In vitro and in vivo reactivity and specificity of fractions from sonicates of Mycobacterium tuberculosis separated by gradient acrylamide gel electrophoresis.

Fractions of cell sonicates from the H37Rv strain of Mycobacterium tuberculosis prepared by gradient acrylamide gel electrophoresis (GAGE) were examined by immunoelectrophoretic analysis and tested for biologic reactivity and antigenic specificity. Each was tested for the ability to elicit delayed type hypersensitivity reactions in the skin of guinea pigs sensitized with M. tuberculosis, M. kansasii, M. intracellulare (Battey), M. scrofulaceum (Gause), and M. fortuitum. Peripheral lymphocytes from persons infected with mycobacteria were also exposed to fractions, and the increase in [3H]thymidine incorporation into DNA was used as an index of stimulation (lymphocyte transformation). Only 4 fractions (6, 11, 12, and 16) were found to be significantly more specific than the unfractionated sonicate, and none was monospecific for M. tuberculosis. The results of fused rocket immunoelectrophoresis suggested that incomplete separation of antigens and/or shared group-specific multideterminant antigens may have accounted for the observed cross-reactions.

HLA-Bw15 and tuberculosis in a North American black population.

Peripheral blood lymphocytes from 60 black patients with Mycobacterium tuberculosis disease proved by culture and 100 healthy black persons were HLA phenotyped using a standard lymphocytotoxicity test. Statistical analysis of the results showed that only Bw15 antigen had a significantly increased frequency among patients with tuberculosis when they were compared to healthy persons. Furthermore, examination of disease manifestations among patients with tuberculosis demonstrated that patients with Bw15 had significantly more far-advanced pulmonary disease with cavitation than did patients without Bw15. These observations suggest that Bw15 may influence both susceptibility to tuberculosis and the course of the disease.

Cell-mediated immune responses in sarcoidosis.

In an attempt to understand divergent observations regarding the in vitro lymphocyte responsiveness to antigens and mitogens, the frequency of cutaneous reactivity, and the relationship of these variables to clinical features of sarcoidosis, we examined cell-mediated immune responses in 75 untreated patients. In vitro lymphocyte responses to phytohemagglutinin and concanavalin A were significantly decreased among the group of patients with chronic active disease. In vitro lymphocyte responses to streptococcal antigen paralleled the patient's cutaneous reactivity; however, when compared to healthy persons, patients with sarcoidosis responded significantly less often to streptococcal skin tests. Twelve of 13 persons expected to be tuberculin positive by clinical history were found to be dermally reactive, and 11 these 12 exhibited positive in vitro lymphocyte responses to purified protein derivative. Among the remaining 62 patients, failure to respond to all stimuli in vivo and in vitro occurred in only 10. No evidence was found to support the general view that sarcoidosis is characterized by anergy to specific antigens. The patterns of responses to mitogens were neither characteristic nor unique for sarcoidosis. In patients in whom diminished specific cellular immune reactivity was observed, no correlation was evident with specific clinical features of the disease.

Comparison of antigens in sonic and pressure cell extracts of Mycobacterium tuberculosis.

Comparisons were made of the yield, chemical content, and biological activity of filtrates and extracts obtained by sonic and pressure cell disruption of bacilli from 4- and 8-week-old Proskauer and Beck cultures of the H37Rv strain (TMC no. 102) of Mycobacterium tuberculosis. The culture filtrates were dialyzed, freeze-dried, reconstituted in saline, and sterilized by membrane filtration. The viable bacilli were washed and resuspended in distilled water and subsequently disrupted either by sonication in the cold for 15 or 30 min or by treatment at 20,000 or 40,000 lb/in2 in a pressure cell. The resulting extracts were clarified by centrifugation, concentrated, and sterilized by filtration. All preparations were adjusted to contain 10 mg of solids (dry weight)/ml and were analyzed quantitatively for protein, deoxyribonucleic acid, ribonucleic acid, polysaccharide, and lipid content. Separation patterns obtained by gradient acrylamide gel electrophoresis, as well as by one- and two-dimensional immunoelectrophoresis, provided the basis for qualitative comparisons of the culture filtrates and cell extracts. Three-point dose-response curves also were used to compare the preparations for skin test reactivity in BCG-vaccinated guinea pigs. It was concluded that, although there were no consistent differences in chemical content or biological activity between the preparations, a 15-min sonic treatment appeared to be the most suitable method for preparation of bacillary extracts based on yield of active components and ease of preparation.

