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PubMed2005

Diphtheria, tetanus and pertussis antibodies in 10-year-old children before and after a booster dose of three toxoids: implications for the timing of a booster dose.

UNLABELLED: In an open study, 502 10-year-old children, who had received primary vaccination against diphtheria and tetanus in infancy and had varying histories of pertussis disease and vaccination, were vaccinated with diphtheria-tetanus vaccine (DT) alone or with the addition of 20 microg or 40 microg of pertussis toxoid. Diphtheria toxin neutralising antibodies, pertussis toxin IgG and tetanus toxoid IgG antibodies were measured before and 1 month after the booster. All toxoids were highly immunogenic. In pertussis toxoid recipients, median levels of pertussis toxin IgG increased to 16.5 U/ml (DTaP20) and to 36 U/ml (DTaP40) in children with non-detectable (<1 U/ml) antibodies before vaccination and to >400 U/ml in children (both DTaP20 and DTaP40) with detectable antibodies before vaccination. A total of 60 children (12%) with non-detectable (<0.01 IU/ml) diphtheria antibodies and 36 children (7%) with non-detectable (<0.01 IU/ml) tetanus antibodies before the booster had lower median antibody concentrations post-vaccination than children with detectable antibodies before the booster (diphtheria: 5.12 vs. 20.48 IU/ml; tetanus: 4.0 vs. 10.0 IU/ml). There were no differences in diphtheria and tetanus antibodies after vaccination between children who did and did not receive pertussis toxoid. CONCLUSION: 10-year-old children with non-detectable diphtheria and tetanus antibodies before the booster had lower post-vaccination antibodies than those with detectable antibodies before the booster indicating a poor immunological memory. Addition of pertussis toxoid to diphtheria-tetanus vaccine did not affect the antibody responses to diphtheria and tetanus toxoids when the three toxoids were combined as a booster. Even though immunity to diphtheria and tetanus was only estimated by surrogate markers (serum antitoxin antibodies) the results indicate that a lower age for the booster dose of diphtheria-tetanus vaccine or diphtheria-tetanus acellular pertussis vaccine should be considered.

Antibodies, Bacterial↗
PubMed1991

Tetanus: pathophysiology, management, and prophylaxis.

As tetanus has become a rare disease in the developed world, physicians have become less comfortable with its diagnosis and management. The extent of adequate antitetanus immunity in the adult population, especially the elderly, is waning, in great measure because primary care physicians have not made prophylaxis a priority in their routine encounters with patients. Furthermore, as the population of immunocompromised hosts grows, an increasing percentage of our patients may not respond to standard active immunization routines. Unless these trends are reversed, we face a substantial increase in the incidence of this dread disorder. Tetanus is also of interest as a relatively simple model of disordered motor control that can instruct us in the management of the many more common causes of neurogenic muscular rigidity. The toxin produced by Clostridium tetani finds increasing use in laboratories investigating brain function as well. Clinical tetanus is divided into four symptomatic types: generalized tetanus, local tetanus, cephalic tetanus, and neonatal tetanus. This monograph discusses the diagnostic aspects of each type of tetanus, its pathophysiology, diagnosis, differential diagnosis, and treatment. Preventing tetanus should be a high priority for all primary care physicians. Active immunization with tetanus toxoid is remarkably effective and safe. Passive immunization with human tetanus immune globulin is indicated in certain circumstances, which are discussed below.

Adult↗
PubMed1992

Lack of early antitoxin response to tetanus booster.

Tetanus immune globulin (TIG) continues to be recommended in persons with tetanus-prone wounds who have incomplete or unknown tetanus immunization status. The aim of this study was to determine whether, following a booster dose of tetanus toxoid in adults who had not been immunized in the previous 10 years, there was an antitoxin response to tetanus toxoid booster within 4 days. Thirty-one adults were investigated, baseline levels for tetanus antitoxin assayed using an ELISA technique, and an injection of adsorbed tetanus toxoid (0.5 ml) given. Blood samples for tetanus antitoxin levels were taken at daily intervals for the 4 days following immunization. Tetanus boosters following the primary course but before the present study did not significantly increase the levels of pre-study tetanus antitoxin and following the study booster there was no difference between the preboost levels and the levels on days 1 to 4. This finding indicates that the present recommendations for the use of TIG in tetanus-prone wounds are appropriate.

