Kehl Markley

Effect of thermal trauma on numbers and function of T and B cells from mouse spleen.

Swiss-Webster female mice were given a moderately severe burn, and studies were carried out on the number and function of T and B cells from the spleens of burned and normal mice. The results showed a significant decrease (p less than 0.05) in the number of T and B cells for 2-3 days after burning with a rapid return to normal and a subsequent rise above normal at 14 and 21 days postburn (p less than 0.05). In the test for function, burned mice had a significant decrease in spontaneous mitotic activity of both T and B cells during the 21-day postburn period. When spleen lymphocytes were incubated with purified mitogens, both T and B cells showed a significantly diminished mitotic response in most of the burned animals.

The role of histamine in burn, tourniquet and endotoxin shock in mice

The release of histamine and mortality was studied in mice after various types of experimental shock. In burn shock, serum histamine rose significantly after injury, but there was no correlation between increased serum histamine and high mortality as a consequence of several therapy regimens. For example, after treatment with histamine or Compound 48/80 before burning, there was a rise of serum histamine, yet shock mortality fell significantly. Although separate administration of antagonists of H1 - or H2 - histamine receptors had no effect on mortality, pretreatment with both diphenhydramine and burimamide significantly increased shock mortality. In tourniquet shock, serum histamine rose significantly, and treatment with both antagonists before trauma produced a significant elevation of shock mortality. In endotoxin shock, prior treatment with one or both drugs did not change mortality. These results suggest that endogenous histamine is not a lethal factor in burn and tourniquet trauma, but rather it appears to have a compensatory, beneficial effect.

The effect of thermal trauma in mice on cytotoxicity of lymphocytes.

Summary The effect of thermal trauma on cellular immunity was studied in inbred mice using an in vitro assay for cytotoxic lymphocytes from spleen, lymph node, and peripheral blood. Burned BALB/c mice sensitized with EL-4 tumor cells from C57BL/6N mice showed a significant decrease in cytotoxicity of spleen, lymph node, and peripheral blood lymphocytes. This depression of cytotoxicity occurred in spleen lymphocytes when the burn was given as early as 7 days before sensitization or as late as 13 days afterwards. Similar results were obtained with peripheral blood lymphocytes, but in the case of lymphocytes from lymph nodes diminished cytotoxicity was observed only when the animals were burned after sensitization. No evidence was found for an inhibitor of cytotoxicity in serum from burned mice or for “suppressor” cells in the spleen after thermal injury. The reduced cytotoxicity of lymphocytes in burned animals could be of critical importance in the impairment of some manifestations of cellular immunity after burn trauma. We wish to thank Dr. John R. Wunderlich of the National Cancer Institute for his helpful suggestions and discussion during the course of this work and Ms. Susan Sharrow of the National Cancer Institute for her advice on technical matters.

On the Mechanism of Action of Phytohemagglutinin in Cellular Immunity

Summary To study the mechanism of action of phytohemagglutinin in cellular immunity, preparations of phytohemagglutinin were made to separate its mitogenic, erythroagglutinating, and immunogenic properties. Boiled phytohemagglutinin (PHA-P, Difco), which had lost its mitogenic and erythroagglutinating activities, was used to test the immunogenic properties of phytohemagglutinin. A purified mitogenic fraction of phytohemagglutinin was isolated chemically which stimulated the lymphoid system in vivo. Untreated PHA-P was able to prolong graft rejection and block the delayed hypersensitivity reaction. Neither boiled PHA-P nor the mitogenic fraction of phytohemagglutinin was able to mimic the actions of the untreated compound. The mechanism of action, therefore, does not appear to rest in either of these properties alone. In the delayed hypersensitivity reaction, intraperitoneal injection of N-acetyl-D-galactosamine and D-galactose was able to block partially the effect of phytohemagglutinin on the reaction. These sugars, however, were not similarly effective in blocking the action of phytohemagglutinin on graft rejection.

