Studies on the mode of action of diphtheria toxin. II. Protein synthesis in primary heart cell cultures.

Primary heart cell cultures of embryonic guinea pigs and the neonatal rat were established and incubated with purified diphtheria toxin. The rat heart cell cultures were refractory to the effects of the toxin; protein synthesis proceeded normally as measured by the incorporation of tritiated leucine into cell proteins; beating heart cells continued to contract; and the cell monolayers remained intact after exposure to the toxin for periods as long as 72 hr. These findings are compatible with the species resistance of the rat to diphtheria toxin. The guinea pig heart cell cultures were found to be extremely sensitive to the toxin. Protein synthesis was inhibited by approximately 50% after incubation with small quantities of toxin for 3 hr. Increasing the concentration of the length of exposure to the toxin did not increase this inhibition significantly. In addition, diphtheria toxin exerted a true cytopathic effect on the guinea pig heart cells. Monolayers were destroyed when incubated with the toxin for 2 to 3 days. The results show that the heart cells reflect species resistance or sensitivity to diphtheria toxin in the absence of neural or endocrine influences and suggest further that the toxin exerts a direct toxicity to muscle cells of the heart. It is not yet possible to explain in biochemical terms why the toxin seems to act specifically on cardiac tissues.

Studies on the mode of action of diphtheria toxin. III. Effect on subcellular components of protein synthesis from the tissues of intoxicated guinea pigs and rats.

The effect of diphtheria toxin on subcellular components of protein synthesis was determined. Polyribosomes prepared from intoxicated guinea pigs functioned normally in an in vitro assay system, while the activity of soluble enzymes (transferases) from toxin-treated animals was significantly reduced. At high toxin dosages, this reduction was widespread, but when levels of toxin comparable to those which might be generated in a natural infection were given, inhibition of soluble enzyme activity was found only in extracts from heart and skeletal muscle. Possible nonspecific inhibition in the assay system due to interference by free toxin or by a serum component was eliminated. Since it was possible to demonstrate reactivation of soluble enzyme activity with nicotinamide and toxin, it was suggested that diphtheria toxin acts in the intact sensitive animal in a manner analogous to its action in tissue culture or in cell-free systems. It was hypothesized that the lethal biochemical lesion of the toxin in sensitive animals was the inactivation of transferase enzymes, principally in the heart. It was also suggested that the lethal lesion induced in diphtheria-sensitive and resistant species may not be identical.

Studies on the mode of action of diphtheria toxin. I. Protein synthesis in guinea pig tissues.

The effect of crystalline diphtheria toxin on protein synthesis in vivo was evaluated in guinea pigs and mice. By two independent methods of analysis (microdensitometry of tissue radioautograms and radioactivity of tissue proteins), it was established that inhibition of protein synthesis was not a widespread metabolic effect of diphtheria toxin. In the sensitive guinea pig, only the heart and the pancreas showed any demonstrable reduction in the quantity of tritiated leucine incorporated into proteins following challenge of the animals with the crystalline toxin. No such inhibition was noted in mice which are resistant to the action of diphtheria toxin. The effect on the pancreas involved a decrease in the synthesis of pancreatic enzymes and their subsequent secretion. For reasons discussed, it was concluded that this lesion was not as significant as the inhibition of protein synthesis in the heart tissues. Although the rate of protein turnover in heart muscle is relatively low, an inhibition of 73% was noted when the exchange period with the tritiated leucine was 6 hr. It was suggested that the inhibition of protein synthesis in heart tissues could provide a biochemical rationale for the site and mode of action of diphtheria toxin in the sensitive mammalian host. An attempt was also made to correlate the biochemical heart lesion described here with past clinical evidence of cardiac failure and tissue pathology noted in many cases of fatal diphtheria infections of humans.

Enhanced metabolism of Leishmania donovani amastigotes at acid pH: an adaptation for intracellular growth.

Amastigotes (tissue forms) of Leishmania donovani isolated from infected hamster spleens carried out several physiological activities (respiration, catabolism of energy substrates, and incorporation of precursors into macromolecules) optimally at pH 4.0 to 5.5. All metabolic activities that were examined decreased sharply above the optimal pH. Promastigotes (culture forms), on the other hand, carried out the same metabolic activities optimally at or near neutral pH. This adaptation to an acid environment may account in part for the unusual ability of amastigotes to survive and multiply within the acidic environment of the phagolysosomes in vivo.