Adult↗
PubMed1991

Cluster survey for poliomyelitis and neonatal tetanus in Ilorin, Nigeria.

The incidence of poliomyelitis and neonatal tetanus in Ilorin Local Government Area was estimated using a 40-cluster, retrospective survey of neonatal tetanus deaths and lameness from poliomyelitis in March and April 1988. The survey identified 19 neonatal tetanus deaths among 1601 livebirths, giving an incidence rate of 14.9/1000 livebirths. Some 31 residual paralytic polio cases were identified among 4576 children aged five to nine years, giving a prevalence rate of 6.8/1000 children in this age group and an annual incidence rate of 38.3/100,000 general population. Immunization of pregnant women with tetanus toxoid, had a greater impact on mortality from neonatal tetanus than delivery in hospital or birth at home attended by a traditional birth attendant. Antenatal coverage with two doses of tetanus toxoid was 62.6%. Poliomyelitis and neonatal tetanus still constitute important causes of childhood morbidity and mortality in Ilorin and antenatal tetanus immunization coverage is not yet sufficient to control neonatal tetanus in this population. The target age for immunization with tetanus toxoid should be extended to include all women of childbearing age. Further retrospective surveys from other Nigerian states are needed to determine the magnitude of the neonatal tetanus problem throughout the country.

Child↗
PubMed2006

Measurement of tetanus antitoxin in oral fluid: a tool to conduct serosurveys.

BACKGROUND: Serosurveys that measure tetanus antitoxin can complement immunization coverage surveys to allow evaluation of immunization services in developing countries. Measurement of IgG tetanus antitoxin in oral fluid was investigated as a practical and noninvasive alternative to and correlate of serum antibodies. METHODS: Serum and oral fluid were collected from Malian infants, toddlers and adults (males without a history of tetanus vaccination). Specific IgG tetanus antitoxin was measured by enzyme-linked immunosorbent assay in serum (S-ELISA) and oral fluid (OF-ELISA). RESULTS: One hundred forty-two pairs of serum and oral fluid samples were collected from infants, 35 pairs from toddlers and 35 pairs from adults. IgG tetanus antitoxin titers measured by OF-ELISA were 100-fold lower than those measured by S-ELISA but they correlated strongly (r = 0.90, P < 0.001). All 35 toddlers who had received 2 or 3 doses of diphtheria-tetanus-pertussis (DTP) vaccine (100%) had serum tetanus antitoxin levels >or=0.15 IU/mL and 28 of 35 (80%) had oral fluid values >or=0.0015 IU/mL. Among adults lacking a history of tetanus immunization, only 6 of 35 (17.1%) had serum titers >or=0.15 IU/mL and 4 of 35 (11%) had oral fluid titers >or=0.0015 IU/mL in oral fluid. CONCLUSIONS: IgG tetanus antitoxin in oral fluid correlates well with levels in serum. OF-ELISA values >or=0.0015 IU/mL constitute protection against tetanus and in subjects >12 months of age imply multiple prior contacts with immunization services. IgG tetanus antitoxin measured by OF-ELISA provides a logistically practical alternative for performing seroprevalence surveys.

Body Fluids↗
PubMed1999

[Tetanus in 1997].

In 1997, 37 cases of tetanus were registered in Poland. The incidence rate of tetanus was 0.1/100,000 population. No neonatal tetanus cases were recorded. The last neonatal tetanus case had been reported in this country 14 years ago and the last death from neonatal tetanus over 30 years ago. Two tetanus cases occurred in persons aged 20 and 28, who underwent a full course of tetanus vaccination and received their last booster dose of tetanus vaccine not more than 10 years ago. 70% of cases occurred in persons aged 60 and over. Case-fatality rate associated with tetanus was 27%. By the end of 1997, 97.8% of children born in 1996 have been immunized with at least three doses of DTP vaccine. High vaccine coverage rates in Poland maintained since many years are an effective means of eliminating tetanus in the population. Since many years a decreasing number of tetanus cases are registered.