THE ROLE OF BACTERIA IN BURN MORTALITY

In summary, the evidence for the role of bacteria in burn mortality is: A majority of severely burned mice showed positive blood cultures during shock period; gram‐negative bacteria predominated.

Protection against burn, tourniquet and endotoxin shock by histamine, 5-hydroxytryptamine and 5-hydroxytryptamine derivatives.

1 5‐Hydroxytryptamine (5‐HT), tryptamine, 5‐methyltryptamine, 5‐methoxytryptamine, N‐methyltryptamine, 5‐hydroxy‐N,N‐dimethyltryptamine, and histamine markedly protect mice subjected to burn, tourniquet and endotoxin shock. All of these compounds protect when given 30 min before the production of shock, but not when administered afterwards. 2 The above compounds, as well as purines and purine derivatives have a similar chemical structure. Protection requires the compounds to contain a 5‐membered ring with one unsubstituted N atom and a side chain with a basic N atom three atoms from the ring. 3 All other biological amines tested without this chemical structure did not protect. 4 Since the simplest compound containing all the prerequisites for protection is histamine, this compound may play the key role in protection, for both 5‐HT and purines release histamine from tissues. 5 Protective doses of 5‐HT and histamine prevent swelling of the injured area after tourniquet trauma and produce an increased bleeding volume and lower haematocrit value after burning. These actions of the drugs on the circulation may account for the increased survival after thermal trauma.

Effect of Burn Trauma in Mice on the Generation of Cytotoxic Lymphocytes

Summary The mechanisms of the impaired cellular immune response after a moderately severe burn in mice was studied. T cells from spleens of burned BALB/c mice showed decreased cytotoxicity (P < 0.05) during the first 9 days postburn compared with nonburned controls. In the one-way MLC, T cells from spleens of the majority of burned BALB/c mice collected on the second, third, and fourth days postburn incubated for 6 days with irradiated spleen cells from C57BL/10J mice also had decreased cytotoxicity (P < 0.05). On the other hand, mixed populations of cells obtained from spleen or peripheral blood of burned BALB/c mice demonstrated no defect in sensitization, either in responding or stimulating abilities, in the MLC. Other experiments in which EL4 cells from burned donor C57BL mice were used to sensitize nonburned BALB/c mice showed decreased immunogenicity in recipient mice. These results indicate that the mechanism of the impaired cellular immune response after burn injury, when assayed by cytotoxicity, probably involves defects in the sensitization process, compromising both responding and stimulating capabilities. The changes produced in T cells by burning to cause this phenomenon are not clear, but no evidence was found for humoral factors in the serum of burned animals or for the presence of suppressor cells which could affect sensitization in the MLC.

The effect of diet protein on late burn mortality.

Summary The effect of protein in the diet on late mortality, food intake, and weight changes of severely burned mice was studied. Mice were fed ad libitum diets containing low (6%), adequate (20%) or high (40%) casein protein diets supplemented with carbohydrate and fat to make caloric contents equivalent. Three diet regimens were used: (a) adult mice fed the diets after thermal injury; (b) weanling mice reared on the diets which were continued after burning; and (c) adult mice fed the diet for 3 weeks prior to thermal trauma and continued on the same diet afterward. The results show that on all diet regimens a low-protein diet produced a significantly higher late mortality in burned mice and an increased weight loss when compared with burned mice fed an adequate protein diet. In contrast, a high-protein diet did not prove more beneficial by the same criteria than an adequate-protein diet. In all groups compared, food intake was not significantly different. When various amounts of methionine (also reported to be beneficial in experimental burn shock and wound healing) were added to the low-protein diet, survival did not improve.

Demonstration of a Circulating Endotoxin Hapten in Burned Mice

Summary A quantitative hemagglutination-inhibition test was developed for the determination of endotoxin (hapten) in serum of mice. When the blood sera of conventional mice given a minimal or severe burn were tested for circulating endotoxin, significantly elevated titers of E. coli endotoxin (or hapten) were found in the sera. There is as yet no conclusive evidence, however, that the circulating endotoxin is biologically active.