Tissue culture methods have been but little used in the elucidation of the various problems encountered in bacterial hypersensitivity. The few studies reported have dealt with tissues from tuberculin sensitive animals. Rich and Lewis (1) showed that tuberculin in proper concentration had a selective toxic effect on cells from tuberculous animals. They believed that the tuberculin sensitivity was inherent in the cells, and that the cytotoxic effect was the result of an antigen-antibody reaction. Aronson (2) confirmed this work and also demonstrated the cytotoxic specificity of various tuberculins on sensitive cells as compared with an indifferent effect of extracts made from other acid-fast organisms. In another series of experiments he (3) compared the reaction of tissues from animals sensitized to proteins with that of tissues sensitive to tuberculin when the respective antigens were added to the culture media. Horse serum added to cultures of tissues from horse serum sensitized animals produced no demonstrable cytotoxic effect; this was in marked contrast to the cytotoxic action of tuberculin on tissues from tuberculous animals; he thus showed that there is a fundamental difference between the two types of hypersensitive tissues. We (4) have also noted that cells from animals sensitized with horse serum, egg albumin or beef lens were not specifically inhibited when the respective antigens were added to tissue cultures. As a preliminary to an analysis of other types of bacterial hypersensitive states it was thought advisable to make a detailed study of tuberculin allergy since this is a prototype. This communication deals, therefore, with a partial repetition of previous work with amplifications and extensions.
1. Most isolated guinea pig mononuclear exudative cells in tissue culture become typical migrating macrophages, but a small proportion take on fibroblastic characteristics, and produce pure colonies of fibroblasts. These fibroblasts maintain their morphological characteristics through repeated subcultures. 2. It is suggested that the subsequent development of individual mononuclear cells in tissue culture is conditioned at the time of explantation. 3. Apposition with other cells is not necessary for the initiation of mitotic cellular division. 4. There is a definite optimal relationship between the bulk of the medium, the number of explanted cells and the extent of proliferation. The presence of other cells in the vicinity enhances cellular division. 5. Mitosis in the isolated explanted cell is preceded by a latent period. The rate of division varies in different colonies of fibroblasts. 6. Admixed erythrocytes in the mononuclear suspension definitely inhibit proliferation of fibroblasts in tissue culture. The inhibiting factor in disintegrating erythrocytes is apparently present in the stroma.
Observation of the specific toxic effect of bacterial products on living sensitive cells in tissue culture offers a direct approach to a study of certain phases of bacterial allergic or hypersensitive states. In a preceding study (1) tuberculin was shown to have a specific toxic effect on sensitive cells from animals infected with several strains of tubercle bacilli having various degrees of virulence.This communication presents the results of an investigation by the tissue culture technique, of hypersensitive states associated with infection by a different species of microorganism, namely the hemolytic streptococcus. This permits of a comparison of cutaneous reactivity, antibody production and the development of cellular susceptibility to the toxic action of certain bacterial products in vitro. A comparison with some features of tuberculin allergy can thus be made. E~ER~NTALAnimals.--Albino guinea pigs, mostly males, weighing between 300 to 450 gin.were used throughout, because the strains of hemolytic streptococci employed were natural pathogens for these animals.Hemolytic Streptococci.--Three strains of hemolytic streptococci, K 104, K 64 and J 20, all isolated from epidemics of spontaneous guinea pig lymphadenitis, and all belonging to group C of Lancefield's serological classification (2) were used. In most experiments strain K 1041 was employed. The inoculum consisted of 0.1 cc. of an 18 hour broth culture injected subcutaneously in the inguinal region.
1. Mononuclear exudative cells, obtained from tuberculous guinea pigs by the intrapleural injection of parowax, exhibited characteristic sensitivity to the toxic action of tuberculin when tested in tissue culture. 2. Experiments with these cells, practically free of body fluids, show conclusively that sensitivity to tuberculin is an inherent characteristic of mesenchymal cells from tuberculous animals. 3. Fibroblastic growths which developed from mononuclear exudative cells derived from a tuberculous animal showed persistence of sensitivity to the toxic action of tuberculin on repeated transplantations over a prolonged period in vitro.
Numerous studies have been made on the relation of fractional blood protein changes to bacterial infection and immunity. The subject is of especial importance in view of the renewed interest regarding the association of immune bodies with the globulins and recent studies in regard to the effects of blood protein changes and plasma viscosity on specific and non-specific agglutination reactions (1, 2). Since the early experiments of Langstein and Mayer (3), it has been generally found that a relative or absolute increase of the globulin fraction and a decrease in the albumin fraction occur during and after various bacterial infections. Glaessner (4) and later Hurwitz and his co-workers (5) showed that increases in globulin may occur during the course of immunization as well. They believed that the globulin increase indicated disturbed metabolism and that no direct parallelism existed between the amount of globulin and the degree of immunity. There was no constant relationship between the increased amount of globulin and the antitoxic potency of prepared sera. Similar findings were noted by Schmidt and Tuljtschinskaja (6) and others. Studies on fractional blood protein changes in malaria, typhoid fever and after typhoid vaccination were made by Lloyd and Paul (7). They observed a marked decrease in the albumin fraction, an increase in the euglobulin and slight or no changes in the total globulin during the acute stage of typhoid fever and malaria. Similar but less marked changes followed vaccination with typhoid bacilli.Many studies have been made on the blood protein changes in lobar pneumonia. Kumpf (8) collected data from 29 reports. It was generally agreed that the total protein was lowered, the fibrinogen and globulin increased and the albumin decreased in amount. No particular attention, however, was given to the speed with which changes in plasma proteins occur after the onset of infection or the time required before the normal equilibrium was regained.Increase of plasma globulin and fibrinogen during infections causes a decrease of the suspension stability of the blood and consequent reduction of the sedimentation time of erythrocytes. The correlation between plasma protein changes and sedimentation time has been demonstrated 589
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