Richard D. Stoner

The effects of single L-amino acid substitutions on the lethal potencies of the microcystins.

Microcystin-LR and -LA are more toxic than microcystin-LY and -RR in adult mice. They induce different degrees of thrombocytopenia and leukopenia, and the lethalities of their binary and ternary mixtures are addictive. Postnatal mice are resistant to doses of microcystin-LR that are lethal to adults but they are susceptible to higher doses. Substitution of a single L-amino acid for another in a microcystin markedly affects the dosimetric potency, but not the pathophysiology of its toxicity.

INCORPORATION OF TRITIATED NUCLEOSIDES AND AMINO ACIDS INTO LYMPHOID AND PLASMOCYTOID CELLS DURING SECONDARY RESPONSE TO TETANUS TOXOID IN MICE

It has become evident in recent years that almost all antibody-containing cells formed within four days after secondary stimulation with soluble antigen arise from precursor cells that synthesize DNA during this period.’ The kinetics and exact localization of this proliferative activity are not clearly ascertained.2 Renewed interest has focused on germinal-center cells as possible precursors of plasma c e k 3 There is lack of agreement as to the life cycle of germinal centers, their possible dependence on antigenic stimulation, their role in antibody synthesis, and their function in plasmocytopoiesis and/or lymphopoiesis.‘ The present report is concerned with the incorporation of tritiated nucleosides and amino acids into lymphoid and plasmocytoid cells of a regional lymph node during the secondary response to tetanus toxoid. The findings will be correlated with changes in cellular composition and histological appearance of the node. Emphasis is placed on growth and behavior of germinal centers in relation to the secondary tetanus antitoxin response. The following basic experimental conditions were established in order to evaluate cellular and histological changes during the secondary response to the specific antigen given at a time remote from primary stimulation: Fluid tetanus toxoid (without adjuvant) was used as the test antigen since nonimmunized mice do not have detectable specific antitoxin. Antitoxin titers may be determined very precisely based on the capacity of the serum to neutralize active tetanus toxin.5 A six-month time-interval between primary and secondary antigenic stimulation was sufficient to allow the primary response to subside. The site of the second injection of antigen differed from the site of primary immunization so as to avert local remnants from the primary response usually detectable in the regional lymph node. Secondary stimulation was given via injection into the footpad because, in nonstimulated mice, the popliteal lymph nodes as the first regional lymphatic “stations” contain very few germinal centers and plasma cells. I .

Pathophysiology of cyanoginosin-LR: in vivo and in vitro studies

Cyanoginosin-LR, one of the group of virulent cyclic heptapeptide toxins (cyanoginosins) isolated from some strains of the cyanobacterium, Microcystis aeruginosa, kills mice within 1-2 hr after iv or ip injection. Although the liver is a target organ of the toxin, the rapidity of lethality is incompatible with metabolic death from failure of hepatocellular function. However, disintegration of sinusoidal endothelium causes massive intrahepatic hemorrhage. The loss of the structural integrity of hepatic sinusoids provides a previously undescribed mechanism for embolization of disintegrating cells from the liver to the lung. No injury to either cultured bovine pulmonary artery endothelial cells or mouse peritoneal macrophages was observed following prolonged incubation with high concentrations of the toxin, and there was no increase in vascular permeability to 125I-labeled albumin detected before intrahepatic hemorrhage. However, plasma fibronectin increased transiently after toxin injection. Acute, severe thrombocytopenia, a characteristic of cyanoginosin-LR toxicity, remains unexplained since platelets did not concentrate in the lungs, liver, or spleen. There are similarities between the effects of cyanoginosin-LR and of the lipopolysaccharide endotoxins, such as elevations of plasma levels of thromboxane B2 and 6-keto-prostaglandin F1 alpha.

Pathophysiologic effects of a toxic peptide from Microcystis aeruginosa

Toxin-LR, a hexapeptide produced by Microcystis aeruginosa, causes marked hepatic vascular congestion, thrombocytopenia, microscopic pulmonary thrombi and death in 50-70 min when injected into mice. Although it is considered an hepatotoxin, we report that sublethal hepatocellular damage produced by CCl4 given 24 hr prior to toxin-LR administration prevents the acute deaths. However, CCl4-treated mice surviving toxin-LR acute effects often died during the subsequent three days. Pretreatment of mice with the microsomal enzyme inhibitors SKF 525A or cobaltous chloride did not alter the acute lethality of toxin-LR, but pharmacologic doses of hydrocortisone prevented both the acute and delayed deaths. X irradiation-induced thrombocytopenia or thrombocytopenia and leukopenia did not significantly affect the toxins lethality. In vitro platelet aggregation or lysis did not occur during incubation with toxin-LR, nor was a humoral aggregating factor detected in plasma from toxin-injected mice.

Incorporation of C14-labeled amino acids by Trichinella spiralis larvae.