Crystal structure of a biologically inactive mutant of toxic shock syndrome toxin-1 at 2.5 A resolution.

Toxic shock syndrome toxin-1 (TSST-1) is one of a family of staphylococcal exotoxins recognized as microbial superantigens. The toxin plays a dominant role in the genesis of toxic shock in humans through a massive activation of the immune system. This potentially lethal illness occurs as a result of the interaction of TSST-1 with a significant proportion of the T-cell repertoire. TSST-1, like other superantigens, can bind directly to class II major histocompatibility (MHC class II) molecules prior to its interaction with entire families of V beta chains of the T-cell receptor (TCR). The three-dimensional structure of a mutant (His-135-Ala) TSST-1 was compared with the structure of the native (wild-type) TSST-1 at 2.5 A resolution. The replacement of His 135 of TSST-1 with an Ala residue results in the loss of T-cell mitogenicity and toxicity in experimental animals. This residue, postulated to be directly involved in the toxin-TCR interactions, is located on the major helix alpha 2, which forms the backbone of the molecule and is exposed to the solvent. In the molecular structure of the mutant toxin, the helix alpha 2 remains unaltered, but the His to Ala modification causes perturbations on the neighboring helix alpha 1 by disrupting helix-helix interactions. Thus, the effects on TCR binding of the His 135 residue could actually be mediated, wholly or in part, by the alpha 1 helix.

Efficacy of combined immunostimulation and chemotherapy in experimental visceral Leishmaniasis.

A regimen of combined immunostimulation and chemotherapy for the elimination of Leishmania donovani amastigotes was evaluated. An in vitro experimental model utilized cultured peritoneal macrophages from C57B1/6 mice infected with L. donovani tissue forms. Partial or complete activation of macrophages as judged by killing of tumor cells significantly enhanced the efficacy of sodium antimony gluconate (Pentostam). The quantity of drug required for elimination of parasites from immunostimulated cells was considerably lower than that required to achieve comparable amastigote killing in thioglycolate-elicited macrophages. In contrast, amphotericin B cleared infected cells of amastigotes at comparable drug levels when tested with immunostimulated and unstimulated macrophages. Several drugs tested inhibited the conversion of amastigotes to promastigotes in vitro but were ineffective in killing of intracellular tissue forms. Allopurinol and difluoromethylornithine (DMFO) blocked amastigote conversion significantly. These drugs at high concentrations, however, exerted only minimal toxicity for amastigotes residing within macrophages. Efficacy of combined therapy was also demonstrated in vivo. Immunoenhancement of L. donovani-infected mice with Corynebacterium parvum vaccine combined with a regimen of sodium antimony gluconate was significantly more effective than was immunotherapy or drug therapy alone.

Enhancement of Glucantime therapy of murine Leishmania donovani infection by a synthetic immunopotentiating compound (CP-46,665-1).

The therapeutic efficacy of CP-46,665-1, a synthetic lipoidal amine with proven immunomodulatory and anti-tumor properties, in combination with chemotherapy was evaluated in L. donovani-infected C57Bl/6 mice. Immunostimulation and drug treatment resulted in a 10-fold lesser infection level than in untreated mice, while animals treated with Glucantime alone exhibited only a modest amelioration of the infection. We also studied the capacity of CP-elicited peritoneal macrophages of C57Bl/6 mice cultured alone or in combination with Glucantime and/or lymphokine to eliminate intracellular L. donovani amastigotes. When CP-elicited cells were incubated with Glucantime, they exhibited a significantly higher killing potential than did drug treated thioglycollate-elicited cells. CP-macrophages stimulated with lymphokine alone or in combination with antimonial drug, killed amastigotes more rapidly and efficiently than similarly treated thioglycollate-elicited macrophages. In vivo and in vitro results of this study show that a combined regimen of immunostimulation with CP and antimonial drug is more effective in treatment of L. donovani infection than either treatment alone.

Acquired immunity against mouse typhoid: genetic restriction and comparative efficacy of ribosomal and conventional vaccines.

