Cellular immunity in vitro. Clonal proliferation of antigen-stimulated lymphocytes.

When sensitive lymphocytes are cultured with the appropriate antigen, lymphoblasts appear after 24-48 hr of incubation and the number of these increases steadily from the 2nd to the 6th or 7th day. Our problem was to discover, at a cellular level, how this increase takes place; whether it is a massive response of many cells, stepwise recruitment of cells into the lymphoblast class, or simply repeated division of a few cells to form clones. In these experiments lymphocytes were incubated with antigen in culture tubes for 2-4 days and then a few cells, usually less than 200, were transferred to special microchambers for further culture. In these microchambers the cells could be viewed continually with a microscope and their fate recorded over the next 3-5 days by time-lapse cinemicrography. Examination of the film produced in this way showed that lymphoblasts divided and redivided to produce clones of 64 cells or more. It was possible to measure generation times from the film for 301 cells; the majority were between 8 and 13 hr but the range was 7.5-38.0 hr. There was no clear difference between generation times of human lymphocytes stimulated with tuberculin, streptokinase-streptodrnase, extract of the American pokeweed, or in the mixed leukocyte reaction. Similar times were also found for rat cells in the mixed leukocyte reaction. While these observations show that clonal proliferation does occur and could reasonably account for all the increase of lymphoblasts in lymphocyte cultures, the experiments, because of their design, do not exclude the possibility that other mechanisms such as recruitment may play a role as well, particularly during the first 48 hr after contact between sensitive cells and antigens.

Requirement of precommitted cells as targets for the augmentation of lymphocyte proliferation by leukocyte dialysates.

After our initial report tha leukocyte dialysates containing transfer factor augment the thymidine incorporation of antigen-stimulated lymphocytes, we have adapted the system to microleukocyte cultures. This modification permits both (a) the simultaneous assay of a single dialysate on the cells of multiple individuals, and (b) the assay of multiple dialysates on the cells of a single individual. The data thus secured, demonstrate that dialysates from both skin-test-positive and -negative donors produced similar degrees of augmentation whether the data are expressed as an arithmetic difference or as a ratio. When expressed as an arithmetic difference, the amount of augmentation is increased in proportion to the level of thymidine incorporation of the assay cells when they were stimulated by antigen alone. When expressed as a ratio, however, the degree of augmentation is independent of the response of the assay cells. An analysis of the ability of dialysates to engage previously uncommitted lymphocytes and thus to augment thymidine incorporation, revealed that precommitted cells were required. In these experiments, antigen-reactive cells were deleted from populations of peripheral blood lymphocytes by incubation with purified protein derivative of tuberculin, diphtheria toxoid, or streptokinase-streptodornase in the presence of [3H]thymidine of high specific activity. This deletion depressed or abolished the effect of dialysate on the residual population when it was recultured with the same antigen, but the effect on the response of the remaining lymphocytes to other antigens was unaltered. In this study, leukocyte dialysate appeared to augment nonspecifically the thymidine incorporation of an antigen-specific precommitted clone of lymphocytes. The relationship of these adjuvant effects on peripheral blood lymphocytes in vitro to the specific and nonspecific activities of transfer factor in vivo remains to be elucidated.

Histocompatibility studies in a closely bred colony of dogs. V. Mechanisms of cellular adaptation in long-term DL-A identical radiation chimeras.

20 Cooperstown beagles of known DL-A genotypes (B" dogs) were exposed to supralethal total body irradiation and received a bone marrow allograft from a DL-A identical donor (A" dog); the resulting chimeras have survived uneventfully for 882, 1466 days, with no evidence of secondary disease, and have been tolerant to kidney and skin allografts obtained from the donor of marrow. Treatment of "A" dogs with serum obtained from their long-term "B" chimeras had no significant effect upon the ability of the recipients to reject "B" skin allografts...

Lymphocyte stimulation: transfer of cellular hypersensitivity to antigen in vitro.

Tuberculin-sensitive human lymphocytes cultured for 36 hours with tuberculin elaborate a soluble material which causes nonsensitive lymphocytes to respond to tuberculin in vitro by transformation and proliferation.

Erythrocytes in human transplantation: effects of pretreatment with ABO group-specific antigens.

