Transmembrane diffusion channels in Mycoplasma gallisepticum induced by tetanolysin.

The permeability properties of Mycoplasma gallisepticum cells treated with a purified preparation of tetanolysin were investigated by determining the initial swelling rates of cells suspended in an isoosmotic solution of electrolytes or nonelectrolytes. The swelling, initiated by the tetanolysin, depended on the tetanolysin concentration and was markedly affected by the molecular size of the various osmotic stabilizers utilized. Thus, the initial swelling rates in an isoosmotic solution of monosaccharides were much higher than those in isoosmotic solutions of di-, tri-, or tetrasaccharides. Cell swelling induced by tetanolysin was much lower with energy-depleted M. gallisepticum cells, with arsenate-treated cells, or when the membrane potential (delta psi) was collapsed by valinomycin (10 microM) plus KCl (100 mM). Swelling was not affected by the proton-conducting ionophore carbonyl cyanide-m-chlorophenylhydrazone (1 to 10 microM) or by nigericin (5 microM). These results support the concept that the damage induced by tetanolysin is due to the formation of water-filled pores within the membranes of energized M. gallisepticum cells. Such pores allow the diffusion of hydrophilic molecules into the cells and may vary in size, depending on the tetanolysin concentration utilized.

Acellular and whole-cell pertussis vaccines in Japan. Report of a visit by US scientists.

Since the introduction of acellular pertussis vaccines in Japan late in 1981, more than 20 million doses have been administered, mostly to children 2 years of age and older. Clinical studies indicate that mild local and febrile reactions are less frequent after administration of acellular pertussis vaccines than after whole-cell vaccines. Serious adverse events with sequelae occurred in 2-year-old children at approximately the same low rate during the period 1975 through August 1981, when whole-cell vaccines were used, and during August 1981 through 1984, when acellular vaccines were used exclusively. Five household contact studies have yielded vaccine efficacy estimates ranging from 78% to 92% in children 1 year of age or older. In addition, there has been a continuing decrease in reported pertussis incidence from the epidemic peak in 1979. Additional data on the safety and efficacy of acellular pertussis vaccines administered to infants would be useful in consideration of acellular pertussis vaccine licensure in the United States.

Tetanus toxin in dissociated spinal cord cultures: long-term characterization of form and action.

The clinical course of tetanus is notable, in addition to its often dramatic clinical presentation, by the long duration of the neuromuscular symptoms. Survivors may have tetanic manifestations for several weeks after the onset of the disease. In this article we correlate the duration of specific electrophysiologic effects produced by tetanus toxin with the degradation of cell-associated toxin in primary cultures of mouse spinal cord neurons. From these studies we can conclude that the toxin has a half-life of 5-6 days. Both the heavy and the light chains of tetanus toxin degrade at similar rates. Labeled toxin, visualized by radioautography, is associated with neuronal cell bodies and neurites, and its distribution is not altered during a 1-week period following toxin exposure. Blockade of synaptic activity persists for weeks at the concentration of radiolabeled toxin used in these studies. This blockade of transmission is reversed as the toxin is degraded, suggesting that degradation of toxin may be a sufficient mechanism for recovery from tetanus.

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.

Serum antibody responses of juvenile and infant rhesus monkeys injected with Haemophilus influenzae type b and pneumococcus type 6A capsular polysaccharide-protein conjugates.

