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I.v botulinum toxin after 60-90 min abolished the dose-response relationship between pentagastrin and gastric acid secretion in anesthetized rats and guinea-pigs. The toxin reduced but did not abolish the acid stimulatory effect of histamine. As expected, the acid response to vagal stimulation was abolished and that to methacholine in rats was unaltered by the toxin.
Small doses of botulinum toxin can produce partial blockage of transmitter release at the nerve--muscle junction. 2. Subthreshold e.p.p.s, 3--10 days after poisoning, show a distribution of amplitudes that is fitted by Poisson statistics. Successive e.p.p.s. in a short train show a marked facilitation. 3. Two weeks or more after poisoning with a dose of toxin that paralyses the whole muscle, when nerve--muscle transmission is in course of recovery, subthreshold e.p.p.s have an amplitude distribution that is fitted by binomial statistics. This property of transmission is similar to those described in newly formed nerve--muscle junctions, during embryogenesis or regeneration. 4. Muscle fibres with subthreshold transmission in the 5--10 day group of muscles were all supersensitive to ACh, as were a number of fibres in which nerve stimulation still produced an action potential. 5. Two weeks or more after poisoning, muscle fibres with subthreshold transmission had lost their extrajunctional ACh-sensitivity, as had many fibres with m.e.p.p.s of roughly normal frequency but no response to nerve stimulation. 6. In diaphragm muscles poisoned with botulinum toxin between 1 and 4 days previously, the rate of fast axonal transport of radioactively labelled proteins down the phrenic nerve is not greatly affected, but the amount of materials carried is reduced to about one quarter of normal. These labelled proteins accumulate in the intramuscular portion of the phrenic nerve, in or near the nerve terminals, to a much greater extent than in controls, showing that the normal release of some of these materials has been prevented by the toxin. 7. It is concluded that the blockage of the trophic effects of nerves by botulinum toxin is due to a blockage of release of trophic factors other than ACh. 8. The muscle nerve cannot maintain a muscle in its normal state simply by activation of contraction, and a regenerating nerve terminal can restore a muscle towards its normal state before it can release enough ACh to produce muscle contraction.
Highly purified preparations of Clostridium botulinum toxins were administered to chickens by various routes. Chickens were highly susceptible to type A toxin, but relatively resistant to toxins of other types. Type C toxin (12S) at a dose of 1 X 10(7) mouse ip LD50 failed to kill the chicken by the oral route. Oral administration of 10 or more of type A, C, or D spores killed normal chickens, whereas cecoligated chickens were insusceptible to oral administration of 10(6) spores. These results show that the site of production and absorption of botulinum toxin in chickens is the cecum. Peroral administration of spores of a type C strain cured of its prophages and producing the C2 factor only also killed normal chickens. Chickens appeared to the more susceptible to the C2 factor than to the C1 toxin. The C2 factor, therefore, may play more important role in chicken deaths from toxico-infection with type C organisms. The optimum temperature for growth of C. botulinum types C and D was found to be 40-42 C. Type C and D toxins were significantly more stable than type A toxin in the cecum contents with pH above 7. These characteristics and the high density of distribution of type C spores in the environment may explain prevailing cases of type C botulism among broiler chickens.
The ultrastructural effects of local injection of botulinum toxin into the left half of the tongue of the rat, were studied quantitatively 35 days postoperatively in the left hypoglossal nucleus. The results showed (1) a decrease in somatic and neuropil bouton numbers because of loss of boutons with symmetrical synapses and clear spherical synaptic vesicles, (2) a decrease in the numbers of dendrite profiles in the neuropil, (3) an increase in the proportion of dendrites and boutons with unusual inclusions, suggestive of profile retraction, (4) an increase in the proportion of profiles which were unusually electron-dense, (5) an increase in the amount of astrocyte, and a growth of astrocyte sheaths around bouton-free neurone surfaces, (6) the presence of occasional microglia, and (7) subastrocytic subsurface cisterns. Control rats injected with boiled toxin had no responses except (3) and (4) above, and then only to a modest extent, possibly due to mechanical damage of a few axons or terminals at the time of injection, or to insufficient inactivation of the toxin by boiling. The results were compared with those at 35 days after axotomy, and it was concluded that botulinum toxin, which interrupts neuromuscular transmission, elicits the same responses in the hypoglossal neurones, as does transection of the hypoglossal nerve, even though earlier studies had discovered no glial replication after botulinum toxin, in contrast to axotomy.
