Otto J. Plescia

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.

SUBVERSIVE ACTIVITY OF SYNGENEIC TUMOR CELLS AS AN ESCAPE MECHANISM FROM IMMUNE SURVEILLANCE AND THE ROLE OF PROSTAGLANDINS

Mice bearing a syngeneic tumor become increasingly immunodepressed during growth of the tumor, being unable to develop both cellular and humoral immunity to a histoincompatible tumor allograft and to reject the allograft. This failure to reject a strongly antigenic tumor allograft suggests that immunodepression associated with growth of a weakly antigenic syngeneic tumor provides the syngeneic tumor with an escape mechanism. This immunodepression is also manifest by the suppression of the response of spleen cells to mitogen stimulation by syngeneic tumor cells, both in vivo and in vitro. T cells that are stimulated by PHA, a T-cell mitogen, are the primary targets, and their suppression is the result of the direct subversive activity of the tumor cells. Subversion of T cells by tumor cells seems to be mediated through the prostaglandin pathway, because the prostaglandin PGE2 is itself suppressive, and an antagonist of PGE2 and an inhibitor of prostaglandin synthetases both inhibit the subversive activity of tumor cells. Several tumor cell lines tested, of different etiology and histologic type, all were subversive. This suggests that this subversive activity may be a general property of tumor cells and may be a key element in their ability to thwart the immunological system of the host. For this reason, any therapeutic regimen of cancer, based on immunostimulating drugs, should include drugs that can inhibit active subversion of the immune system by the tumor itself. Antagonists of prostaglandins and inhibitors of prostaglandin synthetases show promise in this regard.

Inhibition of HIV replication by liposomal encapsulated amphotericin B

This report shows the potential of using a liposomal encapsulated preparation of amphotericin B (a polyene macrolide antibiotic) for the in vitro inhibition of HIV. There was no significant difference between the effective doses of the free form of drug when compared to the liposomal encapsulated preparation in inhibiting the growth of HIV. Virus expression was suppressed at a concentration of 5-10 micrograms/ml of the drugs. The liposomal preparation showed greatly reduced cytotoxicity in experiments using cultures of murine leukocytes. These results show the potential usefulness of liposomal encapsulated drugs in the treatment of patients with AIDS or AIDS related complex.

Comparative toxicities of amphotericin B and its monomethyl ester derivative on glial cells in culture.

Amphotericin B (AmB) is a potent antifungal polyene macrolide antibiotic and is the drug of choice for the treatment of deep-seated mycotic infections. Its use is limited, owing to its nephrotoxicity, and it must be dispersed in deoxycholate for parenteral administration. In contrast, AME (the monomethyl derivative of AmB) is water dispersible, is appreciably less cytotoxic than AmB toward a variety of cell types, and is reportedly active against the acquired immunodeficiency syndrome virus (human immunodeficiency virus type 1). The latter activity has generated interest in AME as an antiviral drug. However, AME is perceived to be neurotoxic, based on the outcome of a human clinical trial of AME as an antifungal drug. AmB is not regarded as neurotoxic, presumably because any neurotoxicity in vivo is precluded by its nephrotoxicity. It was important, therefore, to determine the potential for neurotoxicity of the two agents in comparative tests, assessing the effects of their direct action against neural cells in culture. Rat cortical cells, comprising astrocytes and oligodendrocytes, were used. AME was at least 10 times less toxic than AmB and equally less toxic against several other nonneural cell types also included in these tests. Equally important, AmB disrupted the myelin sheath in these cultures, and it inhibited its generation. AME did not, even at a concentration 10 times greater than the toxic concentration of AmB. AmB is, therefore, potentially more neurotoxic than AME, contrary to current perception. AME is effective as an antifungal and antiviral drug at a concentration far below its toxic concentration for neural cells. Also, AME does not cross the blood-brain barrier appreciably, so that a therapeutic level in blood can be expected without encountering neurotoxicity. Images

An assessment of changes in the complement level of myasthenic sera.

