Arthur G. Johnson

Regulation of the immune system by synthetic polynucleotides. IV. Amplification of proliferation of thymus-influenced lymphocytes.

Abstract The role of thymus-influenced antigen reactive cells (ARC) in the immune response was studied with the aid of a potent adjuvant to both antibody formation and cell-mediated immunity, polyadenylic-polyuridylic acid complexes (poly A:U). The polynucleotide complex increased the anti-sheep erythrocyte rosette forming cell response of irradiated mice after thymus cells were exposed to poly A:U in vivo or in vitro prior to injection of bone marrow cells. This direct stimulatory action resulted in an enhancement of the rate of proliferation of ARC in response to antigen and a shortening of the interval between antigenic challenge and the initiation of division in immunocompetent cells. No stimulatory effect of poly A:U on bone marrow derived antibody forming precursor cells was observed. It is suggested that ARC play an important role in the regulation of the length of the induction period, as well as the rate of increase of antibody forming cells in response to some antigenic stimuli.

Radioautographic and Electron-Microscopic Evidence of Rapid Uptake of Antigen by Lymphocytes

Iodine-125-labeled ferritin molecules were detected by radioautography in the sinuses of the rat popliteal lymph node shortly after injection into the foot pad; they appeared to be taken up by macrophages and phagocytic reticular cells. Electron microscopic examination of the same tissue also revealed ferritin molecules within small lymphocytes as early as 5 minutes after injection. The antigen appeared to be taken up by the process of pinocytosis and was distributed throughout the cytoplasm and nucleus. While the number of ferritin molecules observed in the lymphocyte was much less than that taken into the inacrophage, the observation is significant in understanding the role lymphocytes play during the early phase of antibody response.

Regulation of the immune system by synthetic polynucleotides. Vii. Suppression induced by pretreatment with poly a.u.

Abstract Pretreatment of mouse spleen cells with polyadenylic-polyuridylic acid complexes (poly A:U) either in vivo or in vitro 24 hr prior to addition of antigen, resulted in a substantial time dependent decrease in anti-SRBC PFC. Enhancement was observed 6 hr after poly A:U, while inhibition did not become evident until 24 hr after pretreatment. Inhibition of the PFC response appeared to result from poly A:U activation of a nylon wool adherent, T suppressor cell, capable of diminishing the response of normal spleen cells exposed to antigen on co-culture.

STIMULATION OF THE IMMUNE SYSTEM BY HOMOPOLYRIBONUCLEOTIDES

Stimulation of the immune response by adjuvants, such as endotoxins, nucleic acids, and smaller molecular weight oligonucleotides, has been under investigation in our laboratory (Johnson et al., 1968), with the hope that heretofore subliminal events leading to primary antibody synthesis would be magnified. The mechanism of action of such stimulation would likely be clarified more easily through the use of defined oligonucleotides, particularly since it was time-consuming to purify the products, active as adjuvants, following breakdown of nucleic acids with nucleases (Johnson and Hoekstra, 1967). Accordingly, when homopolyribonucleotides (polyadenylic acid complexed in vitro with polyuridylic acid, termed poly A: U) were shown to be effective adjuvants (Braun and Nakano, 1967), we initiated an in-depth study of their general properties and the cell type(s) affected. Our results to date are as follows.

Enhancement of Antibody Formation by Nucleic Acids and their Derivatives

It is common knowledge today that antibody forming cells can be either suppressed or enhanced by various manipulative procedures. Our objective in this presentation is to describe experiments in which nucleic acids and their derivatives were found to act as adjuvants by decreasing the induction period and increasing the concentration of circulating antibody following a single injection of antigen.

Lymphocyte Cytotoxicity in Human Liver Disease Using Rat Hepatocyte Monolayer Cultures

Isolated rat hepatocytes were used to determine the cytotoxicity of peripheral blood mononuclear cells (PBM) from patients with alcoholic liver disease (ALD) and chronic active hepatitis (CAH). The specificity of the cytotoxic effect on liver cells was monitored using rat kidney cells. PBM from patients with CAH and ALD showed increased spontaneous cell-mediated cytolysis (SCMC) for hepatocytes. The SCMC against kidney cells was comparable for the patient groups and controls. Incubation of liver cells with antibody to human liver-specific protein significantly increased the cytotoxic activity of control PBM but did not block cytotoxicity by PBM of patients with CAH. Incubation of PBM from untreated CAH patients with thymosin fraction 5, a polypeptide extract of the thymus gland, significantly lowered cytotoxicity. Our findings suggest that rat liver cells provide a model for studies of cell-mediated immunity in human liver disease and that thymosin fraction 5 decreases the cytotoxic activity of sensitized lymphocytes in CAH.