Upper limb spasticity is a common and disabling complication of stroke. Botulinum toxin type A (BoNT-A) is widely used for focal spasticity treatment, but naturally derived products may present limitations related to immunogenicity and manufacturing variability. Recombinant botulinum toxin type A, produced by genetic engineering without complexing proteins, may provide improved product consistency. This Ib/II study evaluated the safety, tolerability, and preliminary efficacy of recombinant botulinum toxin type A in adults with post-stroke upper limb spasticity. This multicenter, seamless Ib/II clinical study included an open-label dose-escalation Ib phase and a randomized, double-blind, placebo-controlled II phase. Adult patients with post-stroke upper limb spasticity received a single intramuscular injection of recombinant botulinum toxin type A or placebo. The primary endpoint in Phase II was the change from baseline in the Modified Ashworth Scale (MAS) score of the primary target muscle group at Week 4. Secondary endpoints included MAS and Tardieu scale changes in individual muscle groups, Disability Assessment Scale (DAS), Physician's Global Assessment (PGA), and immunogenicity. The Ib phase showed improvements in MAS, DAS, and PGA, indicating an early efficacy signal. In Phase II, recombinant botulinum toxin type A produced a significant reduction in MAS score of the primary target muscle group at Week 4 compared with placebo, with effects sustained through Week 12. At Week 4, the PGA score in the Eveotox® group showed a statistically significant improvement compared with the placebo group. While MAS and PGA scores showed significant improvement, DAS functional scores did not differ statistically from the placebo group at week 4. The treatment was generally well tolerated, and low incidence of antibodies were observed. Recombinant botulinum toxin type A was safe and effective in reducing post-stroke upper limb spasticity after a single administration. These results support further Phase III clinical evaluation.
1. Structural changes which normally accompany transmitter release at frog neuromuscular junctions are visualized with the freeze-fracture technique. The effects of brown widow spider venom and botulinum toxin were evaluated in terms of their ability to block or produce these structural changes. Changes produced by these neuropoisons were correlated with their known effects on neurotransmitter release. 3. Fusion of synaptic vesicles with the presynaptic plasmalemma, normally evoked by electrical stimulation, was abolished at neuromuscular junctions from frogs treated with botulinum toxin. 3. The concentration of large intramembranous particles in the presynaptic plasmalemma, an indication of the excess of synaptic vesicle fusion over recovery of synaptic vesicle membrane, was increased by treatment with brown widow spider venom, even in the presence of botulinum toxin. 4. When external calcium was present, sites of vesicle fusion induced by brown widow spider venom, as well as by electrical stimulation, were located mainly in the active zone. In the absence of external calcium, many plasmalemmal deformations, also though to be sites of vesicle fusion, were more evenly dispersed over the presynaptic surface of nerve terminals. 5. Botulinum toxin decreased the number of vesicle fusion sites in the active zone induced by spider venom in the presence of external calcium but had little effect on the number of fusion sites induced by spider venom in the absence of external calcium. 6. Nerve terminals soaked in a sodium-free Ringer solution were partially depleted of vesicles. Addition of spider venom to this Ringer did not cause additional depletion of vesicles. 7. Formation of cation-permeable channels in the presynaptic membrane could account for these effects of spider venom on the frog neuromuscular junction. Botulinum toxin blocks vesicle fusion by some means which is not yet understood.
A new method of isolation of highly purified Cl. botulinum toxin of E type from the cultural fluid of strain 188 centrifugates was developed. The method allows to isolate the toxin both in a precursor and in activated forms with a yield of 10--15%. The method includes fractionation by ammonium sulfate, ultrafiltration and subsequent column chromatography on DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex A-50. The preparations were found homogeneous during polyacrylamide gel electrophoresis and immunoprecipitation in agar with antitoxic horse serum. The potential specific toxicity of the preparations is 1--1,2.10(7) DLM/mg of protein. The molecular weight of the toxin is about 160 000; the molar extinction coefficient is equal to 278 nm. The isoelectric point lies around pH 6.0. The highly purified Cl. botulinum toxin of E type was found stable upon storage.
The injection of botulinum toxin type A into the hind-leg of adult rats causes complete paralysis of the leg lasting for several weeks. In the extensor digitorum longus (EDL) muscle transmitter release is reduced to a level of less than 1% of normal. Tetraethylammonium (TEA) and guanidine in concentrations of about 3 mM restore, in EDL muslces in vitro, neuromuscular transmission to about the normal level, provided that the external calcium concentration is 4 mM or higher. 4-Aminopyridine (4-AP) has similar restorative effect but is about 20-30 times more potent. Unlike TEA and guanidine, 4-AP is effective when the ambient calcium concentration is 2 mM; this drug is therefore also active in vivo. The intravenous injection of 4-AP (5 mg/kg body weight) restores neuromuscular transmission from complete paralysis by botulinum toxin to a normal level as shown by the recording of almost normal twitch and tetanic tensions in the EDL muscle. In rats paralysed by a lethal dose of botulinum toxin, the intraperitoneal administration of 4-AP restores general motor activity, the effect lasting 1-2 hours. A study of the effects of these drugs on spontaneous and evoked transmitter release suggests that all three compounds increase the level of free calcium inside the nerve terminals. In botulinum poisoning the transmitter release mechanism appears to be intact, but a reduced sensitivity to calcium has been shown (Cull-Candy et al. 1976), and this could explain why the drugs restore evoked transmitter release in botulinum poisoning.