Adult↗
PubMed2003

Management and prevention of tetanus.

The World Health Organization was committed to eliminating neonatal tetanus by 1995. Three years after this date, the infection killed over 400,000 babies a year, even though a safe, effective vaccine had been available for most of this century. The frequency of tetanus in the developing world epitomizes the healthcare disparity between the developed and the developing world. Consequently, the priority of the medical profession must be prevention, with the development of simpler immunization schedules with longer protection. Consequently, the purpose of this collective review is to provide an overview to the management of tetanus as well as to review the immunization strategy that will prevent this potentially deadly illness. Tetanus is caused by Cloistridium tetani, which is an obligate anaerobic, gram-positive rod that is motile and readily forms endospores. Although C. tetani is located everywhere, the disease is encountered largely in underdeveloped, overcrowded, and economically disadvantaged countries. C. tetani is widespread in the feces of domestic animals and humans, while spores of C. tetani are abundant in soil and in the environment surrounding the habitation of humans and animals. Tetanus usually follows deep penetrating wounds where anaerobic bacterial growth is facilitated. Three basic forms of tetanus may be distinguished: local, cephalic, and generalized. At least 80% of the cases are the generalized form. In the adult patient, the most characteristic sign of generalized tetanus is lockjaw, or trismus. The diagnosis of tetanus is most frequently made on clinical manifestations, rather than on bacteriologic findings. The three objectives of management of tetanus are: (1) to provide supportive care until the tetanospasmin that is fixed in tissue has been metabolized; (2) to neutralize circulating toxin; and (3) to remove the source of tetanospasmin. Because there is essentially no immunity to tetanus toxoid, the only effective way to control tetanus is by prophylactic immunization.

Adult↗
PubMed2003

Tetanus immunization shortage in the United States.

The purpose of this study is to determine the effect of the tetanus immunization shortage on EDs and the EPs understanding of the prioritization of persons needing tetanus immunization. A survey consisting of questions about knowledge of the tetanus shortage, prioritization of immunizations, incidence of tetanus infections, and understanding of CDC recommendations was mailed to a random sample 20% of the US ED medical directors. The results of the survey were input into the SPSS program (SPSS, version 10, Chicago, IL). The survey was returned by 618 of the 1,375 (44.9%) ED medical directors in the United States. Almost all (97.2%, 601 of 617) were notified about the tetanus shortage and 58.3% (360 of 617) reported a shortage. A total of 42.2% (199 of 472) gave tetanus toxoid (TT), instead of tetanus and diphtheria toxoids adult type (dT) when indicated. Only 11.6% of those surveyed (56 of 482) established a patient callback system. Routine vaccination was stopped in 37.5% of the reporting hospitals, most often for adults and children (57.5%, 69 of 120). Twelve hospitals (1.9%) reported they had an increase in tetanus. Although 87.5% of the respondents (539 of 616) stated they were familiar with the CDC's prioritization for tetanus immunization, only 1.8% (11 of 616) got the prioritization correct. Although EM directors uniformly know about and are experiencing the tetanus shortage, few correctly reported the tetanus immunization priority. Few EDs had a patient callback system.

Adult↗
PubMed1959

The relationship of toxin and antitoxin injection site to tetanus development in the rat.