Erythrocyte group antigens A and B can act as potent and group-specific transplantation antigens in man. ABO group-incompatible recipients pretreated with such antigens have rejected skin allografts obtained from donors incompatible for the same antigens in an accelerated (4-5 days) or white graft manner. Skin grafts applied to the same recipients from ABO-compatible donors were accorded first-set survival times. Intact erythrocyte suspensions and antigens isolated from hog (A substance) and horse (B substance) stomachs, were equally capable of inducing this type of allograft sensitivity. The latter observation broadens the spectrum of heterologous antigens capable of inducing allograft sensitivity in the mammalian host and provides a readily available, heat-stable, and water-soluble source of antigens for further studies of allograft rejection mechanisms in man.

In vitro epidermal cell proliferation in rat skin plugs.

Full-thickness skin plugs from immature and adult rats have been shown to incorporate -3H-thymidine in vitro in a semisynchronous fashion for up to 54 hr. DNA synthesis is minimal at 16 hr post sacrifice but increases again at 24 and 48 hr, and cells in S phase at 48 hr have passed through at least one mitosis in vitro (between 32 and 39 hr). Advantage can be taken of this semisynchronous burst of DNA synthetic activity to test the effects of potential inhibitors of skin cell proliferation, and to determine at which phase of the cell cyclic these inhibitors act. High concentrations of epinephrine or isoproterenol (10--5 M) are required to cause inhibition but the effect is specific for the G2 phase of the cell cycle. Propranolol does not demonstrate beta-antagonism in this system, since it is a very potent inhibitor acting in G1 phase. Dibutyryl cycle AMP and theophylline have two effects: one is a specific inhibitory action on cells in the G2 phase and the second is a short-term action limiting thymidine uptake by cells in S phase without affecting the transition of cells from G1 to S.

A microculture system for the measurement of antigen-induced murine lymphocyte proliferation: advantages of 5% horse serum and 5 X 10(-5) M mercaptoethanol.

Short term microculture systems which measure murine lymphocyte proliferative responses to mitogens are well established. We demonstrate here that these microculture methods are not suitable for antigen-induced responses because of the high levels of murine lymphocyte proliferation in control cultures associated with the use of fetal calf serum or human serum. We also show that this problem can be eliminated with the use of a combination of 5% horse serum and 5 X 10(-5) M mercaptoethanol. We describe an antigen-induced murine lymphocyte proliferation microculture system in which good stimulation indices are achieved and the lymphocyte proliferation in control cultures remain at a low level throughout the 7 day culture period.

A micromethod for evaluating the phagocytic activity of human macrophages by ingestion of radio-labelled polystyrene particles.

Studies in vitro of human macrophage function in health and disease have been impeded by the difficulty of obtaining such cells in sufficient number. Unlike animal species, the only readily available source of human macrophages are circulating monocytes. Herein, a method is described whereby the phagocytic rate of small numbers of glass-adherent mononuclear cells can be accurately measured. The method utilizes the ingestion by macrophages of technetium labelled polystyrene particles; both the radiolabel and ingestible substrate are readily available and the labelling process simple and efficient. The phagocytic rate can be expressed as radioactive counts per microgram of cell protein; data is also presented showing that the number of particles ingested per cell can be accurately derived.

Fatal nonpowder firearm wounds: case report and review of the literature.

A previously unreported fatality due to a head wound from a CO2-powered BB pistol is described. In a review of the English language medical literature (primarily forensic), ten previous reports of fatality due to nonpowder firearms were found. A common mechanism is identified in the head wounds leading to fatality. Modern day technology has elevated the BB gun from toy to weapon. Often appearing trivial, BB and pellet gun injuries must be considered in the same class as those from small-caliber low-velocity powder firearms. A patient with a nonpowder firearm injury must be evaluated with a high index of suspicion for injuries that are not apparent during a general physical examination.

Emergency department management of the intoxicated adolescent.

It is widely recognized that many young people will have experiences with alcohol and with psychoactive recreational drugs before emerging from the adolescent years. It is estimated that before leaving ninth grade, one half of the total student population will have had experience with alcohol, and approximately one third with marijuana. Cocaine also is more available to adolescents. For these reasons, the emergency physician is more likely to see teenage patients for drug-related problems.