Abstract 1. 1. Carbon-14-labeled amino acids were fed to mice infected with Trichinella spiralis to test the ability of immature larvae and encysted larvae to incorporate C 14 from the host. The C 14 amino acids were fed beginning 14, 56, or 180 days after infection. 2. 2. Immature larvae (14 days) incorporated higher levels of C 14 activity (C 14 per gram of dry tissue) from glycine-2-C-14 and dl -alanine-2-C-14 than the amounts found in muscle tissue. 3. 3. When the 7-day period of C 14 feeding began 56 days after infection, the encysted larvae incorporated C 14 activity indicating that the larvae were exchanging metabolites with the host. The specific activity of the carbon in the larvae exceeded that found in the muscles of the host. 4. 4. The presence of encysted Trichinella larvae in the muscle of 56-day and 180-day infected mice did not alter the incorporation of C 14 activity into muscle protein when compared with noninfected muscle. 5. 5. The encysted larvae in the 180-day infected mice incorporated significant levels of C 14 activity from the tissues of mice fed glycine-1-C-14 and dl -alanine-1-C-14 diets showing an active metabolism by well-encapsulated Trichinella spiralis larvae.

Incorporation of DL-tyrosine-2-C-14 and DL-tryptophan-2-C-14 by encysted Trichinella spiralis larvae.

1. 1. Trichinella spiralis larvae incorporated significant levels of C14 activity when dl-tyrosine-2-C-14 and dl-tryptophan-2-C-14 labeled diets were fed to mice with infections of 56 and 180 days duration. 2. 2. In the 6-month-old infections the well-developed cyst wall surrounding the larvae retains sufficient permeability to tyrosine and tryptophan or to products of metabolism of these amino acids by the host. 3. 3. The larvae from the 62 and 186-day infected mice that were fed a dl-tyrosine-2-C-14 diet incorporated 86 and 92% of their total C14 content into protein (defined as material precipitable by tungstic acid). 4. 4. When the dl-tryptophan-2-C-14 diet was fed, the 62 and 186-day-old larvae incorporated 86 and 85% of their total C14 content into larval protein. 5. 5. Incorporation of total C14 into muscle protein was reduced in 186-day infected mice fed dl-tyrosine-2-C-14 as compared to muscle proteins obtained from noninfected animals. 6. 6. With the dl-tryptophan-2-C-14 diet, a 10-fold increase in C14 activity was found in 186-day infected muscle protein over that observed in noninfected muscle. This may indicate a significant change in tryptophan metabolism of the host during the course of a Trichinella infection in mice.

Specificity of enhanced immunological sensitization of mice following injections of antigens and specific antisera.

Summary An enhanced antibody response to bovine serum albumin and fluid tetanus toxoid was found in mice when these antigens were injected in slight antigen excess with specific heterologous antisera. The specificity of the enhanced response was limited to specific antigens in complex. An enhanced response was also demonstrated to rabbit gamma globulin by testing the capacity of passively immunized mice to degrade I131-labeled RGG at an accelerated rate.

In vitro metabolism of DL-tyrosine-2-C-14 and DL-tryptophan-2-C-14 by Trichinella spiralis larvae.

Abstract 1. 1. Trichinella spiralis larvae incorporated C14 when maintained in vitro in a serum-Krebs-Ringer medium containing either dl -tyrosine-2-C-14 or dl -tryptophan-2-C-14. Incorporation of C14 from labeled tryptophan was also observed in Krebs-Ringer medium without serum. 2. 2. Uptake of the C14-labeled amino acids was progressive through 3-to 48-hour periods of incubation. 3. 3. The larvae incorporated somewhat more C14 activity (cpm/mg carbon) from the dl -tryptophan-2-C-14 media than from the dl -tyrosine-2-C-14 medium. 4. 4. Of the total C14 taken up by the larvae cultured for 24 hours in the labeled-tyrosine-medium, 43% of the C14 was incorporated into proteins (defined as material precipitable by tungstic acid). 5. 5. With dl -tryptophan-2-C-14 labeled media the larvae incorporated 35% of their total C14 into protein when serum was present. In the serum-free medium the larvae incorporated 54% of their total C14 into protein material.

De novo Formation and Rapid Growth of Germinal Centers During Secondary Antibody Responses to Tetanus Toxoid in Mice

In considering the kinetic behavior and possible functions of germinal centers during immune responses, experimental conditions wherein de novoformation and growth of these structures can be followed are of special interest. It has been demonstrated (Cottieret al., 1964; Cottier et al., 1966) that a delimited situation exists during secondary antibody responses to tetanus toxoid in mice under the following conditions: 1. a time interval of several months is used between the first and second injection of antigen; 2. the site of secondary stimulation differs from the site of primary immunization; and 3. the booster injection is given at a site where the draining lymph nodes in non-stimulated animals contain no, or only a few, germinal centers.

In vitro Metabolism of DL-AIanine-2-C-14 and Glycine-2-G-14 by Trichinella spiralis Larvae.

Summary 1. Trichinella spiralis larvae incorporated C14 when cultured in vitro in a serum-Rrebs-Ringer medium or Rrebs-Ringer medium containing either dl-alanine-2-C-14 or glycine-2-C-14. 2. Carbon-14 analysis of the larvae revealed a progressive uptake of the C14-labeled amino acids in both types of media through 48 hours of incubation. 3. The larvae incorporated more C14 activity (cpm) from glycine-2-C-14-labeled media than from dl-alanine-2-C-14-labeled media. 4. Of the total C14 incorporated by larvae cultured 48 hours in glycine-2-C-14-la-beled media about 70 to 84% was precipitable by tungstic acid, and was presumably chiefly in the proteins. Larvae cultured in the dl-alanine-2-C-14-labeled media incorporated about 32-57% of their total C14 content into material precipitable by tungstic acid.