The enzyme linked immunosorbent assay using the so-called "double-sandwich technique" has been applied to determine botulinum toxin type A. By this assay, 50-100 mouse ip LD50 of toxin type A can be detected. No cross-reaction occurs with botulinum toxins of other types tested. In all probability this is due to the high specificity of the antiserum prepared against the toxic component of type A toxin.
1. Botulinum toxin (BoTx) type A partially blocks spontaneous transmitter release from nerve terminals in the rat. Minature end-plate potentials (m.e.p.p.s) are present at all end-plates, initially with a low frequency but increasing with time after posoning. Their amplitude distribution is at first skew with a predominace of very small m.e.p.p.s but, after a few days, larger than normal m.e.p.p.s appear. 2. Tetanic nerve stimulation, Black Widow Spider Venom, the Caionophore A 23187 or mechanical damage to nerve terminals increases the frequency of m.e.p.p.s and alters the amplitude distribution of m.e.p.p.s towards a normal Gaussian one; the m.e.p.p. size approaches that seen at normal end-plates. This was seen at any time after poisoning. 3. Nerve stimulation gives rise to end-plate potentials (e.p.p.s) of low amplitude and high failure rate. Statistical analysis indicates that evoked release is quantal in nature and follows Poisson statistics, quantum size being initially very small, but after a few days approaching normal size. Short-term tetanic nerve stimulation reversibly increases the quantum content of e.p.p.s and during early stages of paralysis long-term (2 hr) stimulation causes an apparently permanent increase in quantum size. 4. Raising the extracellular Ca concentration from 2 to 16 mM increases the frequency of m.e.p.p.s in normal muscle but not in BoTx poisoned ones. K-free medium or ouabain, which are believed to raise the intracellular Ca concentration in nerve terminals, similarly increases m.e.p.p. frequency in normal but not in poisoned muscles. When the Ca-ionophore A 23187 is used together with high extracellular Ca (greater than 4 mM) massive release of transmitter occurs from poisoned terminals. 5. The extracellular Ca concentration which causes a certain level of transmitter release in reponse to nerve impulses is considerably higher at BoTx poisoned end-plates than at normal ones. The slope value for Ca dependence of transmitter release is about 1-5 compared with about 3 at normal end-plates. 6. Tetraethylammonium (TEA) greatly increases the amount of transmitter released by nerve impulses and restores neuromuscular transmission during all stages of poisoning, although it has not effect on spontaneous transmitter release. In the presence of TEA the power relation between Ca concentration and quantum content at the BoTx poisoned end-plate is similar to that seen at normal end-plates. 7. It is suggested that in BoTx poisoning the mechanism for transmitter release has a reduced sensitivity to Ca, and the level for activation by intracellular Ca is elevated. Once the intracellular concentration of Ca is raised to this level, by tetanic nerve stimulation, mechanical injury to nerve terminals, the Ca-ionophore or the prolongation of the nerve action potential with TEA, augmented transmitter release occurs, similar to that which occurs in normal nerve terminals at a lower level of Ca.
In order to determine the role of acetylcholine (ACh) transmission in neurotrophic regulation of dynamic properties of muscle, the effects of botulinum toxin treatment were compared with those of denervation. The extensor digitorum longus (EDL) and soleus muscles of rats were either denervated or injected with botulinum toxin. At times up to 25 days the isometric properties of these muscles were determined. 2. Both botulinum treatment and denervation produced progressive slowing of the time to peak of the twitch (TPT) and half-relaxation time of the twitch (1/2 RT), which was more pronounced in the EDL than in the soleus. 3. Both treatments produced slowing of the relaxation curve following tetanic contraction, more marked in the EDL than in the soleus muscle. This indicates a slowing of relaxation, and suggests a prolongation of the active state of the muscle. 4. The maximum rate of rise of the tetanus did not change significantly in the EDL and soleus muscles after botulinum treatment or denervation. This suggests that there is no major change in the speed of contraction under conditions of botulinum treatment or denervation. 5. The changes produced by botulinum treatment and denervation were virtually identical in all parameters tested. This is interpreted meaning that cholinergic transmission (including muscle usage), or some other factor closely related to cholinergic transmission, accounts for the motor nerve's trophic influence in maintaining these dynamic properties of skeletal muscles.
