Production of antibodies to amanitins as the basis for their radioimmunoassay

Summary The production of antibodies against amanitins is described. By means of these antibodies, a radioimmunoassay was developed which allows detection of as little as 0.5 ng of amanitins in 1 ml of serum. By this method, the clearance of α-amanitin from the blood of poisoned mice was measured. Acknowledgments. We thank ProfessorT. Wieland and Dr.H. Faulstich (Heidelberg) for their generous gift of β-amanitin and [³H]O-methyl-demethyl-γ-amanitin. This work was supported by grants from C.N.R., Rome.     

Enhanced inhibition of RNA synthesis by amanitins in in vitro cultured cells

Summary The inhibition of RNA synthesis by -amanitin on vitro cultured cells is very slow. The action of various analogues of the toxin was tested and some of them proved more effective. Moreover pretreatment of cell cultures with DEAE-dextran greatly enhanced the effect of -amanitin.Acknowledgments. We thank ProfessorTh. Wieland and Dr.H. Faulstich (Max-Planck-Institut, Heidelberg) for the generous gift of amanitins. This investigation was supported by grant No. 74.00637.65 from C.N.R. (Roma).     

TSH-receptor antibodies, HLA B8 and thyroid autoantibodies in patients with Graves' disease in therapeutically induced euthyroidism.

The prevalence of TSH-receptor antibodies and of thyroid autoantibodies was studied in 48 HLA-typed patients with Graves' disease, who were in an euthyroid state after antithyroid therapy with methimazole. TSH-receptor antibodies, which were found in 35% of the patients, did not correlate with the positivity of HLA B8. By contrast the persistence of thyroid microsomal antibodies was significantly associated with HLA B8.     

TSH-Receptor antibodies,HLA B8 and thyroid autoantibodies in patients with Graves' disease in therapeutically induced euthyroidism

Summary The prevalence of TSH-receptor antibodies and of thyroid autoantibodies was studied in 48 HLA-typed patients with Graves' disease, who were in an euthyroid state after antithyroid therapy with methimazole. TSH-receptor antibodies, which were found in 35% of the patients, did not correlate with the positivity of HLA B8. By contrast the persistence of thyroid microsomal antibodies was significantly associated with HLA B8.     

Antibodies from patients with liver diseases and from normal human or animal sera against glutaraldehyde-polymerized albumins: lack of species specificity

By the use of glutaraldehyde-polymerized albumins of different species (human, rabbit, bovine, goat and mouse) it was demonstrated that anti-albumin antibodies in sera of patients with liver diseases and in normal human and animal (rabbit, mouse) sera are not species specific.     

Nachweis von Nahrungsmittel-Antikörpern mittels der Hämagglutination

Summary By means of ahaemo-agglutination test using erythrocytes sensitised with tannic acid, we were able to demonstrate the presence of cow's milk antibodies in humans. The use of the haemo-agglutination test enables us now to give exact information on the titer value of the antibody in human serum. Previously this was limitately possible using the method of antibody demonstration by means of the complement-compound reaction.We have well-established reasons to assume that other antibodies caused by various antigens, can be determined by means of the haemo-agglutination test.     

Nachweis von organspezifischen Antikörpern

Summary Using a new nephelometric method, species-specific and organ-specific antibodies have been demonstrated. By means of the absorption technic organ-specific components of species-specific antibodies of high specificity have been determined in the serum of rabbits sensitized with various rat organs (heart, liver, kidney, tumor).

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Die Arbeit wurde mit Hilfe des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung durchgeführt.     

Hachweis von individualspezifischen Gewebsantikörpern

Summary Injection of homologous tissue or tissue extracts and homologous transplantation of skin in rabbits provoke the production of antibodies. By means of a new nephelometric method, acquired antibodies against homologous tissue circulating in the serum were found. This tissue-antibody seems to be an individual-specific one, reacting with all organs of the donor. The antibody is thermolabile and not dialysable.

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Die Arbeit wurde mit Hilfe des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung durchgeführt.     