Strains of mice immunised with a ribosomal preparation of Salmonella typhimurium varied in their ability to survive an intraperitoneal challenge of virulent S. typhimurium. Immunised nude (nu/nu), heterozygous (nu/+) mice, strain C57Bl/6J and strain CBA/J succumbed to lethal infection whereas strains C3D2F1/J, B6D2F1/J and A/J, and Swiss mice were fully protected. Strains DBA/2J and C3H/HeJ were partially protected. Enumeration of the systemic bacterial population after challenge with S. typhimurium indicated that all immunised mouse strains were able to reduce the infectious load. S. typhimurium was rapidly inactivated in the peritoneal cavity of immunised mice, effectively reducing the challenge and thereby limiting the number of organisms available to seed the systemic circulation. This response was also obtained in immunised athymic mice and was therefore attributed to a T-cell independent antibody response. Organisms that escaped destruction in the peritoneal cavity multiplied rapidly in the reticuloendothelial organs. Only mice from strains genetically capable of developing an effective cell-mediated immune response to the antigenic stimulus provided by the challenge organism itself survived infection. The efficacy of ribosomal immunisation was compared with immunisation by heat-killed bacteria, viable attenuated and viable virulent organisms by enumeration of the systemic bacterial population after intravenous challenge with S. typhimurium. Vaccination with ribosomal preparations or heat-killed organisms provided limited protection whereas immunity provided by viable organisms was far superior.

Studies on the mode of action of diphtheria toxin. V. Protein metabolism in a guinea pig model simulating chronic diphtheritic toxemia.

An experimental model of "chronic" diphtheria intoxication in the guinea pig was developed. Adult guinea pigs, subjected to a regimen of multiple sublethal doses of purified diphtheria toxin (total of 1.4 minimum lethal doses divided equally in four daily doses), developed a toxemia which terminated in death between 6 and 8 days. During an advanced stage of illness, de novo protein synthesis was assessed by in vivo incorporation of tritiated leucine into tissue proteins. With the exception of pancreas and skeletal muscle, protein synthesis in healthy, control and toxin-treated guinea pigs was comparable. The absence of notable impairment of protein synthesis in this experimental situation is discussed in terms of the mode of action of the diphtheria toxin and the pathophysiology of the disease.

Absorption of botulinal toxin from the gastrointestinal tract.

Oral toxicity of botulinal toxin is manifested when the toxin is absorbed from one or more anatomic regions of the intestinal tract and reaches target neurons. Toxin is absorbed primarily in the small intestine, although in infants the large intestine may be a site of absorption. Nanogram amounts of toxin that escape proteolytic digestion in the intestine may be sufficient to produce neurologic symptoms. It is estimated that approximately 10(11) molecules of toxin reaching peripheral nerve endings is sufficient to cause clinical botulism in adults, although the amount required to cause infant botulism is probably less. Absorption of toxin from the intestine is achieved by means of an endocytic mechanism, as is the absorption of nutritional proteins. Specific toxin receptors may be involved in the movement of toxin from the gastro-intestinal epithelium to target neurons across cellular barriers.

Cell-mediated immune phenomena induced by lymphokines from splenic lymphocytes of mice with chronic staphylococcal infection.

Splenic lymphocytes from normal mice and from mice displaying delayed hypersensitivity to Staphylococcus aureus were cultured in the presence or absence of specific staphylococcal antigens. The cell-free supernatant fluids from these lymphocyte cultures were assessed for their ability to alter the functional capacities of normal macrophages. It was found that supernatants from staphylococcus-immune cells cultured in vitro with antigen possessed migration inhibitory factor activity and also were capable of stimulating the incorporation of [14C]glucosamine into macrophage membrane glycoproteins. In addition, the lymphokine-containing supernatants were capable of inducing activation of normal macrophages so that they inhibited the multiplication of intracellular Listeria monocytogenes. Although it was not possible to snow any significant enhancement of intracellular killing of S. aureus by the activated macrophages, evidence is presented that suggests that cell-mediated immune responses to S. aureus may significantly enhance pahgocytosis of staphylococci and, thereby, may provide for their rapid clearance from extracellular fluids.

A role for oxygen-dependent mechanisms in killing of Leishmania donovani tissue forms by activated macrophages.

