Dennis W. Watson

Influence of Stress on Distribution of Endotoxin in RES Determined by Fluorescein Antibody Technic.

Summary 1. Deposition of endotoxin of Salmonella typhosawas followed in normal and stressed rabbits by the fluorescein tagging technic. 2. Pretreatment with cortisone and x-irradiation did not affect initial phagocytosis of toxin but inhibited degradation and elimination of toxin by the RES. 3. Pretreatment with thorotrast and preliminary injection of toxin caused depression of initial phagocytic functioning of the RES to a subsequent injection of toxin. 4. Our findings in their possible relation to the generalized Shwartzman reaction were discussed.

Role of antibodies in reactions to gram-negative bacterial endotoxins.

Recently we have given evidence for the presence of two interdependent activities in the endotoxin molecule.’’2 The intrinsic or primary toxicity is believed to be associated with the lipid portion of the molecule. The secondary toxicity results from the acquisition of hypersensitivity by the host to some portion of the molecule, usually as a result of contact with intestinal flora or clinical and subclinical infections. We have continued to stress the interdependence of the primary and secondary toxic activities and the importance of taking into consideration the immunologic state of the host in any evaluation of the mechanism of endotoxin action. The ubiquity of so-called “natural antibodies”, in nearly all animals, specific for the 0-determinant of Gram-negative bacterial endotoxins, and the similarity of some toxic manifestations of endotoxins with those associated with antigen-antibody reactions have led some investigators to suggest that endotoxins have no intrinsic or primary toxicity. Attempts have been made to explain all of the toxic reactions in terms of classical antigen-antibody reactions. In our study on the ontogeny of the immune response using germ-free neonatal colostrum-deprived piglets4 we have shown that this host has no detectable immunoglobulins. This was an ideal model, therefore, to test the importance of antigen-antibody reactions as mediators of primary toxic reactions by endotoxins. In the present discussion, evidence will be presented using this model to demonstrate a true intrinsic toxic reaction by endotoxins in the absence of detectable antibodies. Also, we have recently reviewed5 the immunological aspects of pyrogenic tolerance to endotoxins and presented evidence2 that this tolerance results from the acquisition of classical immune mechanisms and not as a result of nonspecific increase in the reticuloendothelial system alone or of other nonspecific mechanisms. Immunoglobulins of the 19s class associated with the 0specific antibodies, but distinct from them, can passively immunize animals both to the pyrogenic and lethal activities of endotoxins. We have stressed5 the fact that the sugar determinants of the 0-specific polysaccharide are not functional in the toxophore group of endotoxins, and therefore antibodies directed toward these determinants will not neutralize the 3

A Specific Soluble Substance Involved in Nephrotoxic Nephritis.

Summary A specific soluble substance which will combine with the nephrotoxic antibody of anti—rat kidney serum was obtained in solution. The material was liberated into solution by digesting rat kidney with trypsin at pH 8. This substance was assayed by its ability to absorb antibodies from nephrotoxic serum. It protected a total of 24 rats without failure. Control experiments excluded trypsin activity as being the protective substance. Further characterization should be possible now that this specific material has been obtained in solution.

Effect of antipyretics on group A streptococcal pyrogenic exotoxin fever production and ability to enhance lethal endotoxin shock.

Summary The fever response of rabbits to group A streptococcal pyrogenic exotoxin (SPE) type C was effectively reduced by pretreatment of the animals with the antipyretics, indomethacin (25 mg/kg), acetylsalicylate (200 mg/kg), and cortisone (5 mg on each of three preceding days and 2 hr before given SPE). Indomethacin and acetylsalicylate also significantly reduced the fever response of rabbits to SPE type C if given near the time of maximum fever response (4 hr after SPE). None of the antipyretic agents protected the rabbits from SPEs capacity to enhance susceptibility to lethal endotoxin shock. These data suggest that SPE fever production required prostaglandin synthesis, probably PGE1 or PGE2, and that the mechanism of fever production by SPE may be different from the mechanism underlying the enhancement of lethal endotoxin shock. Studies are presently being done to elucidate further the mechanisms of fever production and enhancement of lethal endotoxin shock by SPE.

Biogenic amine involvement in pyrogenicity and enhancement of lethal endotoxin shock by group A streptococcal pyrogenic exotoxin.

