibodies (mAbs) were raised against human C3a, C3b, C5a, and C5b after immunization of BALB/c mice with the native components C3 and C5. Using different combinations of these mAbs we have developed four sensitive sandwhich-enzyme-linked immunosorbent assays (ELISAs) for the detection of native C3 or C5 in samples with low concentrations of these proteins, e.g., in cell culture supernatants or synovial fluids and cerebrospinal fluids (CSF) and for the detection of the anaphylatoxic peptides (AT-peptides) C3a or C5a in human EDTA-plasma. The C3- and C5-ELISAs were found to be specific for the uncleaved complement proteins. Two different anti-C3a or anti-C5a mAbs were combined for the C3a- and C5a-ELISA. Before assaying a sample in the C3a- or C5a-ELISA a precipitation step to eliminate uncleaved C3 and C5 was necessary. The sensitivity and specificity of the four ELISAs were tested with purified antigens and EDTA-plasma or Cobra venom factor-activated EGTA-plasma samples as a source of C3a and C5a. The detection limits were 1 ng/ml for C3, 1 ng/ml for C3a, 2 ng/ml for C5, and 100 pg/ml for C5a. Plasma samples from patients undergoing cardiopulmonary bypass (CPB) surgery were used as a source of pathological material.

Interaction of meningococcal group B monoclonal antibody and its Fab fragment with α2-8-linked sialic acid polymers: Requirement of a long oligosaccharide segment for binding

Mouse monoclonal IgG2a antibody (735D4) and other antibodies to the capsular polysaccharide of group B meningococci have been shown to require an unusually long segment of the alpha 2-8-linked N-acetylneuraminic acid polymer for binding. This property may be due to a conformational nature of the polysaccharide epitope recognized, or alternatively due to the requirement of bivalent binding of the antibody to the polysaccharide. In order to study the binding requirements, Fab fragments were prepared from the monoclonal antibody and their binding to alpha 2-8-linked sialic acid polymers of different lengths was studied. Both the intact antibody and its Fab fragment bound to sialic acid poly- and oligomers to similar extents, the critical chain length being about 10 sialyl units for both molecules. This excluded bivalency as the explanation for the requirement of a long oligosaccharide segment for binding. Although the binding was enhanced with increasing chain length, the first 10 monosaccharides were calculated to contribute to more than 90% of the total binding energy. This is in agreement with an oligosaccharide segment with defined conformational epitope binding to the antibody combining site. The antibody preparations also bound polysialic acid containing glycopeptides isolated from developing human and rat brain, suggesting, in quantitative binding assay, an average chain length of 10 or more sialic acid residues. The interaction of the antibody with both the bacterial and the tissue derived polysialic acids suggests that the conformational epitope critical for the interaction is formed by both classes of compounds.

A safe and efficient method for elimination of cell culture mycoplasmas using ciprofloxacin

The antibacterial activity of ciprofloxacin, a 4-fluoroquinolone antibiotic, in the control of mycoplasma contamination in experimentally infected cell lines has been investigated. Seven mycoplasma species, including M. hyorhinis, M. gallisepticum, M. orale, M. salivarium, M. hominis, M. fermentans, and M. arginini, which had chronically infected the murine plasmocytoma line X63-Ag8 653, were eradicated with 10 micrograms/ml ciprofloxacin. Wild type laboratory infections of two human cell lines, HL-60 and U-937, were eliminated by 12 days of such treatment. Mycoplasma decontamination of cell cultures was monitored by the cultivation method 4 weeks after treatment. No side effects were seen in cell cultures and complex proliferation assays with cells of human and murine origin, using ciprofloxacin in doses up to 2.5 times the usual bactericidal concentration.

Comparative study on biological activities of various anaphylatoxins (C4a, C3a, C5a): Investigations on their ability to induce platelet secretion

Several anaphylatoxic substances (human C3a, guinea pig C3a, human C4a, guinea pig C5a, and a synthetic C3a-related hexapeptide) were compared with regard to their ability to induce secretion of [3H]serotonin from guinea pig platelets. Functional identity of the C3a preparations, C4a, and the hexapeptide was demonstrated by the phenomenon of crossed desensitization. Whereas C3a of human and guinea pig origin proved to be qualitatively and quantitatively identical, C4a expressed only 3% of the activity of the C3 fragments on a molar basis. Investigations with goat anti-guinea pig C3a demonstrate that human and guinea pig C3a possess one antigenic determinant in common; however, this determinant is not the C-terminal amino acid sequence. Addition of the anaphylatoxins with low doses of thrombin led to a potentiation of [3H]serotonin release from the platelets. Under these conditions C3a concentrations of 1.5×10−10μmol/liter (65 pg of C3a) could be detected. Thus the platelet system represents the most sensitive in vitro assay known for evaluation of biological activity of the C3a anaphylatoxins.

Platelet Activation: a New Biological Activity of Guinea‐pig C3a Anaphylatoxin

3H‐serotonin‐release from labelled gp‐platelets is established as a sensitive method for testing a new biological activity of gp‐C3a anaphylatoxin in an autologous situation. Time‐, dose‐ and temperature‐dependent release reactions as well as specific inhibition by carboxypeptidase Band anti‐C3a antibodies show that C3a is a potent and specific inducer of platelet activation. Inactive C3a does not induce 3H‐serotonin‐release but specifically inhibits the action of C3a on platelets.

Monoclonal antibodies to polysialic acid reveal epitope sharing between invasive pathogenic bacteria, differentiating cells and tumor cells

Monoclonal antibodies (mAb) for rapid diagnosis and detection of invasive bacteria and identification of pathogenic factors in infectious diseases are equally important in medical microbiology and clinical pathology and may even provide a breakthrough in basic medical and cell biology research. Such a situation evolved from the application of a unique mAb against the poorly immunogenic homopolymers of α2,8-linked sialic acid ofEscherichia coli K1 and meningococci group B capsules which could be derived from immune-hyperreactive NZB-autoimmune mice. The cross-reactivity of this mAb with identical polysialic acid (polySA) units of the neural cell adhesion molecule (N-CAM) revealed antigenic mimicry as the basis for the escape of the above-mentioned bacteria from host immune response and immune defense. The mAb proved to be a specific and sensitive diagnostic reagent as well as a very efficient therapeutic agent in experimentalE. coli K1 and meningococcal group B infections in mice. Furthermore, the mAb was found to react exclusively with long-chain polySA units characteristic of the embryonic form of N-CAM. This led to the discovery that the embryonic form of N-CAM is present outside neural tissue in the mesodermally derived kidney where it is specifically expressed during embryonic organ differentiation and reexpressed under conditions of malignant growth in nephroblastoma. Therefore, the embryonic form of N-CAM represents an onco-differentiation antigen in kidney.