1. The common peroneal nerve was implanted into soleus in the mouse and 2 weeks later a sublethal dose of botulinum toxin injected causing a block of neuromuscular transmission at the terminals of the soleus nerve. Most muscle fibres became innervated by the common peroneal nerve. 2. Recovery of neuromuscular transmission at the soleus nerve terminals was delayed in the common peroneal nerve implanted muscles. 3. Stimulation of the soleus nerve after botulinum-evoked subthreshold end-plate potentials (e.p.p.s) in virtually every fibre tested in unoperated muscles. In common peroneal nerve-implanted muscles stimulation of the soleus nerve failed to evoke e.p.p.s in about 40% of fibres tested and where e.p.p.s were recorded their amplitudes were generally smaller. 4. When the common peroneal nerve was cut 2 months after botulinum, neuromuscular transmission at soleus nerve terminals occurred after 4 weeks. When the common peroneal nerve was cut 6 months after botulinum, transmission was found at soleus nerve terminals within 1 week. 5. Recovery of transmission at soleus nerve terminals from the effects of botulinum toxin is delayed if the muscle fibres become innervated by the common peroneal nerve and a proportion of soleus nerve terminals cease to release acetylcholine (ACh) until after the peroneal nerve has been cut.
1. The central action of botulinum toxin A (BTA) on the cholinergic transmission at Renshaw cells (RCs) and on the RC-induced inhibition of Ia inhibitory interneurones (IaINs) was studied in anaesthetized cats. BTA was administered by application directly into the spinal cord, injection into a ventral root (L7) and/or injection into the triceps surae (GS) muscle. 2. A direct application of BTA into the spinal cord led to a decrease of the early and the late response of RCs. 3. When the neurotoxin was injected into the GS muscle, the RC activity remained unaffected during the test period (33-46 h after application). 4. No effect appeared up to 10 h after an injection into the ventral root L7. 5. The RC-induced inhibition on IaINs, when tested in animals with local botulismus, remained intact during the test period. 6. From the present results it is suggested that on the spinal level the central action of botulinum toxin predominantly passes on the motoneurones.
Serotonin inhibits the muscular contractions elicited by indirect stimulation; injected before Botulinum toxin it prevents the paralysis induced by this toxin.
In order to detect minimal amounts of Clostridium botulinum toxins in animal tissue or food specimens it is necessary to use an extraction method which results in concentration of the botulinal toxins. In the present examinations, artificially contaminated canned beans were used to develop a suitable procedure for extraction and concentration of botulinal toxins A-E. The procedure consisted of 4 steps: 1. Canned beans were diluted 1:2 with 0.1 m phosphate buffer pH 6.0. 2. The diluted material was homogenised with an "Ultra-Turrax" homogeniser for 20 sec. 3. The monogenised material was centrifuged at 4000 rpm for 30 min. 4. 15 ml of supernatant was concentrated using a "Millipore ultrafiltration chamber" (with a membrane capable of excluding all material with a molecular weight above 25,000). A pressure of 1.5 atmospheres was applied until the terminal volume was 0.5 ml. Following extraction and concentration, the samples were assayed for botulinal toxin in mice. Using this assay the concentration of the five toxins were shown to be as follows: Type A toxin: 19.0-fold toxin concentration Type B toxin: 14.8-fold toxin concentration Type C toxin: 20.6-fold toxin concentration Type D toxin: 28.2-fold toxin concentration Type E toxin: 112.2-fold toxin concentration
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Naturally-occuring antibodies against Clostridium botulinum toxins were found in Cathartes aura (turkey vultures), Canis latrans (coyotes) and Corvus brachyrhynchos (crows) by the passive hemagglutination (PHA) test and verified by the serum neutralization (SN) test. The prevalence of IHA antibodies was 18 of 20 vultures (90%), 5 of 12 crows (42%) and 25 to 110 coyotes (23%). Vultures and coyotes were seropositive by the PHA test against A, B, C, D, and F toxins. The highest antibody titer 1:8192 was in vulture serum against type C. In descending order, the highest antibody levels were against type C, D, F, E, A and B toxins.
A sulfhydryl-dependent protease (SHP) was purified from a culture of Clostridium botulinum type F. The enzyme can activate type E progenitor toxin completely but type B progenitor toxin only partially. This may suggest that SHP by itself could completely activate the toxin of proteolytic C. botulinum types A and F in culture. The toxicity of type E progenitor toxin potentiated by the treatment with SHP persisted, whereas that of derivative toxin decreased rapidly by further incubation with SHP. This may indicate that only the progenitor toxin, the complex of the toxic and nontoxic components, activated by SHP withstands the subsequent exposure to the enzyme in cultures of proteolytic C. botulinum.