I n 1954, Nastuk, Plescia and Osserman initiated a study of hemolytic complement ((2’s) in sera of patients with myasthenia gravis on the basis of what seemed reasonable similarities in the properties of serum C‘ and cytolytic serum factors thought to be associated with myasthenia gravis. The results of this study’ showed tha t the level of C’ in myasthenic sera tends to lie outside the established normal range, and tha t the level often falls with exacerbation of the disease and rises with remission, exceeding normal values sometimes. How are we to explain this observed deviation from normal? The first thought is that C’ is “fixed” in vivo by antigen-antibody complexes which might be involved in the pathogenesis of the disease. I n fact, it was this consideration that led Strauss and his collaborators to look for serum factors, presumably antibody, with a specific affinity for muscle. They obtained clear evidence tha t such serum factors do exist and are capable of “fixing” C’ when bound to muscle.’ This observation, however, is only suggestive and does not constitute proof that “fixation” of C‘ in vivo is responsible for abnormal levels of C’ in myasthenic sera. Proper assessment of the changes in the level of C’ requires consideration of the several factors that are known to influence the titer of C’ in serum. These include: (1) the dependence of the C‘ titer on the composition of the C’ system which is multicomponent; (2) the relative concentration of specific inhibitors of C’ generally present in serum; and (3) the possible presence of serum factors, in addition to specific inhibitors, that cause inactivation of C’. Consideration of these factors prompted us to undertake a more detailed analysis of a large number of myasthenic sera, made available to us by W. H. Nastuk. These sera were assayed for individual components of C’ and for inhibitors of C‘ with the expectation that the results might give greater insight as t o the causes for the abnormal levels of C’. This report is a summary of these results and deals with possible interpretations.

Regulation of antigen induced changes in cyclic nucleotide levels in NZB/WF1 Mice

Abstract Normal C57BL/6J mice respond to the iv injection of antigen with an increase in splenic cAMP at 2 min. NZB/WF1 mice are predisposed to autoimmune and immunological disorders upon aging. The ability of NZB/WF1 mice to respond to antigen with an increase in their splenic cAMP level was found to diminish with age. This loss of responsiveness is antigen specific and not due to a loss of adenylate cyclase activity in spleen cells of old NZB/WF1 mice. The adoptive transfer of spleen cells from unresponsive old mice into responder young mice inhibited the cAMP response to antigen by the recipients. Spleen cells from young responsive mice, on transfer into old nonresponsive NZB/WF1 recipients, resulted in restoration of the cAMP response to antigen. In both cases, the activity of donor cells was dependent on the transfer of T cells. These results indicate that populations of T cells participate in the regulation of the cAMP response to antigen by NZB/WF1 mice. The response of old mice could also be restored by treatment with indomethacin, and also the spleen cells of such treated donors failed to suppress the cAMP response of young recipients. Together, the results suggest a role for prostaglandins in regulating the cAMP response to antigen.

Methylated Bovine Serum Albumin as a Carrier for Oligo- and Poly-Nucleotides

We initiated an immunochemical study of DNA in 1956, at a time when there was little decisive data dealing with the antigenicity of DNA (Blix et al., 1954; Lackman et al., 1941) and no evidence that chemically pure DNA was capable of eliciting antibody formation in a suitable host. For this reason, we used in our initial experiments DNA-rich preparations extracted from Brucella abortus by a phenol procedure (Braun et al., 1957). These preparations contained appreciable protein and carbohydrate material that could not be dissociated easily from the DNA.

CURRENT STATUS OF RESEARCH ON MYASTHENIA GRAVIS

In this monograph on myasthenia gravis many of the papers presented have been devoted to basic aspects of the structure and function of the neuromuscular junction. Other papers have been centered on clinical problems and the management and treatment of myasthenic patients. All of these subjects are of interest both to clinicans and to research workers. The clinician must utilize such fundamental information in devising rational lines of treatment which will more effectively alleviate the symptoms of myasthenia gravis and which may arrest or even reverse its course. In making our remarks on the current status of research on myasthenia gravis, our hope is to serve the interests of all working in this field. In thinking how we might do this, we realized that it was not practical to summarize and review in detail all of the papers presented in this monograph. We, therefore, decided to direct our remarks toward the following two essential questions: (1) What is the intimate nature of the pathological defects which occur in myasthenia gravis? (2) By what mechanisms are these pathological defects produced? In dealing with these questions, we have drawn on fundamental and clinical evidence, much of which was presented in this monograph.

Fixation of Complement to Fragments of Antibody

A specific precipitate of ovalbumin and its rabbit-serum antibody, after fixing human serum complement, was digested with papain. The digest was analyzed immunochemically for complexes of antigen, antibody fragments, and components of complement. The results indicated that complement is not bound to Porter fragment III, but very likely is bound to fragments I and II.

Fractionation and Immunological Properties of a DNA-rich Preparation from Brucella abortus.

Summary A DNA-rich preparation, obtained from Brucella abortus by extraction with 0.5% phenol, was fractionated by differential ultracentrifugation, and its immunilogical properties were studied. The antigens that precipitated antibodies from the sera of rabbits injected with the crude DNA preparation were concentrated in a fraction consisting essentially of DNA and only about 2% protein. This fraction did not elicit the formation of precipitating antibodies, whereas a protein-rich fraction with no reactive antigens did. Treatment of the reactive antigens with DNase altered some of their properties. On the basis of present evidence, it is suggested that the DNase-sensitive antigens are complexes containing DNA.