Hans Zinsser

Bacterial allergy and tissue reactions.

The bacterial allergies, though they are phenomena of relatively specific hypersusceptibility, differ in certain fundamental respects from true protein anaphylaxis. Our own investigations on this subject have dealt chiefly with the tuberculin reaction in regard to which it seems safe to state that: 1. Tuberculin allergy as manifested by the intracutaneous reaction may occur without general anaphylaxis to tuberculoprotein, and vice versa. 2. The active principle of tuberculin which elicits reactions in the sensitive subject is not a protein. 3. Tuberculin sensitiveness seems inseparably associated with the reactions of the animal tissues to the tubercle bacilli or their constituents; in other words, after prolonged experimentation, we arrive again at the recognition that there is no tuberculin sensitiveness without a tubercle. Here again the fact that the tuberculin reaction is merely the classical example of the bacterial allergies in general is apparent in that our own work with other bacteria as well as especially that of Fleischner and Meyer with bacillus abortus has indicated that bacterial allergy is associated with infection rather than with the type of immunization with soluble bacterial substances which leads to antibody formation without necessarily active focal inflammatory reactions. In the case of the tubercle bacillus Petroff and the writer have shown that the injection of the dead bacilli induces tuberculin allergy quite effeotively, probably because of the insolubility of these organisms, in consequence of which the dead organisms lead to tubercle formation essentially analogous to that induced by the living. This point, apart from its possible practical imterest, is theoretically significant in that it indicates that, in the association of bacterial allergy with infection rather than with irqmunization, it is the tissue reaction which is of importance rather than any differences in the products which pass into the infected animal from dead and living bacteria respectively.

The chemotherapy of urease- and citrase-producing bacteria of the urinary tract.

Excerpt INTRODUCTION Beginning with the premise that magnesium ammonium phosphate stones formed in the urinary tract were primarily the result of the enzymatic urea-splitting capacities of some typ...

Agar Slant Tissue Cultures of Typhus Rickettsiae (Both Types)

For some time the writers have been engaged in extorts to obtain typhus fever Rickettsise cultures on a scale sufficiently large to permit vaccine-production. While the X-rayed-rat technic, elsewhere published by one of the writers with Castaneda, 1 is entirely adequate for vaccine-production with the murine strains, it has consistently failed with the classical European type. Tissue-culture vaccine-production would undoubtedly be the most practical method of producing vaccines on a large scale against the European disease, and the effectiveness for immunization of formalin-killed tissue-culture organisms has already been demonstrated, both by Kligler and Aschner 2 and by one of the writers with Macehiavello. 3 Work done on the physiology of Maitland cultures by one of the writers with Schoenbach 4 indicated that whereas virus agents multiply in such cultures only during the period of active tissue metabolism—the Rickettsise begin to multiply most profusely after metabolism has slowed down or ceased. This encouraged the hope that it might be possible to develop a method of Rickettsia multiplication in which larger amounts of tissue—and consequently a larger yield of the organisms—could be used. The problem was to find a technic in which the tissue could be held to a minimum of metabolic activity under circumstances of controlled pH and retardation of autolytic change. Along these lines of effort we have been able to obtain large yields of Riekettsise of both the murine and classical (European) types by the following method : A 4% agar solution in water is mixed with an equal volume of double strength Tyrodes solution to which 50% horse serum has been added before filtration. Phenol red is added in order that the reaction of the tubes may be observed. The initial reaction should be approximately pH 7.4 to 7.6. Guinea pig tunica is minced, as for Maitland culture, and is heavily inoculated by dropping the centrifugalized concentrate of a preceding culture on the tissue in a Petri dish. After 5 or 10 minutes at room temperature, this tissue is then “buttered” on the surface of the agar, and the tubes are closed with a rubber stopper through which there has been passed a glass tube drawn to a capillary, as shown in Fig. 1. The purpose of the glass tube is that by either breaking or sealing the capillary and allowing any formed carbon dioxide to escape or to accumulate, the reaction can be to some extent controlled, if that becomes necessary. The most important single point in the technic essential to success is reaction-adjustment, and it seems that in most cases, if the initial reaction is as stated, adjustment by the capillary is not necessary in the course of the 8 or 10 days necessary for development of the cultures. At the end of 6 days, multiplication is already apparent and after 10 days smears from the tissue on the surface of the agar show growth of Rickettsise. The organisms not only crowd many of the cells which are reasonably preserved, but considerable numbers of scattered Rickettsise are found throughout the tissue detritus. Whether or not these actually grow in the diffused cell material or represent the yield of ruptured cells has not yet been determined. At any rate, a single culture of this kind gives a better total yield than three or four of the larger Maitland flasks. It is still to be determined why large amounts of tissue on slanted agar do not inhibit Rickettsial growth in the same way in which even a slight excess of tissue prevents multiplication in liquid cultures, even when reaction-changes are controlled. It is probable that since active metabolism seems to delay rather than favor Rickettsial multiplication, the cells laid on the surface of agar may, from the beginning, exhibit a very low level of metabolic exchange. In consequence, changes of reaction and the possible production of inhibiting substances take place to a slight degree and slowly, and the products diffuse away from the cells into the agar. Since heavy inoculation is necessary in order to obtain yields suitable for vaccine production—which is the ultimate purpose of the work—we have had to carry these cultures to the 5th agar generation and a dilution, in terms of tissue used—of the original Maitland culture of approximately 1 to 700—before we could be sure we were not dealing with organisms carried over. We are at present engaged in improving methods of controlling the reaction and determining the suitability of embryonic tissue and other cellular material more easily procurable in considerable quantities than guinea pig tunica. Although probably not in its final form, the method appears to us to offer hope of solving in a simple manner the problem of vaccine production for human protection against both the murine and the classical European varieties of typhus fever. It is not out of question that it may be applicable to virus cultivation as well since the cells are no doubt to some extent active for 48 hours.

