Fate of tetanus toxin bound to the surface of primary neurons in culture: evidence for rapid internalization.

We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material at or near the dye front, which was lost when the cells were delipidated before electrophoresis. Gangliosides purified from the lipid extract were separated by thin-layer chromatography and the chromatogram was overlaid with 125I-toxin. The toxin bound to gangliosides corresponding to GD1b and GT1b. Similar results were obtained with brain membranes; thus, gangliosides rather than glycoproteins appear to be the toxin receptors both in vivo and in neuronal cell cultures. To follow the fate of tetanus toxin bound to cultured neurons, we developed an assay to measure cell-surface and internalized toxin. Cells were incubated with tetanus toxin at 0 degree C, washed, and sequentially exposed to antitoxin and 125I-labeled protein A. Using this assay, we found that much of the toxin initially bound to cell surface disappeared rapidly when the temperature was raised to 37 degrees C but not when the cells were kept at 0 degree C. Some of the toxin was internalized and could only be detected by our treating the cells with Triton X-100 before adding anti-toxin. Experiments with 125I-tetanus toxin showed that a substantial amount of the toxin bound at 0 degree C dissociated into the medium upon warming of the cells. Using immunofluorescence, we confirmed that some of the bound toxin was internalized within 15 min and accumulated in discrete structures. These structures did not appear to be lysosomes, as the cell-associated toxin had a long half-life and 90% of the radioactivity released into the medium was precipitated by trichloroacetic acid. The rapid internalization of tetanus toxin into a subcellular compartment where it escapes degradation may be important for its mechanism of action.

The structural gene for tetanus neurotoxin is on a plasmid.

A pool of synthetic oligonucleotides was prepared based on the amino terminal amino acid sequence of tetanus toxin. This probe hybridized to plasmid DNA isolated from three toxigenic strains of Clostridium tetani but not to plasmid DNA from a nontoxigenic strain. These results show that the structural gene for the toxin is on the plasmid. The pCL1 plasmid from one of the toxigenic strains spontaneously deleted 22 kilobase pairs of DNA to form pCL2. Strains harboring this deleted plasmid are nontoxigenic. However, the probe mixture hybridized to pCL2, indicating that the DNA encoding the amino terminus of the toxin had not been deleted. Restriction endonuclease cleavage maps of pCL1 and pCL2 were constructed and indicate the approximate location and orientation of the structural gene for tetanus toxin.

The effects of interleukin 2 and alpha-interferon administration on hepatic drug metabolism in mice.

We have administered the cytokines interleukin 2 (IL-2), alpha-interferon (IFN-alpha), and gamma-interferon (IFN-gamma) to mice and measured the alterations in hepatic drug-metabolizing enzyme activities. For comparative purposes and to understand the mechanism of diphtheria and tetanus toxoids and pertussis (DTP) vaccine-induced inhibition of drug metabolism, we also studied the effects of vaccine administration in mice. The administration of IL-2 alone or in combination with IFN-alpha or IFN-gamma causes dose-dependent increases in hexobarbital-induced sleep times. These increases correlate well with the inhibition of specific microsomal mixed-function oxidase activities. Sublethally irradiated mice and athymic nude mice receiving injections of IL-2 or IL-2 plus IFN-alpha do not show the inhibition of drug metabolism seen in normal mice. However, the inhibition of drug metabolism in DTP vaccine-treated mice was similar in all three groups. These observations indicate a possible role for immune cells (probably T-lymphocytes) in the inhibition of drug metabolism caused by administration of these cytokines, which is different from the inhibition of drug metabolism caused by DTP vaccine.

Cholesterol-dependent tetanolysin damage to liposomes.

Tetanolysin caused membrane damage, resulting in release of trapped glucose from liposomes containing cholesterol. Maximum glucose release occurred from liposomes that contained 50 mol% cholesterol. At higher or lower levels of cholesterol, glucose release was reduced and glucose release did not occur at all below 40 mol% cholesterol. The apparent activity of tetanolysin was not influenced by temperature (24 degrees C compared to 32 degrees C) or by liposomal phospholipid fatty acyl chain length. We conclude that tetanolysin caused cholesterol-dependent lysin-mediated damage to liposomes, possibly by means of a pore consisting of a complex of toxin and cholesterol.

Tetanus toxin interactions with the thyroid: decreased toxin binding to membranes from a thyroid tumor with a thyrotropin receptor defect and in vivo stimulation of thyroid function.