The pattern of development of symptoms was studied following injection of 3 MLD (minimum lethal dose) of tetanus toxin in each of the following sites in the rat: vein, muscle, endoneurium, epineurium, spinal cord, subarachnoid space, and subdural space. Similar observations were made when 3 MPD (minimum protective dose) of antitoxin were injected into each of the above sites a few minutes after the intramuscular injection of toxin. Local tetanus followed intramuscular, endoneurial, and epineurial injection of the toxin; blood-borne tetanus also appeared in the latter instance. Tetanus dolorosus followed intraspinal injection of toxin. Both dorsal tetanus and blood-borne tetanus developed after subdural administration of toxin. Blood-borne tetanus, alone, appeared following intravenous and subarachnoid injection of toxin. In no case did injection of antitoxin at the various sites listed above prevent local tetanus from developing after intramuscular injection of toxin. Of the various sites injected with antitoxin, the intramuscular, intraspinal, and intravenous were the most effective. It is suggested that the endoneurial tissue spaces serve as a conduit for tetanus toxin from the muscle to the CNS. The perilemma of the peripheral nerve trunk may act as a selectively permeable membrane which permits tetanus toxin to pass from the epineurium to the endoneurium. Outward diffusion of toxin from the endoneurial spaces is apparently markedly reduced. The antitoxin probably is prevented from permeating this barrier in either direction. The importance of absorption of tetanus toxin by the lymphatic and blood circulation is discussed.

Animals↗
PubMed1990

Prevention by the NMDA receptor antagonist, MK801 of neuronal loss produced by tetanus toxin in the rat hippocampus.

1. The behavioural and neuropathological effects of tetanus toxin, microinjected directly into the hippocampus, were studied in rats. 2. A single dose (1000 minimum lethal doses, MLDs) of tetanus toxin, injected unilaterally into the hippocampus produced a time-dependent neuronal loss in the CA1 pyramidal cell layer. In comparison with the contralateral, untreated side these effects became statistically significant (P less than 0.05) 7 days (22.0 +/- 1.1% reduction) and 10 days (29.2 +/- 1.7% reduction) after the injection. No significant changes were observed 7 days after treatment with 500 MLDs whereas a reduction of 37.5 +/- 3.1% in the CA1 area cell number was produced 4 days after the injection of 2000 MLDs. 3. Behavioral stimulatory effects were also induced by tetanus toxin (1000 MLDs) within 48 h of the injection and these culminated in generalized convulsions 5-7 days later. Convulsions were observed after a shorter period of latency in rats receiving 2000 MLDs tetanus toxin whereas 500 MLDs were ineffective. 4. No behavioural and neuropathological effects were observed in rats treated with neutralized tetanus toxin (1000 MLDs), bovine serum albumin or phosphate buffer. 5. Pretreatment with MK801 (0.3 mg kg-1, i.p., given 1 h before and after the injection with tetanus toxin and then once daily for 4 or 7 days) prevented the behavioural and neuropathological effects induced by tetanus toxin (1000-2000 MLDs). In addition, such treatment fully protected the animals from the lethal effects induced by 1000 MLDs tetanus toxin. In addition, such treatment fully protected the animals from the lethal effects induced by 1000MLDs tetanus toxin. By contrast, pretreatment with diazepam (3.Omgkg-1, i.p.) using the same schedule as for MK801 did not antagonize the effects of tetanus toxin (1000-2000 MLDs). 6. In conclusion, the present experiments have demonstrated that the intrahippocampal injection of tetanus toxin produces in rats a dose- and time-dependent behavioural stimulation and neuronal loss in the CAl pyramidal cell layer which can be prevented by the non-competitive NMDA antagonist, MK801.

Animals↗
PubMed1999

Neuronal binding of tetanus toxin compared to its ganglioside binding fragment (H(c)).

