Werner Braun

Antibody Formation: Stimulation by Polyadenylic and Polycytidylic Acids

Complexes of polyadenylic and polyuridylic acids, or of polycytidylic acid and methylated bovine serum albumin, enhance the early rate of increase in numbers of antibody-forming spleen cells in mice immunized with sheep red blood cells or other particulate antigens. Polyadenylic and polycytidylic acids, respectively, appear to be the source of the critical stimulators which, as demonstrated by others in bacteria, may act by influencing nucleotide kinase activity. The stimulated antibody response, but not the normal response, is antagonized by kinetin riboside and by an adenosine derivative occurring in sRNA.

Interferon Preparations as Modifiers of Immune Responses

Summary Within certain dose ranges, preparations of mouse serum interferon or tissue-culture interferon show a modest capacity to increase the number of antibody-forming spleen cells in mice as measured by the Jerne plaque technique. When low levels of polyadenylic polyuridylic acid (= poly A:U), i.e., an amount that causes only modest stimulation, were used together with amounts of the interferon preparations that also by themselves are ineffective, a good stimulation of early appearance of antibody-forming cells was produced. High concentrations of IF preparations reduced antibody formation, and the potent immunoenhancing effects of chlorite-oxidized amylose (COAM) were almost completely abolished in the presence of low concentrations of interferon preparations. These findings suggest certain similarities between the effects of interferon preparations and those of agents known to modify the cyclic AMP system. Such suspected relationships are supported by preliminary data demonstrating a capacity of IF preparations to elevate adenyl cyclase activity and cAMP levels in mouse spleen cells.

Nucleic Acids as Antigens

Publisher Summary Not only can antibodies against DNA be found in various autoimmune diseases, but DNA, RNA, various oligonucleotides, and even mono-nucleosides can be immunogenic under suitable conditions or when complexed with carriers such as methylated bovine serum albumin. This chapter reviews the development of the antigenicity of nucleic acids from the beginning of exploratory studies when the single point in question was whether nucleic acid-specific antibodies could be induced in suitable hosts by any means. To obtain antibodies with which nucleic acids and their components could react specifically, it was necessary to find the appropriate carrier for these haptens. Two major types of approach proved successful are the use of chemically defined, artificial conjugates or complexes and the use of naturally occurring complexes such as ribosome. The antigenicity of nucleic acids has been decided in the affirmative, the problem becomes to establish the feasibility of preparing homogenous populations of antibodies with defined and desired specificity, to extend the utilization of such antibodies in studies on structure and function of nucleic acids, and to explore the usefulness of this system of antibody production to an understanding of general mechanisms of antibody synthesis. The chapter also discusses some implications of findings for the analysis of structure and function of nucleic acids and for understanding antibody synthesis.

Cyclic AMP effects on antibody formation and their similarities to hormone-mediated events.

Summary Cyclic AMP enhances antibody formation in vivo and in vitro as judged by tests in which sheep red blood cells served as antigen and the effects were measured by determining the early rate of increases in the number of antibody-forming spleen cells of CFW mice. Furthermore, theophylline, a known stabilizer of cAMP, was found to enhance the stimulatory effects of poly A:U on antibody formation. These observations form the basis for a discussion of apparent parallels between immunological events and known events in hormone-controlled activations of cells.

Influence of oligodeoxyribonucleotides on early events in antibody formation.

Summary The number of hemolysin-forming cells, assayed by Jernes technique in spleens removed from AKR mice, 48 hours after immunization with heterologous red cells, is significantly higher when antigen is administered in conjunction with an enzymatic digest of calf thymus DNA. This stimulation by oligodeoxyribonucleotides is not matched by comparable effects of oligoribonucleotides; mixtures of monodeoxyribonucleotides or -sides are inactive. The stimulation appears to involve a stimulated multiplication of early appearing, or early activated, antibody-forming clones, and is more difficult to discern as the interval between immunization and assay of spleen cell populations increases. Oligodeoxyribonucleotides do not stimulate early immune responses unless specific antigen is administered concurrently; possible reasons for this requirement of antigen, in contrast to a lack of such requirement in comparable stimulations produced by bacterial endotoxins, are considered. The influence of dosage and route of administration of DNA digest have been analyzed and an effect on secondary as well as primary responses has been demonstrated. Kinetin riboside abolishes the stimulatory effects of oligodeoxyribonucleotides without influencing the basic, non-stimulated immune response, whereas Actinomycin D interferes with the immune response in the absence and presence of oligodeoxyribonucleotides. Possible relationships to problems of natural and adjuvant-elicited stimulations of antibody production have been discussed.