Normal rat thyroid membranes adsorb neurotoxicity when incubated with purified tetanus toxin. Membranes from a rat thyroid tumor with a thyrotropin receptor defect adsorb very little neurotoxicity when similarly evaluated. This inability of the tumor membranes to adsorb neurotoxicity is correlated with a defect in their ability to bind both 125I-labeled tetanus toxin and [125I]iodothyrotropin. The effect of tetanus toxin on the release of radioiodine from the thyroids of appropriately prepared mice has been measured by adapting methods used for the bioassay of thyrotropin. One minimum lethal dose of tetanus toxin given sc caused a significant release of radioiodine into the blood of mice 48 h after injection. In mice subjected to the stress of prior bleedings or anesthesia, the release of radioiodine from the thyroid by tetanus toxin was accelerated, i.e., the increase in blood radioiodine could be measured 24 h after injection. These results again suggest that tetanus toxin may interact with thyrotropin receptors on thyroid plasma membranes. The "sympathetic overactivity syndrome" seen in some patients with tetanus and the syndrome characterized as "thyroid storm" in patients with Graves' disease are discussed as they may relate to these observations.

Developmental and neurochemical specificity of neuronal deficits produced by electrical impulse blockade in dissociated spinal cord cultures.

Blockade of spontaneous electrical activity in dissociated fetal spinal cord cultures produced neuronal deficits as measured by biochemical and morphological techniques. Spinal cord cultures exhibited an age-dependent vulnerability to impulse blockade with tetrodotoxin (TTX) or xylocaine. Neuronal cell counts, [125I]tetanus toxin fixation and [125I]scorpion toxin binding indicated that TTX application produced neuronal deficits during the second or third week in culture. Application of TTX during the first or fourth week did not produce a difference in tetanus toxin fixation from controls. Radioautography of [125I]tetanus toxin revealed no obvious change in the label distribution after TTX treatment. Suppression of electrical activity during the first 6 days in culture had no effect on choline acetyltransferase (CAT) activity and no apparent effect on the appearance of the cultures. Application of TTX during the seventh day in culture decreased CAT activity to 68% of control. Chronic electrical blockade produced a progressively greater loss of CAT activity through 21 days in culture. GABAergic neurons, as indicated by high-affinity GABA uptake, glutamic acid decarboxylase activity and [3H]GABA radioautography, were not affected by electrical blockade. These data indicate that there is developmental and neurochemical specificity in the neuronal death produced by blocking spontaneous electrical activity in dissociated spinal cord cultures.

Potency testing of bacterial vaccines for human use.

The potency tests for bacterial vaccines are quite diverse. For some products (pertussis, cholera, anthrax, typhoid and BCG vaccines) these are specified as Additional Standards in the Code of Federal Regulations. For other products (tetanus and diphtheria toxoids, plague vaccine) the testing is done according to so-called Minimum Requirements, which have less regulatory authority than Additional Standards. Still other products (e.g., polysaccharide conjugate vaccines, acellular pertussis vaccine, live oral typhoid) are tested according to individualized criteria that are contained in their specific Product License Applications. For some products there is inadequate knowledge of the pathogenic mechanisms and/or protective factors to design valid in vitro potency tests. In these cases, animal testing with subsequent serologic evaluation or challenge testing is often necessary. Examples would include vaccines such as cholera and plague vaccines. The FDA supports the elimination of animal testing when suitable alternatives are available. Thus, many of the potency tests, especially for newer products, rely on in vitro characterization. For example, the immunogenicity of conventional polysaccharide vaccines is largely proportional to their molecular weight. Potency testing therefore relies heavily on physical characterization in terms of composition, molecular weight, and quantity.

Differential toxicity of chronic exposure to phenytoin, phenobarbital, or carbamazepine in cerebral cortical cell cultures.

The effects of phenytoin (30 micrograms/ml), phenobarbital (64 micrograms/ml), and carbamazepine (24 micrograms/ml) were assessed in cerebral cortical cell cultures. After antiepileptic drug exposure for eleven days, cultures were assayed for total protein, number of neurons, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, activity of choline acetyltransferase, and benzodiazepine binding. Carbamazepine-exposed cultures demonstrated minimal effects, whereas highly significant deficits related to generalized toxicity were observed in cultures exposed to phenytoin or phenobarbital.