A TEXTBOOK OF BACTERIOLOGY. The Application of Bacteriology and Immunology to the Etiology, Diagnosis, Specific Therapy and Prevention of Infectious Diseases for Students and Practitioners of Medicine and Public Health

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Further Studies of Agar-slant Tissue Cultures of Typhus Rickettsiæ.∗

In a preliminary note published in these Proceedings, 1 the writers described an agar-slant-tissue method for the cultivation of Rickettsiæ, both of the murine and of the classical types of typhus fever. Further study of this method has led to modifications and standardizations which are reported herewith in order to facilitate its use in the hands of others. Preliminary experiments by Pinkerton with Rocky Mountain Spotted Fever virus indicate a possibility that the method may be successful with Rickettsiæ other than those of typhus. The medium as now used differs from the original only in proportions of ingredients. We have found that results are most regular when the agar is prepared as follows: A “Tyrode” solution of the ordinary formula is made up double strength throughout, except that only 1 gm. of bicarbonate of sodium is used per liter. The agar used is a Difco granular Bactoagar. A mixture of 150 cc. double strength Tyrode and 150 cc. of horse serum with 8 cc. of phenol red solution is filtered through a Seitz filter.† To the filtrate is added 150 cc. of 4% agar in distilled water, cooled to 45°C.

On the Nature of Virus Agents.

3. Francis, T., Jr. A.J.P.H., 27:211, 1937. 4. Francis, T., Jr., Magill, T. P., Beck, M.D., and Rickard, E. R. J.A.M.A., 109:566, 1937. 5. Francis, T., Jr., and Magill, T. P. Proc. Soc. Exper. Biol. & Med., 36:132. 1937. 6. Francis, T., Jr., and Magill, T. P. Ibid., 36:134, 1937. 7. Francis, T., Jr. Science, 80:457, 1934. 8. Muench, H. Unpublished data. 9. Andrewes, C. H., Laidlaw, P. P.., and Smith, W. Brit. J. Exper. Path., 16:566, 1935. 10. Fairbrother, R. W., and Hoyle, L. J. Path. & Bact., 44:213, 1937. 11. Turner, T. B. In press. 12. Hoyle, L., and Fairbrother, R. W. Brit. Med. J., 1:655, 1937. 13. Smorodintseff, A. A., Tushinsky, M.D., Drobyshevskaya, A. I., Korovin, A. A., and Osetroff, A. I. Am. J. Med. Sci., 194:159, 1937.

