Florence B. Seibert

DMSO AND OTHER COMBATANTS AGAINST BACTERIA ISOLATED FROM LEUKEMIA AND CANCER PATIENTS

Florence B. Seibert,** Frances K. Farrelly and Catherine C. Shepherd Mound Park Hospital Foundation, Inc. St. Petersburg, Fla. INTRODUCTION Bacteria have been found to be present in tumors and in bloods, especially of patients with leukemia, by many investigators, including Diller,”* Werthele-Ca~p6,~ and Alexander-Jack~on,~ and the present report adds further confirmation. An etiological role has been strongly suggested by the data of Diller;’ and therefore a search is urgent for methods to avoid or eliminate their presence from the body. Any such reagent would first have to be proved effective in in vitro destruction of the bacteria. The study will, therefore, describe the properties of such bacteria and the successful search for an effective antibacterial agent, which has proved to be DMSO, a compound reported to be relatively nontoxic to blood elements and body constituent^.^-^ METHODS

An antigenic polysaccharide, “polysaccharide II”, isolated from tuberculin

Abstract 1. 1. A polyglucosan, with a very low nitrogen content, a relatively large particle weight, and forming opalescent solutions in water has been isolated from several strains of human and bovine type tuburcle bacilli. 2. 2. Although it consists mainly of glucose, it is not a glycogen but may be similar to a dextran structurally. It is able to elicit precipitins to itself and it gives a high precipitin titre with a horse antiserum and lower titre with some tuberculous rabbit and human sera. 3. 3. It did not induce a significant immunity in a small number of rabbits, nor did it cause a tuberculin reaction in patients who were sensitive to the protein.

Correlation of Extent of Tuberculosis with Amount of Polysaccharide in the Serum.

An intensive study has been made in recent years of tuberculous sera, with the objective of detecting the presence of antigen, antibody or any substance which may be characteristic of the disease. The primary approach has been based upon observation of the electrophoretic diagrams of sera by means of the Tiselius technic. It was shown 1 that during the development of tuberculosis in the rabbit the beta globulin fraction increased in proportion to the extent of the disease. In the human being, on the other hand, the consistent change 1 was a progressive increase in the gamma globulin. Increases in alpha and beta globulin fractions also frequently occurred but not with the same degree of regularity as was the case with the gamma globulin. An explanation of the cause of the rise in any of these components would be of value in the understanding of the disease. The possibility of the presence of antibodies immediately presents itself in connection with the rise in the gamma globulin, especially in view of the fact that antibodies to the tuberculin protein have been to be present in this fraction in sensitized rabbits. However, there could not be shown to be sufficient antibody to account for the total rise in the gamma globulin. In seeking for other explanations of the rise, one is impressed with the idea that if tuberculin polysaccharide were found free in the serum, it would migrate in electrophoresis with approximately the same mobility as the gamma globulin.3 Analyses were, therefore, made of the polyssacharide content of the albumin and the gamma globulin fractions isolated from several sera by means of electrophoresis. The polysaccharide was determined directly on the protein solutions, without preliminary hydrolysis, by means of the carbazole reaction,4 as outlined previously, 6 using the Evelyn colorimeter.

Electrophoretic Identification of Antibody to Tuberculin Protein.

Conclusion Antibody to tuberculin protein has been identified in serum as a component with an electrophoretic mobility close to α-globulin. This component increases in sera following sensitization to the protein, and is removed in part with the specific precipitate produced by adding antigen to the immune serum.

Pyrogens from an Historical Viewpoint

In 1920 fevers that followed intravenous transfusions posed difficult problems for the surgeon, and the use of triple‐distilled water for the preparation of such solutions was recommended on an empirical basis.

A standardised tuberculin (purified protein derivative) for uniformity in diagnosis and epidemiology