Involution of the mesonephros and differentiation of the epididymis in chickens: immunohistological study]

By means of purified rabbit anti-adult chicken kidney antibodies two types of antigens have been identified in the mesonephros: one, localised in the cells of the proximal segment of the secretory tubules, the other characteristic of the collecting segments derived from the Wolffian duct. During the transformation of certain mesonephric tubules into ductuli efferentes and conjugentes of the epididymis the collecting tubule antigen disappears whereas the proximal secretory tubule antigen not only persists but can also be detected in the parietal layer of Bowman's capsules as they transform before connecting with the rete testis.     

Differentiation antigens in keratinocytes II: cytoplasmic specificity expressed in the basal cell of the external root sheath of hairs but not in the basal cells of epidermis]

By means of indirect immunofluorescence with sera obtained from patients after bone marrow transplantation it has been demonstrated that antibodies were reacting with the cytoplasm of basal cells ((C.B.C.) in the external root sheath of hairs (E.R.S.). Some sera were found to react with C.B.C. of the E.R.S. but not with the C.B.C of the epidermis. The change in the antigenic properties of the basal cells was found to be situated at the orifice of the sebaceous gland duct.     

Skeletin immunoreactivity in peripheral nerves

Summary By use of monospecific antibodies against the cow heart intermediate filament protein, skeletin, an antigenic relationship between skeletin and neurofilament protein of peripheral nerves is demonstrated. Crossreactivity is also demonstrated in the filament-containing Schwann cells. The results are consistent with the existence of several subclasses of related intermediate-sized filament proteins.Acknowledgments. The skilful technical assistence of Mrs Elisabeth Rubing, Miss Inga Johansson, and Miss Marléne Lundström is gratefully acknowledged. This work was supported by grants from Lions Research Fund, the Swedish Medical Research Council (12X-3934) and the Faculty of Medicine, University of Umeå.     

A simple method for the preparation of antibodies to the mitochondrial biotin-dependent carboxylases

Heterologous antiserum to the 3 biotin-dependent carboxylases was prepared by selective removal of these enzymes from human liver on an avidin-sepharose column. A carboxylase-avidin-sepharose matrix was used as an antigen to produce anti-carboxylase antibodies. The resultant antisera can be used to purify the specific carboxylases, to prepare monoclonal antibodies to these enzymes or to study inherited carboxylase deficiencies and biotin-dependent intermediary metabolism.     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B3 to bovine serum albumin. Aflatoxin B3 was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B1 (AFB1) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B1, B2, G1, and G2 when tritiated AFB1 was used as the marker ligand. The concentrations causing 50% inhibition of binding of 3H-AFB1 to the antibodies by unlabeled aflatoxins B1, B2, G1, G2 and B3 were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B1 and G1, two of the most important toxic metabolites produced by Aspergillus flavus and A. parasiticus.     

Antigénicité et pouvoir immunogène des toxines extraites des venins d'Elapidae et d'Hydrophiidae

Summary In this article the results are reported of recent experiments concerning the antigenicity and the immunogenic properties of some basic toxic proteins recently extracted from snake venoms. A serological classification of these substances is proposed. 3 groups have now been determined. As most of the low molecular weight proteins (<10.000) the immunogenicity of the toxin ofNaja nigricollis, taken as an example, is poor. The influence of polymerization of this toxin, and the adjuvant effect of the Freund mixture on the production of specific antibodies, are described. By affinity chromatography, using Sepharose 4B as a carrier, antibodies against this toxin were separated from the other proteins of the antiNaja nigricollis horse immune-serum. They belong to the IgT immunoglobulins. By the same technique, when erabutoxin a fromLaticauda semifasciata is covalently linked to the Sepharose, antibodies reacting with both this toxin and the toxin were separated from the same immuneserum. This confirms that an immunological relationship exists between the toxin of an Elapidae:Naja nigricollis, and the erabutoxin a of an HydrophiidaeLaticauda semifasciata.     

Polyreactive antibodies in adaptive immune responses to viruses

B cells express immunoglobulins on their surface where they serve as antigen receptors. When secreted as antibodies, the same molecules are key elements of the humoral immune response against pathogens such as viruses. Although most antibodies are restricted to binding a specific antigen, some are polyreactive and have the ability to bind to several different ligands, usually with low affinity. Highly polyreactive antibodies are removed from the repertoire during B-cell development by physiologic tolerance mechanisms including deletion and receptor editing. However, a low level of antibody polyreactivity is tolerated and can confer additional binding properties to pathogen-specific antibodies. For example, high-affinity human antibodies to HIV are frequently polyreactive. Here we review the evidence suggesting that in the case of some pathogens like HIV, polyreactivity may confer a selective advantage to pathogen-specific antibodies.     

