Earlier studies (1, 2) on the tubercle bacillus-monocyte relationship were directed towards an understanding first of procedural problems, a solution to which was essential if the "natural history" (in a tissue culture type of environment) of the fate of parasitized monocytes was to be recorded. Preliminary trypsinization of rabbit monocytes and employment of 40 per cent rabbit serum in Tyrode's solution created conditions in which uninfected suspensions of monocytes exhibited little change in cell numbers over a 72 hour period in the Mackaness type of culture chamber. It was then possible to demonstrate clearly that monocytes derived from BCG-vaccinated rabbits bathed in variously derived antisera constituted a system highly immune to the normally destructive action of the virulent tubercle bacilli and further, that whereas the action of the antiserum was relatively non-specific, the resistance evidenced by the monocytes from vaccinated rabbits was of a greater degree of specificity.The present paper describes the cross-immunity conferred on monocytes by immunization of the host against infection either by Mycobacteriurn tuberculosis or Brucella melitensis. Data are presented which describe the ability of the monocytes from specifically immunized and normal animals to handle ingested virulent Brucetla, and finally, observations are described which show that absorption of the agglutinating antibody does not diminish the enhancing action of the anti-Brucella rabbit serum. Materials and MethodsMonocytes.--An adult normal rabbit or one which had been vaccinated with the Brucdla vaccine (3) or with BCG was used as a source of normal or immune monocytes.

Studies of the mechanism of immunity in tuberculosis have focused attention upon a number of interesting but rather controversial issues. One of these is concerned with the relative importance of humoral and cellular factors in immunity.The role of mononudear phagocytes in the destruction of human tubercle bacilli in the natively resistant animal has been reported by Lewis and Sanderson (1). Histological and cultural studies by Lurie (2) of the fate of tubercle bacilli contained in normal and immune phagocytes cultivated in the anterior chambers of the eyes of normal rabbits have suggested a greater capacity of the mononuclear phagocytes of the tuberculous animal to inhibit multiplication of tubercle bacilli. The recent experiments by Surer (3) appear to confirm Lurie's conclusions that the mononudear cells of immune animals are peculiarly and specially endowed with the capacity for handling virulent tubercle bacilli. Investigations by Mackaness (4), on the other hand, revealed no significant differences in the behavior of normal and immune monocytes toward virulent strains of Mycobacterium tuberculosis.Although various reasons may account for these diametrically opposed findings, it seemed likely that a major contributing factor was the absence of any quantitative enumeration of the monocyte population used in the experiments. Since the conclusions are based on the numbers of intracellular bacilli per total monocyte population, it is axiomatic that either a decrease or an increase in cell population would markedly distort the final results.The present investigation is an attempt at a more precise quantitative analysis of the problem. The survival of cell populations when cultivated in vitro under various conditions in the Mackaness type of chamber is described. Data indicating a degenerative effect of virulent tubercle bacilli upon normal and immune cells cultivated in vitro and an inhibitory or delaying effect of sera of animals vaccinated with BCG upon this cell degeneration are also presented.

Earlier work on the bacterium-bacteriophage reaction (1) has stressed the importance of bacterial growth as a conditioning factor for phage production. It was found that the rate of phage formation could be expressed in terms of the rate of bacterial reproduction and from this single differential equation there were derived integral forms predicting satisfactorily the time of lysis, number of bacteria present when lysis begins, etc. In the basic equation the rate of phage production was expressed as a power of the rate of bacterial reproduction; i.e., in a mixture of phage and growing bacteria the ratio of phage to bacteria continually increases. Finally, when a certain threshold value is attained (100-140 activity units per bacterium) the process of cellular dissolution or lysis begins. Other workers have since reported practically identical kinetic mechanisms for other phages and other bacteria (2-3).Despite the fact that the available experimental evidence indicated bacterial growth to be the prime conditioning factor for phage production there remained the possibility that under some conditions the two phenomena might be dissociated. This possibility was confirmed by Scribner and Krueger (4) in an investigation of the reaction between phage and susceptible bacteria in the presence of 0.25 molar NaC1. Under these special conditions a prolonged maximal stationary phase of bacterial growth occurred and during this time phage continued to be produced at the usual rate.We have conducted further experiments dealing with the effect of temperature and pH on the phage-bacterium reaction, and have