Leishmania donovani, the causative agent of visceral leishmaniasis, infects macrophages (M phi ) of susceptible vertebrates. Immunologically activated M phi are leishmanicidal, but the mechanisms involved in the killing process are not well defined. We sought to investigate the role of reactive oxygen intermediates in the killing of L. donovani. Both the free-swimming promastigote and the intracellular amastigote forms were found to be susceptible to killing in vitro by hydrogen peroxide and other oxygen intermediates. Upon phagocytosis by mouse peritoneal M phi, promastigotes elicited a significantly stronger respiratory burst compared with amastigotes as measured by release of superoxide anion. Although amastigotes do not elicit a strong burst of M phi oxidative metabolism during the initial phagocytic event, immunologically activated M phi that acquired leishmanicidal capacity could be triggered to release substantial amounts of H2O2. Hence, the development of leishmanicidal capacity was correlated temporally with enhanced H2O2 generation by the M phi. In contrast, M phi that lost their ability to release significant amounts of H2O2 after several days in culture were unable to eliminate their parasite burden. Catalase markedly inhibited the elimination of amastigotes by lymphokine-stimulated M phi. In toto, the results implicate reactive oxygen intermediates in killing of the tissue form of L. donovani by its host cell, the mononuclear phagocyte.

Staphylococcal enterotoxins fail to disrupt membrane integrity or synthetic functions of Henle 407 intestinal cells.

The potential cytotoxic activity of purified staphylococcal enterotoxins for mammalian cells was evaluated. The effects of staphylococcal enterotoxins A (SEA) and B (SEB) on cell membrane integrity as measured by leakage of labeled cytoplasmic constituents ([3H]uridine), amino acid transport (lysine and aminoisobutyric acid), and macromolecular synthesis (protein, ribonucleic acid, and deoxyribonucleic acid) was evaluated for a human intestinal epithelial cell (Henle 407). No evidence of cytotoxicity by any of these criteria could be detected for cell monolayers incubated with SEA for periods of between 30 min and 24 h. Purified staphylococcal hemolysins (alpha- and delta-toxins) were shown to exert cytotoxicity by the leakage and amino acid uptake assays. In efforts to detect synergistic effects between enterotoxin and the staphylococcal cytotoxins, membrane functions were evaluated after sequential or combined treatment with enterotoxin and alpha-toxin or with enterotoxin and delta-toxin. In no instance could a contribution to cytotoxicity by the staphylococcal enterotoxin be detected. That the assays were sufficiently sensitive to detect synergistic effects was shown by the greater than additive effects achieved with a combination of alpha- and delta-toxins. The data, contrary to previous reports, showed that staphylococcal enterotoxins did not behave as bacterial cytotoxins.

Cyclosporin A treatment converts Leishmania donovani-infected C57BL/10 (curing) mice to a noncuring phenotype.

Cyclosporin A prevents visceralization of Leishmania major infection of BALB/c mice (N. C. Behforouz, C. D. Wenger, and B. A. Mathison, J. Immunol. 136:3067-3075, 1986; W. Solbach, K. Forberg, E. Kammerer, C. Bogdan, and M. Rollinghoff, J. Immunol. 134:702-707, 1986). We report that cyclosporin A exacerbates disseminated leishmaniasis caused by L. donovani in C57BL/10 mice. Normal mice challenged with 5 x 10(6) amastigotes intravenously cleared the infection within several months by spontaneous acquisition of cell-mediated immunity. In contrast, cyclosporin A administered daily intraperitoneally at a dose of 1.25 mg per mouse prevented development of curative immunity and converted C57BL/10 (curing) mice to a noncuring phenotype. A rationale for the contrasting effects of cyclosporin A in the two murine models of leishmaniasis is provided.

Phagocytosis and intracellular killing of Staphylococcus aureus by normal mouse peritoneal macrophages.

Although Staphylococcus aureus is incapable of intracellular multiplication in cultured mouse peritoneal macrophages, it is killed at a much slower rate than the avirulent Staphylococcus epidermidis. In addition to the presence of capsular material which inhibits phagocytosis of specific strains of S. aureus, the data show that a number of cellular and environmental factors affect the functional capacities of mononuclear phagocytic cells. The data obtained by varying the initial level of infection indicate that the number of ingested bacteria may subsequently alter the kinetics of intracellular killing. In vitro maturation of macrophages in culture was also found to exert a pronounced effect on the kinetics of bacterial death.