SPECTRUM AND MODE OF ACTION OF POLY A:U IN THE STIMULATION OF IMMUNE RESPONSES

It is now almost 20 years since we recognized that nucleic acids, apart from their specific informative properties based on nucleotide sequences, also can exert important regulating effects that are basically independent of nucleotide sequences. First in studies with bacteria, and later in studies with mammalian cells (compare Braun and Firshein, 1967), we as well as a few others (for example, Johnson, 1968) observed that oligo- and polynucleotides, in contrast to ineffective mononucleotides, can alter the rate of a number of biosynthetic events including rates of cell multiplication. When synthetic polynucleotides became available we tested their effects in a system that had proved susceptible to stimulation by natural oligo- and polynucleotides, namely antibody formation. At first, using single-stranded polynucleotides, such as poly A, poly U, poly C., and poly G, we found no effects. We were about to discard these homopolymers as inactive, when we made a final trial to determine the possible effectiveness of a mixture of these homo-polymers and discovered, in tests on antibody formation to sheep red blood cells (sRBC) in mice, a pronounced stimulation (Braun and Nakano, 1967). The rest is now history, documented by much of what is being reviewed at this Symposium: Complementary homopolymers, such as double-stranded poly A : U, poly C : G, or poly I : C., influence the behavior of cells involved in immune responses.

Antibodies to DNA and a Synthetic Polydeoxyribonucleotide Produced by Oligodeoxyribonucleotides

Calf-thymus DNA was degraded into small fragments (oligodeoxyribonucleotides); the fragments were treated with methylated bovine serum albumin, and the complexes so formed were emulsified in complete Freunds adjuvant and injected into rabbits. The serums of the immunized rabbits contained antibodies that reacted with homologous and heterologous unfragmented heat-denatured DNA, and also with a synthetic polydeoxyadenylate-thymidylate. Relatively small DNA fragments (of the order of tetra-hexanucleotide) can thus serve as haptens for the production of DNA antibodies; this finding increases the probability of producing antibodies specific for unique sequences of nucleotides.

Stimulation of Antibody Formation by Pyran Copolymer

Summary Pyran copolymer, administered with sheep red blood cells to mice, enhances the early rate of appearance of antibody-forming spleen cells. Although the cytotoxicity and pyrogenicity of pyran and bacterial endotoxins are similar, pyran, in contrast to endotoxin, does not trigger a nonspecific initiation of antibody formation; it only enhances specific responses. The major effect of pyran appears to be on macrophages.

Interactions between mononuclear phagocytes and Brucella abortus strains of different virulence.

Summary Studies on the in vitro interactions between S and R strains of Brucella abortus and monocytes from normal or immune guinea pigs have indicated 1) intracellular multiplication of S bacteria and a lack of, or insignificant extent of, multiplication of R bacteria in normal monocytes, 2) differences in the rate of ingestion of S and R bacteria, 3) differences in the rate of destruction of monocytes by S and R types, 4) differences in the rate of intracellular multiplication among S strains, 5) a significant modification of these events in immune monocytes, and 6) an apparently unique behavior of immunogenic, relatively avirulent strain 19-S bacteria in normal monocytes. The relation of these observations to some problems of host-parasite interactions is discussed. It also has been shown how these observations may provide the basis for a new technic of rapid assessment of the homogeneity or heterogeneity of bacterial populations in regard to properties associated with virulence.

Hemolysin formation in newborn mice of different strains.

Summary Newborn mice of AKR, Ha/ICR and C57Bl strains start to respond to immunization with sheep red blood cells at different ages (as measured by assays on circulating hemolysins and numbers of antibody-forming cells in spleen and lymph nodes). Once competence to respond has been attained, the magnitude of the response, in terms of numbers of antibody-forming spleen cells, is similar for all strains tested. However, there is no correlation between numbers of hemolysin-forming cells in the organs tested and circulating hemolysins. Only the latter parallel the strain differences in responsiveness observed by others in adult animals of these strains. When antigen is administered in conjunction with oligodeoxyribonucleotides, the early immune responses are enhanced, but the time of competence to respond to the antigenic stimulus is not advanced.