Juvenile and infant rhesus monkeys were injected subcutaneously with saline solutions of Haemophilus influenzae type b (Hib) and pneumococcus type 6A (Pn6A) capsular polysaccharides conjugated to either tetanus toxoid (TT), horseshoe crab hemocyanin, or cholera toxin (CT), and the antibody responses of the monkeys to both bacterial components were measured. All three Hib conjugates were immunogenic and elicited booster responses; their comparative immunogenicity was Hib-CT greater than Hib-TT greater than Hib-horseshoe crab hemocyanin. Hib alone did not elicit antibodies in the juveniles. Juveniles responded earlier and with higher levels of antibodies than did infants. TT, as well as diphtheria-tetanus toxoids-pertussis vaccine adsorbed injected concurrently at a separate site, increased both Hib and TT antibody responses in juveniles (P less than 0.05). Concurrent injection of 5 Lf of fluid TT with a nonimmunogenic 5-micrograms dose in infants elicited levels of Hib antibodies comparable to those elicited by 50 micrograms of Hib-TT. Hib antibodies elicited by the conjugates remained at protective levels in both juveniles and infants 2 months after the last injection, were bactericidal, and conferred passive immunity against bacteremia in infant rats. Passive immunization of juveniles with tetanus immune globulin before each injection of Hib-TT did not suppress Hib antibodies. Hib-TT and Hib-CT elicited increases of Hib antibodies of the immunoglobulin M and G isotypes in the infants. The Pn6A-TT conjugate was considerably less immunogenic than the Hib-TT conjugate; only a few of the juveniles or infants responded with protective levels of Pn6A antibodies. Pn6A antibodies from responders conferred protection in mice against intraperitoneal challenge with Pn6A organisms. TT antibodies were elicited in both juvenile and infant animals after one injection of 50 micrograms of Hib-TT and in the infants injected with 5 micrograms of Hib-TT plus 5 Lf of TT; 5 micrograms of Hib-TT and Pn6A-TT in combination alone did not elicit TT antibodies. Hib-CT elicited CT antibodies in both juveniles and infants.

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.

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.

Tetanus toxin: convulsant action on mouse spinal cord neurons in culture.

The effects of direct application of tetanus toxin on fetal mouse spinal cord neurons in culture are described. Tetanus toxin produces increased excitation characterized by paroxysmal depolarizing events (PDE). In contrast to the abrupt onset of convulsant action produced by postsynaptic glycine antagonist strychnine, the convulsant action of tetanus occurs after a dose-dependent latent period. The onset of the convulsant action of tetanus toxin is paralleled by a reduction in observed spontaneous inhibitory synaptic potentials. Excitatory synaptic events can be identified as components of some tetanus-PDE. The toxin does not alter postsynaptic responses to the inhibitory amino acids glycine and gamma-aminobutyric acid. The latency and convulsant action of tetanus toxin are consistent with an irreversible presynaptic membrane interaction that reduces inhibitory transmission, a mechanism of action distinct from those of convulsants that antagonize inhibitory transmitters at the postsynaptic membrane.

Structural characteristics of tetanolysin and its binding to lipid vesicles.

Tetanolysin binding to lipid vesicles was found to depend on the molar ratio of cholesterol to phospholipid, being low in vesicles containing up to 20 mol% cholesterol and high in vesicles containing more than 33 mol%. High concentrations of purified tetanolysin preparations formed arc- and ring-shaped structures. The structures were not readily detectable in diluted preparations unless incubated with lipid vesicles containing high molar ratios of cholesterol to phospholipid. It is suggested that the toxin is concentrated on the vesicles to local concentrations high enough to form the arcs and rings.

Receptor-mediated endocytosis of diphtheria toxin by cells in culture.

The binding and uptake of fluorescently labeled diphtheria toxin by cells in culture has been examined by using epifluorescence video intensification microscopy. Rhodamine-labeled diphtheria toxin retained significant toxicity on bioassay and in cell culture and was tested for uptake by human WI-38 and mouse 3T3 fibroblasts grown in culture. When added to cells at 37 degrees C, toxin was observed to become concentrated and internalized in discrete vesicles in both cell lines. The appearance of fluorescent clusters could be prevented by addition of excess unlabeled diphtheria toxin to the medium or by addition of ATP (which has been shown to block toxin binding to cells), indicating that the rhodamine-labeled toxin was binding to diphtheria toxin-specific cell surface binding sites. When the simultaneous uptake of rhodamine-labeled diphtheria toxin and fluorescein-labeled alpha 2-macroglobulin was monitored, the two proteins appeared in the same clusters indicating that the toxin undergoes receptor-mediated endocytosis. Despite the difference in susceptibility to diphtheria toxin of cells derived from sensitive (human) and resistant (mouse) tissues, the behavior of the rhodamine-labeled derivative in both cell lines was indistinguishable in terms of toxin required for formation of clusters or inhibition by unlabeled toxin or by ATP. These results demonstrate that diphtheria toxin-specific cell surface binding sites occur on both insensitive and sensitive cells and suggest that toxin is processed similarly by both cell types during its initial cell surface binding and internalization by this pathway. The possible involvement of this uptake system in the mechanism of action of diphtheria toxin in cells is discussed.