Rifampin and cell-mediated immune responses in tuberculosis.

To estimate the potential adverse consequences of rifampin therapy on cell-mediated immunity in tuberculosis, we measured in vitro lymphocyte responses to phytohemagglutinin, concanavalin A, pokeweed mitogen, and in vitro and in vivo responses to purified protein derivative tuberculin. Thirty-seven patients treated with therapeutic combinations containing rifampin were compared with 13 persons who had never received the drug. After initial improvement, responses to phytohemagglutinin became depressed in patients receiving rifampin for periods of 4 to 24 months. No significant changes were noted in lymphocyte responses to concanavalin A or pokeweed mitogen. In vitro and in vivo responses to purified protein derivative tuberculin were not altered. Because a favorable therapeutic outcome was achieved in all subjects, we concluded that rifampin does not have clinically significant immunosuppressive activity.

Immunoelectrophoresis of Mycobacterium tuberculosis antigens. Comparative analysis of cell extract and culture filtrate antigens.

Immunoelectrophoretic analyses were performed on a cell extract and culture filtrate of Mycobacterium tuberculosis, H37Rv strain using homologous polyvalent goat antisera. General similarity between the cell extract and culture filtrate antigens was found. Significant variation between two anti-culture filtrate antisera was observed. These antigens and antisera also differed significantly from previously described reference reagents. These findings demonstrate the necessity of using reference materials for precise identification of mycobacterial antigens in comparative studies.

Tuberculin-induced lymphocyte transformation and skin reactivity in monkeys vaccinated or not vaccinated with Bacille Calmette-Guérin, then challenged with virulent Mycobacterium tuberculosis.

The in vitro lymphocyte transformation test was compared to the skin test at intervals after aerogenic administration of bacille Calmette-Guerin vaccine to monkeys, and also at monthly intervals after aerogenic challenge of monkeys vaccinated and not vaccinated with virulent strain H37Rv of Mycobacterium tuberculosis. Individual responses varied, but several consistent patterns of sensitivity development could be discerned. The lymphocyte transformation test was more sensitive, and often positive when the skin test was negative, doubtful, or feeble. Conversion to tuberculin reactivity was detected by lymphocyte transformation in vitro earlier in the disease or after vaccination, and persisted longer after sensitivity to the skin test had waned or after the animals had become anergic by the skin test. Monkeys not vaccinated, but challenged, developed larger in vitro skin reactions and responses than animals that were either vaccinated and challenged or only vaccinated; however, the unvaccinated, challenged monkeys developed anergy to tuberculin and progressive disease more rapidly than other groups, and their cells became less responsive to phytohemagglutinin in vitro.

Mitogen-induced blastogenic responses of lymphocytes from marihuana smokers.

Blastogenic responses in vitro to phytohemagglutinin and pokeweed mitogen were examined in microcultures of peripheral blood lymphocytes from a group of 12 healthy, long-term marihuana smokers and a group of matched control subjects. With either mitogen, no significant difference in cellular incorporation of (3H)thymidine was noted between the groups. These results were interpreted to indicate that the functional status of blood lymphocytes was not altered by long-term smoking of marihuana.

Effects of a morphine-rabbit anti-morphine antibody mixture on guinea-pig isolated ileum.

Incubates of morphine with serum globulins obtained from sera of rabbits immunized with a morphine-bovine serum albumin conjugate produced immediate Schultz-Dale contractions when added to superfused, electrically stimulated guinea-pig ileal strips. Incubates of morphine with Krebs-Henseleit solution produced relaxation and depression of tone, and inhibition of electrically induced contractions. It is concluded that the spasm of guinea-pig ileum produced by incubates containing morphine-binding serum globulins and morphine resulted from transient passive sensitization and an acute anaphylactic type of response.

Technical factors affecting an immunoelectrophoretic reference system for analysis of mycobacterial antigens.

The length of immunoelectrophoretic separation patterns obtained with a reference mycobacterial antigen was reduced significantly when a continuous buffer system was used in place of the recommended discontinuous system. Varying the supporting medium by using 1% concentrations of different agar preparations in the discontinuous system also affected the separation length and resolution of the reference pattern. Measurement of the electrical resistivity of the agar medium provided an explanation of these differences on the basis of current density or field intensity applied per slide. Variation in the reference pattern obtained in differently designed electrophoresis chambers also was attributed to differences in field intensity. To avoid these obvious alterations in the separation pattern of the reference antigen, it was suggested that separation procedures described originally for the reference system be used without modification.