Notes on the Weil-Felix Reaction in Individuals not Suffering from Typhus

In view of the increasing frequency with which the Weil-Felix reaction is being used in the United States for the diagnosis of obscure fevers suspected of Rickettsia origin, and especially because of our own interest in the possibility of latent typhus infection in individuals infected a long time ago, we undertook to carry out a considerable number of Weil-Felix reactions on individuals not at the time suffering from typhus or fevers of any kind, for the purpose of establishing the significance of low titre reactions. We are reporting these briefly because they seem to us helpful in appraising occasional doubtful cases. The reactions were carried out on sera from several groups of subjects. One group was composed of Jewish out-patients furnished us by the Beth Israel Hospital Clinic in Boston, and in these record was made as to whether the individual was born in Russia or in the United States. Since such information had no significant effect on these observations, we abstain from tabulating it. Another group was composed of non-Jewish subjects, another of routine Wassermann sera in which no attention was paid to the race of the patient; and, finally, a small group of 24 Russian-born garment workers. Table I gives the results. It will be seen that there was no significant difference between the groups, and it is rather fortunate in protecting us against false conclusions that the actual percentage of positive reactions in the Russian-born garment workers was rather lower than in the other groups.

Observations on anaphylaxis in lower monkeys

It is always dangerous to apply to any species of animal reasoning or theories deduced from experimental observations upon another species. In no phase of immunological work is such deduction more unjustified than in anaphylaxis where we know that the reactions induced in different species by a reinjection of proteins vary from each other in fundamental, physiological mechanism. It is of course unlikely, therefore, that we can justly draw conclusions from monkey experiments to conditions prevailing in human beings. But some of those who have regarded the occurrence of true anaphylaxis in the human being as at least doubtful, have, at the same time, cited the difficulty of producing anaphylactic reactions in monkeys in analogy. The problem is hardly one warranting a great deal of extensive research, but in connection with other work going on in our laboratory, we have found it important to investigate, for ourselves, the true conditions prevailing in the lower monkeys. The production of antibodies in monkeys has for some time been a matter of controversy. Uhlenhuth 1 injected human serum into Macacus rhesus and found that specific precipitins were formed. Berkeley, 2 in 1913, reinvestigated this question on Macacus rhesus and on a Java monkey, and found that these animals treated with human, horse or dog sera, receiving four injections of 1 to 2 c.c. of these sera, produced neither precipitins nor complement fixing antibodies for the antigens used. He does not believe, therefore, that it would be possible to utilize antisera from lower monkeys for the forensic differentiation of human and monkey sera, as suggested by Uhlenhuth. There has not been a great deal of systematic work published upon monkey anaphylaxis. Yamanouchi 3 was unable to produce active anaphylaxis in the lower monkeys against horse serum, and found that the serum of the lower monkeys did not sensitize guinea pigs passively.

On certain poisonous substances produced in bacterial cultures

In a paper published by one of us last year we called attention to the fact that it was quite likely that not all the toxic substances produced in cultures by bacteria can be peremptorily classified as either exotoxins or endotoxins. The writers working with a number of different organisms, biologically unrelated, have found poisonous substances of moderate potency developed both on fluid and on solid media which they believe should not be regarded at the present time either as specific or antigenic exotoxins, or as endotoxins. Indeed, it seems quite impossible at the present time to definitely classify these substances, for which reason they are referred to in our laboratory, for the present, as the “X” substances.

Enlarged Tissue Cultures of European Typhus Rickettsiae for Vaccine Production

Our methods of producing typhus vaccines with the murine strains of Rickettsiae has depended upon the inoculation of rats in which resistance had been reduced by a variety of procedures, the most consistently successful of which has been preliminary X-ray radiation. These methods have persistently failed to give adequate results with the European virus. This fact in itself constitutes further strong evidence that the two types of Rickettsiae are distinct and biologically fixed varieties. After much effort to apply the “rat” methods to the organism of the classical European disease we were finally persuaded that other methods of approach must be sought for obtaining accumulations of the European Rickettsiae adequate for practical purposes of immunization. In a paper, now in press, the writers have reported the results of experiments in which the active immunization of guinea pigs against the European typhus virus was accomplished, both with the use of formalinized tissue culture vaccines and by methods of sero-vaccination. While this work was going on, Kligler and Aschner 1 published observations on a method of successful animal immunization with formalinized tissue culture vaccine, similar in all important principles to the method which we are using. Since the development of a tissue culture technique for producing vaccines in amounts adequate for practical purposes appeared the most hopeful direction of effort, we have tried for a long time to improve the technique of making such cultures, particularly in regard to increasing the volumes of the tissue cultures themselves. In studying the general problem of tissue cultures, one of the writers (Zinsser) in collaboration with Schoenbach, turned his attention to observations on what may be called the physiology of tissue cultures in general, particularly as regards changes in pH, oxygen consumption and oxydation-reduction potentials.†