Differential neurochemical effects of chronic exposure of cerebral cortical cell culture to valproic acid, diazepam, or ethosuximide.

We have assessed the relative neurochemical effects of valproic acid, ethosuximide, and diazepam on dissociated cultures of mouse cerebral cortex. Cultures were exposed chronically (11 days) to each antiepileptic drug and assayed for number of neurons, total protein, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, choline acetyltransferase activity, and specific and clonazepam-displaceable benzodiazepine binding. Ethosuximide-exposed cultures did not evidence neuronal toxicity; exposure to valproic acid and diazepam resulted in modest neuronal toxicity. However, exposure to each of these drugs resulted in a marked reduction in benzodiazepine binding. This effect may relate to a common mechanism of action of drugs used to treat absence seizures.

Immune response of the llama (Lama glama) to tetanus toxoid vaccination.

An ELISA was developed to measure serum concentrations of tetanus toxoid-specific immunoglobulins. The titers obtained with this assay were compatible with those obtained by the standard mouse toxin-neutralization test. Serum samples from 123 llamas were analyzed for ELISA titers to tetanus toxoid. Of the 82 vaccinated adults, 75 (91%) had titers greater than or equal to 1:50. The vaccination status and titers of weanlings and juveniles (3 to 12 months old) varied; of the 21 vaccinated, 17 (81%) had titers greater than or equal to 1:50 and 7 of 9 (78%) unvaccinated llamas had titers less than 1:50. The ELISA titers of unvaccinated llamas less than 8 weeks old (crias) were matched with the maternal titers. All crias with titers less than 1:50 had dams with titers greater than or equal to 1:50.

Mechanism of tetanolysin-induced membrane damage: studies with black lipid membranes.

Tetanolysin produced similar rates of leakage of K+ and hemoglobin from erythrocytes. When studied by using cholesterol-containing black lipid membranes, this hemolysin induced conductance steps with a broad frequency distribution. These findings are inconsistent with the formation of structural channels and suggest that tetanolysin acts by causing lipid perturbations.

Binding of tetanus toxin to somatic neural hybrid cells with varying ganglioside composition.

125I-labelled tetanus toxin interaction with several somatic hybrid cell lines was investigated. Binding of toxin is most effective in NCB-20, followed by NBr-10A, NG108-C15, and SB21-B1 cells. Specific binding of toxin to NCB-20 and SB21-B1 cells is 7- and 60-fold lower, respectively, in comparison to enriched rat cerebral neuron cultures. The NCB-20, NBr-10A, and NG108-C15 clones display a complex ganglioside pattern, including the presence of [N-acetyl-neuraminyl]-galactosyl-N-acetylgalactosaminyl[ N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1a) and two unidentified [14C]galactose-labelled lipid-soluble compounds, while the SB21-B1 is most abundant in [N-acetyl-neuraminyl]-galactosylglucosyl-ceramide (GM3) and N-acetyl-galactosaminyl-[N-acetyl-neuraminyl]-galactosylglucosyl-c eramide (GM2) gangliosides. None of the cells tested contain measurable levels of [14C]galactose-labelled or resorcinol-positive bands of galactosyl-N-acetyl-galactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1b) and [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GT1b) gangliosides. After 2 h at 37 degrees C a near plateau of toxin association with NCB-20 cells is seen. Binding in low-ionic-strength medium is 1.35-fold higher at 37 degrees C than at 4 degrees C, but is reduced by 21 and 51% at 4 degrees C and 37 degrees C, respectively, in physiologic medium. Treatment of NCB-20 cells with neuraminidase causes a partial loss (29%) of toxin-binding sites. Binding to the hybrid cells is significantly different from that of cerebral cultures with respect to temperature, salt effect, and sensitivity to neuraminidase, suggesting perhaps a different class of receptors for the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Reevaluation of the role of gangliosides as receptors for tetanus toxin.

Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) than in 25 mM Tris-acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris-saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100-fold higher in Tris-saline compared to Tris-acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GD1b approximately equal to GT1b approximately equal to GQ1b greater than GD2 greater than GD3 much greater than GD1a approximately equal to GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris-saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low-salt and -pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.

Binding of nonsubstrate ligands to the glutathione S-transferases.