Summary o (1) The use of tuberculin as a diagnostic agent in epidemiology and clinical practice has increased considerably in recent years. With the further drop in infection incidence that may be expected, a still greater diagnostic value is to be anticipated. (2) In view of this probable increase in the use of tuberculin, standardisation permitting more accurate comparison of data than is at present possible with the variable preparations on the market, seems timely. (3) Old Tuberculin, a sample of which has been arbitrarily selected as an International Standard, logically might receive first consideration as a basic substance for a standard tuberculin, because of its proven specificity and long history of successful application in epidemiology. (4) Old Tuberculin, however, cannot be made uniformly, because of inherent inconstancies in its manufacture. The various preparations of Old Tuberculin are inevitably solutions of unknown strength of an active principle. Therefore it is logical to separate the active principle from Old Tuberculin and use this substance in solutions of known strength. (5) The active principle of Old Tuberculin can be isolated by a method outlined in the text of the is article, and put up in dry form such that the essential requirements of a standard, viz., specificity, high potency, constancy in strength and stability, are ensured. The active principle as it exists in Old Tuberculin is a protein derivative, the exact chemical composition of which is under investigation. In its present refined form it has been designated simply Purified Protein Derivative from tuberculin. (6) Optimum dosages of this substance for epidemiological work and testing of child and adult tuberculous patients, have been established on the basis of several thousand tests. The doses selected are 0·00002 mg. by weight of the Purified Protein Derivative for the initial test, and 0·005 mg, for injection of those failing to react to the first dose. Experience has shown that the small first dose detects all the highly sensitive cases, without danger of excessively severe reactions, while the second dose defects the remainder of positive cases, that is, those whose sensitiveness is such that they would respond to the conventional doses of preparations of tuberculin of strength equal or superior to that of the International Standard. (7) Several series of tests in the United States, England and Wales, are here reported, in some of which comparison with Old Tuberculin and X-ray findings is recorded. The X-ray examinations indicate that the Purified Protein Derivative, like other tuberculins, may fail to defect an occasional case of healed primary tuberculosis with calcified foci, but that cases of clinical significance are not missed. (8) The incidence of positive tuberculin reactions to the Purified Protein Derivative in surveys in England and Wales varied from 38 per cent. in children 16 years of age or less in an orphan asylum, to 100 per cent. in a tuberculosis sanatorium for adults. Almost all adult patients with tuberculosis reacted to the small first dose. (9) In the United States during the fall of 1934 the Purified Protein Derivative was used in testing about eight thousand college students in various parts of the country. The results are significant, because, for the first time, the tuberculin used on various groups of college students was uniform. In the east about 50 per cent, of the students reacted positively in the middle-west 25 to 30 per cent, and on the west coast about 50 per cent.

Nucleic Acids and Their Derivatives in Alcoholic Extracts of Rat Tumors and Normal Muscle.

Summary Spectral absorption analyses on alcoholic extracts of tumors revealed that they had a higher specific density/mg of nucleic acid than did similar extracts of normal muscle or than pure DNA or RNA. This high specific density, as well as type of absorption curve, suggested the presence of guanosine or deoxyguanosine. Paper electrophoresis experiments, followed by study of paper strips for the position of ultraviolet-absorbing components, and for presence of ribosides by an orcinol spray, or of deoxyribosides by cysteine: H2SO4 spray, led to the conclusion that tumor extracts contained chiefly deoxyribosides, while muscle extract contained ribosides. The small amount of deoxyribosides in normal muscle extracts in contrast to tumor extracts was supported by chemical analyses and by highly specific bio-assay methods. Moreover, by these methods the relative amounts of purine deoxyribosides and pyrimidine deoxyribosides in tumor extracts differed from those in normal muscle extracts, suggesting that different nucleic acid derivatives exist in tumor and the host muscle tissue.

Chromatography of α-∊-Diaminopimelic Acid on Starch Columns.

Summary The new amino acid, discovered by Work, α-∊-diaminopimelic acid, is absorbed on and eluted from starch similarly to lysine by the solvents 1:2:1 n-butyl-n-propyl-0.1 N-HCl and 2:1 n-propyl-0.5 N-HCl. It migrates in electrophoresis in barbiturate buffer pH 8.6 between glutamic acid and lysine but close to proline, whose isoelectric point is near to that of the diaminopimelic acid.

Complement Fixation by Tuberculopolysaccharide.

Summary 1) High complement fixation titers with PPD-S were obtained with BCG antisera which showed high precipitating antibodies to PPD-S and to tuberculo-polysac-charide. 2) Polysaccharide fixed no complement even in these sera when incubation was carried out at 37°C. However, at 4°C fixation did take place. 3) When polysaccharide was mixed with PPD-S the complement-fixation titer was decreased from that obtained with the PPD-S alone, suggesting inhibition of fixation at 37°C. However, this decrease appeared to be due to fixation by polysaccharide in the first stage, followed by a probable release of the complement to the red-blood-cell-amboceptor system in the second stage.