Tetanus toxin association with developing neuronal cell cultures. Kinetic parameters and evidence for ganglioside-mediated internalization.

Rat cerebral neurons maintained in monolayer culture accumulate 125I-labeled tetanus toxin. Accumulation is receptor-mediated; i.e. it can be prevented by including unlabeled tetanus toxin, gangliosides, or tetanus antitoxin in the incubation medium but not by including tetanus toxoid, high concentrations of serum, or thyrotropin. Accumulation is time-dependent, reaching a plateau after approximately 3 h when 60% of the added toxin is associated with the cells. It is better at 0 degrees C than at ambient temperature and is significantly higher when 0.25 M sucrose replaces physiological salts in a medium containing 5% serum. Unlabeled tetanus toxin, tetanus antitoxin, and tetanus toxoid do not release the accumulated 125I-labeled tetanus toxin to any significant degree; however, gangliosides (50 micrograms/ml) can release 30% of the accumulated 125I-labeled toxin. Treatment of cells with Triton X-100, under conditions where over 90% of the lipids and 70% of the gangliosides are removed, extracts only 15% of the cell-associated 125I-labeled toxin. Evidence is presented that over 50% of the accumulated toxin is internalized in a cellular compartment which is not in immediate equilibrium with the extracellular environment and which is associated with detergent-insoluble cellular constituents. The tetanus toxin accumulated in this compartment has the same gel electrophoretic pattern as the native toxin and is bioactive. The role of gangliosides as potential shuttle vehicles for tetanus toxin internalization is discussed as are the implications of these data to in vitro studies of the pathogenesis of tetanus-induced neurotoxicity.

Effect of tetanus toxin on the accumulation of the permeant lipophilic cation tetraphenylphosphonium by guinea pig brain synaptosomes.

Accumulation of the permeant lipophilic cation [(3)H]tetraphenylphosphonium (TPP(+)) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K(+) and by veratridine. A further 1:10 to 1:15 decrease in TPP(+) uptake occurs under nitrogen and in the presence of mitochondrial inhibitors such as oligomycin, whereas starvation and succinate supplementation have no effect. These data indicate that, in analogy to intact neurons, there is an electrical potential (DeltaPsi, interior negative) of -60 to -80 mV across the synaptosomal membrane that is due primarily to a K(+) diffusion gradient (K(+) (in)-->K(+) (out)). The data also indicate that mitochondria entrapped within the synaptosome but not free mitochondria make a large contribution to the TPP(+) concentration gradients observed. Conditions are defined in which tetanus toxin binds specifically and immediately to synaptosomes in media used to measure TPP(+) uptake. Under these conditions tetanus toxin induces dose-dependent changes in TPP(+) uptake that are blocked by antitoxin and not mimicked by biologically inactivated toxin preparations. The effect of tetanus toxin on TPP(+) uptake is not evident in the presence of 193 mM K(+) or veratridine but remains under conditions known to abolish the mitochondrial DeltaPsi. Moreover, tetanus toxin has no effect on TPP(+) uptake by isolated synaptosomal mitochondria. The results thus define an in vitro action of tetanus toxin on the synaptosomal membrane that can be correlated with biological potency in vivo and is consistent with the in vivo effects of tetanus toxin on neuronal transmission.