Fluorescence spectroscopy and inhibition kinetics were used to quantitate the affinity of nonsubstrate ligands for the rat liver glutathione S-transferases AA, A, B, and C in the presence of glutahione. The dissociation constants KD, for ligands such as bilirubin, indocyanine green, and hematin were determined by measuring the decrease in the intrinsic fluorescence of the proteins attendant on the addition of ligand. A second technique, used for compounds which absorb strongly at the excitation maxima of tryptophan, was to utilize 8-anilinonaphthalen sulfonate in the formation of protein complex fluorescing at a higher wavelength. The quenching of this complex allowed the determination of the dissociation constants for ligands such as 3,6-dibromosulfophthalein and cephalothin. These data indicate that all four proteins bind these ligands but do so with different affinities. The bilirubin-induced decrease in fluorescence was used to estimate the stoichiometry of binding as 1.2 mol of bilirubin bound/mol of transferase B and 0.7 mol/mol of transferase C. All of the ligands examine are inhibitors of catalytic activity, as tested in a standard assay with GSH and 1-chloro-2,4-dinitrobenzene as substrates. From these studies we conclude that these proteins have a broad specificity not only for their substrates, but for the binding of nonsubstrate ligands as well.

Monoclonal antibodies as probes of tetanus toxin structure and function.

Monoclonal antibodies specific for fragment B, fragment C, and light chain of tetanus toxin were prepared by fusion of P3X63Ag8 BALB/c myeloma cells with spleen cells from BALB/c mice immunized with tetanus toxoid or fragment B. Hybridoma colonies were assayed for antibody production by an enzyme-linked immunosorbent assay. Fourteen positive clones were identified, cloned by limiting dilution, and injected intraperitoneally into mice to obtain ascites fluids. Thirteen of the monoclonal antibodies were of the immunoglobulin G1 subclass and one was immunoglobulin G2. Two of the antibodies were directed against sites on fragment C, nine were directed against the light chain, and three were directed against the portion of fragment B which does not comprise the light chain of tetanus toxin. At least one antibody in each group exhibited significant toxin neutralization activity. However, only one of these neutralizing antibodies strongly inhibited the binding of 125I-tetanus toxin to ganglioside-coated plates. These data indicate that interference with receptor recognition is not the only means of neutralizing tetanus toxin. Monoclonal antitoxins as potential therapeutic and prophylactic reagents are discussed.

Antibodies against the light chain of tetanus toxin in human sera.

Tetanus toxoid elicits protective antibodies against tetanus toxin in humans and animals. It has been reported that antitoxin from immunized humans contains no anti-light chain antibodies, based on immunodiffusion and quantitative precipitin analyses. We confirmed the absence of precipitating anti-light chain antibodies in tetanus immune globulin. However, the presence of antibodies against the light chain of the toxin was shown by direct binding and inhibition analyses, using enzyme-linked immunosorbent assays. Using a neutralization inhibition test, we also found that about one-fourth of the neutralizing antibodies in tetanus immune globulin are directed against the light chain. These results suggest that the light chain of tetanus toxin contains immunogenic determinants and that antibodies directed against it may have a role in the prevention of tetanus or treatment of tetanus or both.

Mechanism for the several activities of the glutathione S-transferases.

The catalyzed reactions of GSH with organic nitrate and thiocyanate esters and with a series of chloronitrobenzene substrates have been investigated and the results used to formulate a mechanism for glutathione S-transferase catalysis. All the homogeneous preparations of the glutathione transferases that have been tested catalyze the reaction of GSH with organic nitrates and thiocyanates. The nature of the reaction with nitrate esters, resulting in the formation of GSSG rather than a thioether, has been investigated further. The presence of an additional nonsubstrate thiol decreased the formation of GSSG to an extent that cannot be explained by disulfide interchange. These results are interpreted to reflect the enzymatic formation of an unstable glutathione sulfenyl nitrite that undergoes subsequent non-enzymatic decomposition. Hammett plots of the catalytic constants of rat liver transferases B and C obtained with a series of 4-substituted 1-chloro-2-nitrobenzene substrates demonstrate a linear relationship with sigma- substituent constants, reflecting the nucleophilic nature of the enzymatic reactions and their strong dependence on the electrophilicity of the nonthiol substrate. These data suggest that the many diverse reactions catalyzed by the glutathione transferases may be formulated as a nucleophilic attack of enzyme-bound GSH on the electrophilic center of the second substrate. The final products observed reflect this primary event and the existence of subsequent nonenzymatic reactions.