Studies of passive transfer of cellular resistance, as manifested by refractoriness to necrotization with virulent tubercle bacilli, have shown that immune histiocytes or immune lymphocytes were effective transferring agents; immune polymorphonuclear leucocytes and immune serum as well as comparable cells from normal animals lacked this capacity. Comparisons of immune histiocytes and immune lymphocytes showed that the former cells were more efficient; this was indicated by (a) the smaller numbers of immune histiocytes needed for passive transfer, (b) the longer duration of cellular resistance in recipients given histiocytes than in those given lymphocytes, (c) the greater capacity of histiocytes to effect serial passive transfer, and (d) the ability of histiocytic but not lymphocytic lysates to transfer cellular resistance. Experiments to establish the mechanism of passive transfer of cellular resistance showed that there was no active induction of resistance in recipients through transfer of bacillary antigens contained in immune histiocytes; in fact, the results of serial passive transfers with immune histiocytes suggested an active replication of the "cell resistance factor."

Despite intensive research on tuberculosis and the tubercle bacillus, the important and basic problems of virulence and resistance remain a challenge.Studies of acquired immunity have revolved around the relative importance of humoral and cellular factors. As previously indicated (1), valid conclusions concerning the role of cells in acquired immunity against tuberculosis require analyses of bacterium-cell relationships during periods when the in dtro cell populations show no change in cell numbers. Under such conditions, it was observed that the virulent H37Rv strain of tubercle bacillus regularly induced degeneration of both normal and immune monocytes (derived from rabbits immunized with BCG) when these were cultivated in vitro in the presence of normal rabbit serum (derived from tuberculin-negative rabbits); in contrast, the serum of rabbits immunized with BCG protected immune but not normal cells against the degenerative effects of virulent tubercle bacilli. The first of these observations posed a possible relationship between virulence and the factor in tubercle bacilli which promoted degeneration of cells cultivated in vitro. The protection of immune monocytes by the serum of rabbits immunized with BCG suggested an hitherto unexplored role for serum components in resistance against tuberculosis.The present paper represents an investigation along the lines suggested by these earlier observations. It describes the different abilities of virulent, attenuated, and avirulent strains of tubercle bacilli to induce degeneration of monocytes cultivated in vitro. It also describes the activity of bacterial culture filtrates in promoting cell degeneration and compares this activity of bacterial filtrates with those of purified protein derivative and old tuberculin. Finally, it presents data which indicate that an element of specificity is involved in the resistance of monocytes against virulent tubercle bacilli and further, that such resistance is mediated by serum components which are seemingly non-specific in nature.

Although various observations (1-5) have failed to relate serum antibody to resistance against tuberculosis, these findings need not preclude a role for humorM factors in an as yet indeterminate and possibly rather complex pattern of resistance. The participation of humoral factors in resistance to tubercle bacilli was indicated by the observation that the sera of animals immunized with the BCG strain of tubercle bacillus contained a substance which protected the monocytes of these immunized animals against the necrotizing action of virulent tubercle bacilli (6). While the protective substance was found in immune and not in normal sera, its activity was seemingly non-specific in nature, for the immune sera of animals immunized with antigens totally unrelated to the tubercle bacillus (Salmonella, Brucdla, ovalbumin) proved equally effective in protecting the monocytes of BCG-immunized animals against virulent tubercle bacilli (7, 8). It was also found that these immune sera protected the monocytes of the BCG-immunized animal but failed to afford similar protection to normal monocytes (6).Studies of cellular resistance showed that it was also not entirely specific, since, in the presence of immune sera, the monocytes of animals immunized with either Mycobacterium or Brucdla proved partially or completely resistant to heterologous as well as homologous infection (8). Despite the seeming lack of specificity suggested by the cross-immunity between the cells of animals immunized with Brucella and Mycobacterium some selectivity of action was demonstrable in cells; thus, while both anti-Brucdla and anti-Salmonella sera contained a substance which protected the cells of BCG-immunized animals against virulent tubercle bacilli, the monocytes of the Brucella-and Salmonellaimmunized animals behaved quite differently in that the cells of the Salmonellaimmunized animal proved completely susceptible to virulent tubercle bacilli (7).

No abstract

The various cellular components of immune rabbit histiocytes have been analyzed for their ability to induce cellular resistance in normal animals. The results of these investigations have shown that the nuclear and mitochondrial fractions were inactive and that the microsomal and ribosomal fractions were active. The importance of ribonucleic acid in induction of cellular resistance was established by isolation of an active ribosomal RNA and by demonstration of inactivation of this material with ribonuclease but not with deoxyribonuclease or trypsin. The possibility that viable bacilli were present in immune ribosomes was tested; the absence of complement-fixing antibodies and of skin reactivity to tuberculin in animals inoculated with ribosomes was considered as partial evidence of absence of living bacilli.