The non-toxin 50 kD C-terminus peptide of the heavy chain of tetanus H(c) contains the ganglioside binding domain of tetanus toxin (TTX). H(c) retains much of the capacity of tetanus toxin for binding internalization and transport by neurons. For this reason tetanus H(c) has been studied as a vector for delivery of therapeutic proteins to neurons. We directly compared H(c) and TTX in the capacity to bind and be internalized by neurons by ELISA. Primary cultures of dissociated fetal cortical neurons were incubated with equimolar amounts of TTX or H(c). Neuronal associated tetanus protein was 4-8 fold greater on a molar basis with tetanus toxin compared to H(c) (1 h incubation). This increase in neuronal tetanus protein was evident with incubation in concentrations from 0.1 microM to 2 microM. There were greater amounts of TTX delivered to the cultured cells at both 0 degrees C (representing membrane bound tetanus protein) and 37 degrees C (bound and internalized tetanus protein). Unlike H(c), TTX showed significant continued accumulation of protein with increasing incubation durations. Neuronal associated TTX increased 2-3 fold over incubation times ranging from 1 to 8 h. Tetanus toxin appears to be clearly superior to the ganglioside binding fragment (H(c)) in the capacity for neuronal binding and internalization. Atoxic tetanus proteins containing additional molecular domains as well as H(c) may be more suitable vectors for linkage with therapeutic proteins and delivery to neurons.

Animals↗
PubMed1981

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.

Animals↗
PubMed1975

New concepts on tetanus immunization: naturally acquired immunity.

Comments were made on misinterpretations concerning the natural resistance and natural immunization against tetanus. Arguments were given explaining why the disease itself did not determine adequate immunity. When, however, adequate conditions appear, tetanus toxin is known to stimulate the immune system and produce detectable humoral antibodies. Various possibilities resulting from the postulated harboring of tetanus bacilli by the human body and their eventual toxin production were analyzed and related to the human tetanus pathology. The existence of natural immunization was unquestionably demonstrated by presence of protective levels of tetanus antitoxin in the blood of the majority of 59 surveyed subjects considering that none of them had ever received any tetanus toxoid and most of them never received a single shot of any drug. The results of this survey originated a few arguments that may support the answer to some still intriguing phenomenona such as: 1. The relatively small number of cases of overt disease among people and animals born and living in large tetanus-risk regions all over the world. 2. The existence of "poor responders" and "good responders" to the primary tetanus toxoid stimulus. 3. The age distribution of tetanus showing evident prevalence among newborns and children. 4. The wide individual variations in the clinical picture of human tetanus as indicated by the localization and limitation of the symptoms and their severity.

Adolescent↗
PubMed1985

The continuing problem of tetanus.

Thirty-eight instances of tetanus were treated during a recent 20 year period at the University of Mississippi and Jackson Veterans Administration Medical Centers. One patient had received a single prior dose of tetanus toxoid and the remainder had never received tetanus toxoid. Sixteen patients sought medical care for their tetanus wound prior to the onset of clinical tetanus, but none received specific antitetanus prophylaxis. The majority of tetanus wounds were located on lower extremities and often were chronic vascular ulcers. The over-all mortality was 37 per cent and survival rate was not affected by patient age, duration, location or severity of the tetanus wound or presence of associated diseases. Aggressive surgical treatment of the tetanus wound was associated with decreased mortality for uncertain reasons. Although low mortality from tetanus is possible with improved intensive care technology, the disease should be virtually preventable by the provision of proper tetanus prophylaxis to all patients at risk.

Adolescent↗
PubMed1997

Immunologic response to tetanus toxoid in geriatric patients.

STUDY OBJECTIVE: Tetanus antibody levels have been shown to be inadequate in 50% of patients older than 65 years. Although immunization recommendations have been made for this age group, the efficacy of this intervention has not been well documented. We sought to determine the difference in tetanus antibody levels after the administration of one tetanus toxoid immunization to geriatric patients without adequate titers. METHODS: Thirty-five patients older than 65 years at a large urban comprehensive care geriatric center who were documented to have inadequate tetanus antibody titers were each given one tetanus toxoid immunization. Repeat titers were obtained at least 2 months after the immunization with the use of enzyme-linked immunosorbent assay (Bindazyme kit; the Binding Site Corporation, Birmingham, England). We considered tetanus antibody levels greater than .17 IU/mL protective. RESULTS: The mean age was 79.4 years; 30 of 35 (86%) were female. Repeat tetanus antibody titers were obtained an average of 123 days (range, 63 to 204 days) after immunization with tetanus toxoid. The mean preimmunization antibody titer was .1 IU/mL (range, .04 to .16 IU/mL). After immunization, antibody titers increased a mean of .61 IU/mL (range, -.01 to 2.23 IU/mL; 95% confidence interval, .35 to .87 IU/mL). Thirty of the 35 patients who received a single injection of tetanus toxoid (86%) developed protective titers. We found no relationship between seroconversion and age, sex, or medical history; nor did we find a relationship between antibody level and time elapsed since immunization when repeat titers were obtained. CONCLUSION: Administration of one tetanus toxoid injection affords protective immunity in many geriatric patients.

