Biomedical subjects
Publications and source records attributed to A H COONS.
As the interval between two antigenic stimuli to mice is lengthened, the magnitude of the response to the second dose increases up to approximately 40 days; thereafter it remains at a fairly constant, high level.
The effect of drugs upon the primary and the secondary antibody response to diphtheria toxoid in mice was studied using an experimental system previously described. Triethylenethiophosphoramide (thio-TEPA), chloramphenicol, 6-mercaptopurine, 8-azaguanine, and versenate were found to inhibit, partially or completely,"priming" for the secondary response. Thio-TEPA, chloramphenicol, and 6-mercaptopurine, in doses exceeding those effective in inhibiting priming, did not cause alteration of the secondary response when given only during the secondary response. However, when chloramphenicol and amethopterin were given for 5 days prior to and at least 5 days after the second antigen injection, slight suppression of peak secondary titers occurred. Therefore, drug dosages effective in suppressing priming had less effect on the secondary response. It thus appears that there is a real difference between "priming" and the induction of antibody synthesis.
Young adult mice were primed with 20 Lf (56 microg) of diphtheria toxoid and given a second injection of the same size 40 days later. This procedure produces a reproducible secondary response which can be used as a standard. Chloramphenicol in maximum dosage prevents the unknown process by which the animal is primed for the second response. To be fully inhibitory, the drug must be given from the hour of the first antigen injection in maximum dosage for 2 weeks. A delay of 48 hours in starting the drug allows completion of the priming process, and shorter delays produce partial inhibition. Hence the initiation of priming is a rapid process sensitive to chloramphenicol. Subsequent changes in the cell population necessary for the full development of priming are not sensitive to chloramphenicol. The secondary antibody response is not inhibited in mice by chloramphenicol at the doses employed.
Cells from lymph nodes of rabbits injected repeatedly with bovine serum albumin were transferred subcutaneously to previously irradiated rabbits, and the recipients were immediately injected with bovine serum albumin. A good antibody response resulted. In a series of such animals killed on successive days, skin samples at sites of cell deposition were removed and examined by immunofluorescence and by light microscopy. In these tissues abundant plasmocytes were found to have multiplied and differentiated in a regular progression from immature, to medium, to mature plasmocytes. During the 6 days of the experiment the small plasmocytes accumulated until they reached 85 per cent of the total plasmocytic population. The mitotic index of the large and medium plasmocytes averaged 11 per cent, implying a generation time of 6.3 hours on the basis of a 1 hour mitotic time. This rate of growth is sufficiently rapid to account for all the plasmocytes on the 6th day as deriving from less than 1 per cent of the population initially transferred. This rate and the orderly progression in the evolution of the plasmocytic population, make it highly improbable that plasmocytes arise from transformation of lymphocytes, but rather indicate that they spring from specific precursors already present among the transferred cells.
The physical presence of the antigen used to stimulate a secondary antibody response was demonstrated in the cells of popliteal lymph nodes. Rabbits previously injected with apoferritin (containing no iron), which was prepared from recrystallized horse ferritin, were given an injection of ferritin 5 weeks later. The antigen was traced by means of the Prussian blue reaction, by specific fluorescent antibody, and by electron microscopy. Antiferritin antibody was localized by immunofluorescence, though it was not possible to test cells simultaneously for antigen and antibody. Horse ferritin induces a rather weak primary antibody response, but a brisk secondary response characterized by the appearance in the medullary cords of numerous plasma cells containing antiferritin. Many intact ferritin molecules were found in the nucleus and cytoplasm of numerous reticular and other phagocytic cells in the sinuses. In decreasing amount, ferritin molecules were also clearly demonstrated in hemacytoblasts (plasmoblasts), and immature and mature plasmocytes.
Human liver sections were stained with anti-human serum albumin and/or anti-human fibrin monomer fluorescent conjugates. Approximately 10 per cent of the hepatic cells stained specifically for human serum albumin,1 per cent for fibrinogen, and 0.1 per cent for both. Approximately 18 per cent of the Kupffer cells stained specifically for human serum albumin and 33 per cent for fibrinogen. Staining of both cell types was mainly cytoplasmic, although albumin was found in the nuclei of some parenchymal cells, depending on the method of fixation. Cytoplasmic granules staining specifically for fibrinogen could be seen just inside the cell membrane facing the bile caniculi in many more parenchymal cells than the 1 per cent showing diffuse cytoplasmic staining. The technical difficulties involved in preparing fluorescent conjugates against these antigens and in the fixation of these antigens for immunofluorescent staining are discussed.
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Rabbits were injected into the hind foot with diphtheria toxoid and bovine serum albumin. Fragments of popliteal lymph node taken from them several months later were placed in plasma-clot cultures with Eagle's medium. When antigen was added to the culture fluid, anamnestic antibody responses occurred regularly. When the antigen was diphtheria, responsiveness remained for 4 days after the beginning of the culture. When it was bovine serum albumin, responsiveness lasted for about 8 days. Once an anamnestic response had begun, antibody formation continued for 4 weeks or more. High concentrations of bovine serum albumin (0.5 mg/ml) did not inhibit the response. When both antigens were used to stimulate the same culture, it was found that the two responses were independent.
When lymph node fragments from previously immunized rabbits were stimulated in vitro to produce a secondary response, the continuous presence of 50 microg/ml (0.15 mM) of chloramphenicol in the medium during the entire incubation period of 15 to 21 days produced nearly complete suppression of the response. Concentrations as low as 5 microg/ml (0.015 mM) produced approximately 80 per cent suppression of the response. When 50 microg/ml of chloramphenicol was present during only the first 6 days of culture, the secondary response was reduced 90 per cent. When it was absent for the first 6 days but present for the next 9 to 15 days, the response was reduced only 40 per cent. Since over 95 per cent of the antibody of the secondary response in most experiments appeared in the medium after the 6th day, chloramphenicol apparently inhibits antibody production by interfering with some early phase of the response. It is suggested that this interference involves messenger RNA and that animal cells have appeared resistant to this drug only because their complement of messenger RNA present when the drug has been added is stable over the short periods during which protein synthesis has usually been studied.
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