Anti-bovine beta-lactoglobulin antibodies react with a human lactoferrin fragment and bovine beta-lactoglobulin present in human milk

Human milk samples react against anti-bovine beta-lactoglobulin rabbit antibodies, as measured by a competitive radioimmunoassay. Immunoreactivity was positive even in milk from mothers consuming a diet free of cow's milk. An increase with a diet rich in cow's milk proteins was detected by immunoelectrophoresis. The human milk fraction cross-reacting with anti-bovine beta-lactoglobulin antibodies corresponds to the 20 kDa fragment from the N-terminal end of human lactoferrin. Three regions of this fragment exhibit sequence homology with a sequence contained in cow's beta-lactoglobulin (between residues 124 and 141).     

The neurosecretory system of the octopus vena cava: A neurohemal organ

Summary Endings of about two million small neurones form a voluminous neuropil inside the vena cava of cephalopods, in direct contact with the blood. These nerve endings are filled with masses of typical neurosecretory granules. By immunocytochemistry we could distinguish three different populations of secretory endings in the vena cava neuropil ofOctopus vulgaris: 1) a population of endings which were immunoreactive with antibodies against the pentapeptide proctolin; 2) a population with oxytocin/vasopressin- and neurophysin-like immunoreactivity; 3) a population immunoreactive with antibodies which were raised against the molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide, against -melanotropin, and against atriopeptin. Extracts of the octopus vena cava stimulated amplitude and frequency of the isolated octopus heart preparation. Similar effects were exerted by peptides with the C-terminal structure-Arg-Phe-amide. Recently, we could isolate and identify in vena cava extracts four peptides; Phe-Met-Arg-Phe-amide, Phe-Leu-Arg-Phe-amide, Ala-Phe-Leu-Arg-Phe-amide and Thr-Phe-Leu-Arg-Phe-amide. Other peptides have not yet been identified. The fact that the peptides against which the immunoreactive antibodies were raised affected, in different organisms, blood volume, blood pressure, renal function and heart contraction suggests that one of the main functions of the neurosecretory system of the vena cava is a hormonal control of circulation.     

Production of monoclonal anti-Schistosoma mansoni antibodies. Preliminary study of their biological activities]

Monoclonal antibodies against Schistosoma mansoni have been produced by fusion of splenic lymphocytes from S. mansoni infected Rats and P3-X63-Ag8 BALB/c cells. In vitro and in vivo studies of the biological activities of these antibodies have led to the identification of IgE antibodies with a high reaginic activity and antibodies which in a complement dependent or eosinophil dependent system were shown to have a marked cytotoxicity for schistosomula in vitro. This methodology seems to open new perspectives for the study of antibody function in immunity against parasites as well as for the isolation of the corresponding target antigens.     

Immunological observations following vasectomy

Summary Lymphocytotoxic antibodies (LCA) against panels of normal lymphocytes and leukemic B-cells were demonstrated in vasectomized men. Since vasectomy is known to induce antibody formation to spermatozoa, the demonstration of these lymphocytotoxic antibodies may be related to antigenic constitutents of spermatozoa such as HLA or B-cell alloantigens. Long term follow-up is needed to clarify the clinical significance of these antibodies.     

Xenoreactive natural antibodies

Shortages of human organs for transplantation have made it necessary to examine the possibility of using nonhuman organs for xenotransplantation the transplantation of tissues between different species. Pigs are now regarded as the most likely species to serve as donors for clinical xenotransplantation. However, rejection of pig tissues and organs, mediated by the host's immune system, remains a major barrier to successful xenotransplantation. The primary immunological hurdle to overcome is rejection mediated by antibodies in the host that recognize antigens present on xenogeneic tissues. Since these antibodies are produced naturally in the host without immunization, they are termed natural antibodies. Here, we review the nature of xenoreactive natural antibodies directed toward pig tissues, and summarize recent progress in the field of xenotransplantation directed at overcoming humoral rejection of porcine xenografts.