Summary Group A streptococcal pyrogenic exotoxin type C (SPE C) was shown to produce fever which in part depended upon norepinephrine and stimulation of α-adrenergic receptors. Intracisternal injection of norepinephrine into rabbits already showing fevers due to SPE C resulted in further heightened fevers. Pretreatment of animals with either α-methyl tyrosine to deplete norepinephrine stores or phenoxybenzamine to block α-receptors depressed SPE-induced pyrogenicity. Pretreatment of animals with P-chlorophenylalanine to deplete serotonin stores accentuated fevers due to SPE and giving serotonin intracisternally to rabbits with fevers resulted in a significant drop in body temperature. This indicated serotonin exerted a negative effect on SPE pyrogenicity. Isoproterenol and propranolol did not affect SPE C fever production. When used alone, none of the drugs prevented the capacity of SPE C to enhance lethal endotoxin shock. However, phenoxybenzamine in combination with fluid replacement increased the survival rate of rabbits. It is proposed that SPE C may alter the endotoxin detoxification system, thus allowing endotoxin to persist in the circulation producing shock. This work was supported by USPHS Grant AI-06487 from the National Institute of Allergy and Infectious Diseases. Dr. Schlievert received stipend support from USPHS Training Grant HLI-07014 from the National Heart, Lung and Blood Institute.

Suppression by Endotoxin of the Immune Response to Actinophage in the Mouse.

Summary Repeated injections of 4 mg endotoxin/kg, subsequent to antigenic stimulation with actinophage MSP8, retarded the early and late immune response in the mouse and resulted in an accelerated loss of preformed antibody. Cessation of endotoxin treatment permitted phage neutralizing activity in stimulated animals to increase.

Antibody production in neonatal chickens following injection of adult cells mixed with antigen in vitro.

Summary Antibody production to Brucella abortus antigen was initiated in neonatal chicks by transfer of adult homologous hen spleen cells mixed with antigen in vitro. The studies indicate a wide variation in response and that a pathologic picture consisting primarily of splenomegaly is regularly associated with good agglutinin titers in the recipient.

Histological Changes of Lymphoid Tissues in Relation to the Ontogeny of the Immune Response in Germfree Piglets

Germfree, colostrum-deprived piglets obtained by aseptic hysterectomy 3 to 5 days prior to term were devoid of immunoglobulins and their tissues were free of “background” antibody forming cells. They were immunologically “virgin”; however, they were highly immunologically competent as demonstrated by an excellent immune response upon antigenic stimulation (1,2,3). In the “true” primary immune response, the first antibody formed was 19SγG followed by 7SγG-immunoglobulins (4). These immunologically “virgin” piglets were used to investigate the cellular origin of the sequential synthesis of 19S to 7S antibody in the ontogeny of the immune response. This report presents briefly the histological changes of the lymphoid tissues in relation to the “true” primary immune response (5,6).

Effect of Massive Antigen Dosage on Antigen Retention and Antibody Response in Rabbits.

Summary Antigen elimination rates for massive doses of bovine albumin from the circulation of rabbits were resolved into two relative groups: (a) those eliminating the antigen rapidly, and (b) those retaining residual antigen for an extended period. All antibody-forming sites were assumed to be saturated with this antigen, and in the latter group antibody formation was suppressed. Data are presented suggesting that the breakdown and/or removal of an antigen from an antibody-forming site may be a necessary prerequisite to antibody formation. The rates of elimination of antigen from the various organs of the host are discussed in relation to postulated mechanisms concerned with serum sickness and the late sequelae of streptococcal infections.

Production of Generalized Shwartzman Reaction with Group A Streptococcal Factors.

Summary A soluble factor from group A streptococcal skin lesions prepared American Dutch rabbits for the generalized Shwartzman reaction. The reaction can be provoked in rabbits prepared with this material not only with toxins from Gram-negative organisms but also by reduced filtrates of streptococcal cultures with a high streptolysin O content. The tissue changes associated with the reaction brought about with these materials include myocardial necrosis and cortical necrosis of the kidney. Although these reactions simulate those observed in the classical generalized Shwartzman reaction, points at difference are discussed. It was possible to immunize rabbits against the preparative activity of the soluble factor by repeated injections of the streptococcal skin lesion extract.