Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

Localization of α1-adrenoceptors in rat and human hearts by immunocytochemistry

The localization of the ai adrenoceptors (₁-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human ₁-AR (AS sequence 192–218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, ₁-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of ₁-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the -adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands.     

Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores ofPhytophthora cinnamomi

Summary The role of tubular mastigonemes in the reversal of thrust of the anterior flagellum ofPhytophthora cinnamomi was analysed using mastigoneme-specific monoclonal antibodies and immunoflu-orescence and video microscopy. Exposure of live zoospores ofP. cinnamomi to the mastigoneme-specific Zg antibodies caused alterations in the arrangement of mastigonemes on the flagellar surface and at Zg concentrations above 0.3 /ml, mastigonemes became detached from the flagellum. As a consequence of antibody binding to the mastigonemes there were concentration-dependent perturbations in zoospore swimming behaviour and anterior flagellum beat pattern. With increasing antibody concentration zoospores swam more slowly and other parameters of their swimming pattern, such as the wavelength of the swimming helix and the frequency of rotation, were also reduced. The effects of Zg antibodies were specific at two levels: control immunoglobulins or antibodies that bound to other flagellar surface components did not have an effect on motility, and Zg antibodies did not interfere with the motility of zoospores of oomycete species to which they did not bind. The effects of antibody-induced disruption of mastigoneme arrangement strongly support previous hypotheses that tubular mastigonemes are responsible for thrust reversal by the anterior flagellum, enabling it to pull the cell through the surrounding medium.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

Summary The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 m long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, -D-mannose and -D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (M_r 133,000 and 146,000). Reductions in the M_r of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.Abbreviations ECM extracellular matrix - BPA Bauhinia purpurea agglutinin - BSA bovine serum albumin - DIC differential interference contrast - FITC fluorescein isothiocyanate - GNL Galanthus nivalis lectin - GSI-B₄ Griffonia simplicifolia isolectin B₄ - HEPES (N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid) - IIF indirect immunofluorescence - IPC isopycnic centrifugation - MAb monoclonal antibody - PEG polyethylene glycol - PBS phosphate buffered saline - PNGase peptideN-glycosidase - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCS tissue culture supernatant - TEM transmission electron microscopy - TFMS trifluoromethane sulphonic acid     

Autoantibody to the nucleosome subunit (H2A-H2B)-DNA is an early and ubiquitous feature of lupus-like conditions

Chromatin, a huge polymer of nucleosomes, has been implicated as an important target of autoantibodies in idiopathic and drug-induced lupus for decades, but the antigenicity of chromatin has only recently been dissected. IgG reactivity with the (H2A-H2B)-DNA complex, a subunit of the nucleosome, is present in the majority of patients with systemic lupus erythematosus, in >90% of patients with lupus induced by procainamide and in individual patients with lupus induced by a variety of other drugs, but is not seen in people taking these medications who are clinically asymptomatic. Anti-[(H2A-H2B)-DNA] accounted for the bulk of the anti-chromatin activity in drug-induced lupus. The earliest detectable autoantibody in lupus-prone mice recognized similar epitopes in the (H2A-H2B)-DNA subnucleosome complex; as the immune response progressed, native DNA and other constituents of chromatin became antigenic. The importance of chromatin-reactive T cells in the anti-[(H2A-H2B)-DNA] response is suggested by the presence of somatic mutations in antibody V_H and V_L regions, their perdominant IgG isotype and the similarity in kinetics of their production to that of conventional T cell dependent antigens. Together with the serologic data from human lupus-like disease, these results are consistent with chromatin being a common stimulant for both B and T cells. While chromatin-reactive antibodies are closely associated with systemic disease and have recently been implicated in glomerulonephritis in SLE, the absence of renal disease in drug-induced lupus indicates that additional abnormalities are required to manifest the serious pathogenic potential of anti-[(H2A-H2B)-DNA] antibodies.Abbreviations APC antigen present cells - DIL drug-induced lupus - ELISA enzyme-linked immunosorbent assay - GBM glomerular basement membrane - [(H2A-H2B)-DNA] an intermolecular complex consisting of DNA and a dimer of histones H2A and H2B - nDNA native (double-stranded) DNA - SLE systemic lupus erythematosus     

Development of human monoclonal antibodies: A review

This article describes the current status in the development of human monoclonal antibodies. Over the last ten years a lot of information about the human immune system has emerged. Combining these with the many new (bio-)technologies it is plausible that the long awaited breakthrough of this technology is close. This paper focuses on the classical cell-biological methods of achieving stable, antibody-producing human cell lines via cell fusion methods or virus derived transformations of human B-lymphocytes, as well as genetic engineering methods e.g. DNA libraries or phage display technology. The available in vitro immunization methods are critically reviewed and their impact on this topic is discussed. Therapeutic applications for cancer treatment or passive immunization against infectious diseases with antibodies derived by both ways are also reviewed.     

Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

Factors inhibiting IFN activity

Several factors may inhibit the activity of IFNs. Some of these occur naturally, others are therapy-induced or artificial. Naturally occurring antibodies appear to have a much broader reactivity than therapy-induced antibodies. Naturally induced antibodies are reported in patients suffering from chronic graft-versus-host disease after bone marrow transplantation. Differences in the reported immunogenicity between interferons may not be due to the minor variation in amino acid sequence. The clinical significance of therapy-induced antibodies has been unclear. In patients treated for chronic hepatitis C, antibody formation is closely related to relapse. In animal studies the efficacy of treatments targeting the IFN receptor interaction has been shown. Soluble IFN- receptor inhibits the development of autoimmune diseases in mice. Monoclonal antibodies to the IFN- receptor protects against allograft rejections in monkeys. Two naturally occurring inhibitors of IFN action were reported. The clinical significance and structure of these inhibitors remain elusive.     

鼠肺支原体单克隆抗体的研究

通过杂交瘤技术建立了３株抗鼠肺支原体单克隆抗体细胞株,它们分别是ＢＡ１１,ＢＤ７和Ｂ６１２．试验表明：３株细胞所分泌的抗体均属ＩｇＧ１亚类,都是鼠肺支原体的特异性抗体,与多种其它支原体无交叉反应。