Aged↗
PubMed1996

Tetanus--still a topic of present interest: a report of 27 cases from a Belgian referral hospital.

OBJECTIVE: To study the clinical presentation, treatment and outcome of tetanus in a European population. DESIGN: A retrospective study. SETTING: A large university hospital with a regional as well as a referral service. SUBJECTS: All patients with tetanus from 1983 till 1993. RESULTS: Twenty-seven patients (13 men, 14 women; mean age 68.5 years) were studied. Seventeen out of 24 wounds (three patients had no history of a recent wound) were described as tetanus-prone. Ten patients had medical wound care, but none received tetanus immunoglobulins despite the absence of tetanus immunity. All patients had the generalized type of the disease. The classical symptoms of trismus, dysphagia and muscular rigidity were present in all patients. Treatment consisted of wound management, neutralization of tetanus toxin by immunoglobulins, antibiotics, treatment of muscle spasm and instability of the autonomic nervous system, and supportive care. Twenty-five patients were admitted to the intensive care unit for a mean duration of 25.7 days. Sixteen patients were artificially ventilated for a mean duration of 27.5 days. Three patients died, 11 patients had a complicated course caused by haemodynamic instability and 15 patients had pulmonary complications. CONCLUSIONS: Tetanus has not vanished. It remains a difficult-to-treat disease with a substantial morbidity and mortality rate. Prevention during wound management of tetanus-prone wounds was inappropriate in many patients. The elderly population may have the highest risk for tetanus since they may not have had tetanus toxoid immunization or regular booster injections.

Aged↗
PubMed2003

Tetanus immunisation in geriatric patients with accidental wounds: how much is needed?

OBJECTIVE: In the industrialised world, the elderly carry the highest risk for tetanus. This prospective serological study was performed to evaluate the reliability of tetanus immunisation histories and the antibody response to tetanus vaccinations in the elderly. METHODS: 40 individuals >65 years with a bleeding trauma were included in the study. Their tetanus vaccination histories were investigated, and accordingly a single booster (group A, n = 7), or a three dose basic immunisation against tetanus (group B, n = 33) was provided. In addition, tetanus antitoxin levels were determined. RESULTS: Age varied between 67 and 95 years. Inconsistencies regarding the vaccination history were found between patients and their physicians in 30% (12/40), between patient statements and vaccination documents in 57.1% (8/14) and between physicians' records and vaccination documents in 35.7% (5/14). Antitoxin titres >0.15 IU/ ml were considered protective, giving a seroprevalence of 92.5%. Sensitivities and negative predictive values for tetanus immunity were 30.6% and 10.7% based on patient histories and 2.8% and 7.9% based on physicians' records. In group A, after the single booster immunisation the median titre rose from 1.2 to 14.2 IU/ml, in group B from 0.9 to 5.3 IU/ml after the first, and to 9.6 IU/ml after the third tetanus toxoid dose (p <0.001 using Friedman's test). CONCLUSIONS: In Switzerland, elderly patients with a tetanus prone wound provide an unreliable vaccination history but their seroprotection against tetanus is high. A single booster for secondary immunisation is therefore sufficient. It is not necessary to take the largely unreliable vaccination history into account.

Aged↗
PubMed1990

Preparation of affinity-purified, biotinylated tetanus toxin, and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells.

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 1:1 to 20:1 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60-80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using 125I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2-3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these "primed" cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.

Adrenal Gland Neoplasms↗