Stimulatory Effect of Polynucleotides on Short Term Leukocyte Cultures

Conclusions and Summary Polyadenylic acid when complexed with polyuridylic acid, exerted an adjuvant effect in short-term human leukocyte cultures activated by purified protein derivative of tuberculin or in mixed leukocyte interactions. In contrast, stimulation of DNA synthesis in leukocytes by phytohemagglutinin was depressed by these homoribopolymers.

Synthetic immunoregulating molecules: A potential bridge between cytostatic chemotherapy and immunotherapy of cancer

The concept of immunomodulation as an adjunct to cancer therapy is based on ample evidence that a variety of agents augment the immune reaction to unrelated antigens in experimental animals and increase their resistance to tumors. The ever-growing list of such agents includes viruses, bacteria, fungi, and their products (Ciba Foundation Symposium, 1973). Following the pioneering work of Math6 with BCG (Math~, 1971), investigators have been very active in this field in recent years (Chedid et al., 1973; Lamensans et al., 1975; Lamoureux et al., 1976; Laucius et al., 1974; Leclerc et al., 1976; Mastrangelo et al., 1976; Old et al., 1959; Weiss et al., 1961; Zbar et al., 1970). Most, if not all of these studies, have been done with viable or killed microorganisms (Hersch et al., 1976; Israel, 1976; Pouillart et al., 1976), cell walls (Chedid et al., 1973; Gray et al., 1975; Yamamura et al., 1976a; Zbar et al., 1972), or very complex fractions such as raethanol extract residue (MER) (Perloff et al., 1977; Yron et al., 1973) or interphase material (IPM) (Lamensans et al., 1975). Some organisms such as BCG and Corynebacterium parvum have been shown to be dramatically effective in certain experimental models, but investigators are well aware of the fact that they contain a great variety of heterogenous antigens, some of which might cross-react with host tissues (Borsos and Rapp, 1973; Bucana and Hanna, 1974; Vandenbark et al., 1975). They are also endowed with undesirable pharmacologic side effects (Werner et al., 1977). Consequently, current attempts at isolating chemically well-defined fractions from whole bacteria have been given a high priority, with the justification that they may lead to preparations that retain important therapeutic effects but with diminished or no toxicity.

The effect of 6-mercaptopurine on antibody production in atopic individuals

Abstract Recent interest in the use of the purine antimetabolites in the treatment of diseases of proved or postulated immune etiology prompted a study of the effect of 6-mercaptopurine (6MP) on antibody formation in atopic individuals. Six prison inmate volunteers with ragweed hay fever and/or asthma (treatment group) were matched according to age, sex, and race with six ragweed sensitive patients who served as untreated controls. Subjects in the treatment group were given 6MP, 1.5 mg. per kilogram per day by mouth for 21 to 42 days. As compared with the control group, subjects in the treatment group demonstrated the following: diminished antibody response following immunization with E. coli Vi antigen; no change in pre-existing titers of ragweed reagin as measured by direct skin test and passive transfer studies; no change in pre-existing hemagglutinin titers to diphtheria toxoid; and no change in pre-existing serum concentration of gamma-1A, gamma-1M, or gamma-2 globulin. Two subjects in the treatment group developed toxicity which necessitated discontinuance of the drug. Bone marrow examinations at the end of the study revealed macronormoblasts in four treated patients. The lack of suppression of pre-existing antibody titers, including reagin, suggests that when used under the conditions studied, and aside from possible nonimmunologic effects, 6MP would be of little value in treatment of the atopic diseases.

TYPE OF ANTIBODY ELEVATED BY 5-FLUORO-2-DEOXYURIDINE AND ENDOTOXIN DURING THE PRIMARY RESPONSE OF THE MOUSE.

Summary Mice stimulated with human gamma globulin formed antibody which was mercaptoethanol sensitive and assumed to be of the 19S variety. Incorporation of bacterial endotoxin with the human gamma globulin stimulated antibody to appear earlier and to higher levels. This increased antibody response was due both to production of mercaptoethanol sensitive antibody and mercaptoethanol insensitive antibody. Injection of 5-fluoro-2-deoxyuridine one hour before the human gamma globulin also resulted in early and enhanced levels of mercaptoethanol sensitive antibody, with an increase in the level of low molecular weight antibody appearing later. Similarly, C58 mice receiving endo-toxin and BGG formed antibody earlier and to greater levels than did those receiving BGG alone. The increased antibody production in this strain also was due to both mercaptoethanol sensitive and insensitive antibody.