Action Of Botulinum Neurotoxin Research Articles

Outpatient Infant Botulism in the United States, 1976-2021

To characterize cases of outpatients with infant botulism (IB) in the US identified by the Infant Botulism Treatment and Prevention Program at the California Department of Public Health from 1976 through2021. Outpatient IB cases were defined as patients presenting with an illness consistent with the known paralyzing action of botulinum neurotoxin and with laboratory confirmation. Outpatient cases were distinguished from the majority of patients with IB by the atypical fact that they did not require hospitalization throughout the course of their illness. Of the 4372 cases of IB identified by the Infant Botulism Treatment and Prevention Program over a 45-year period (1976-2021), 17 (0.4%) were outpatient cases. Most (11/17; 65%) cases occurred in California. The median age at disease onset was 20weeks (range=6 to 55weeks). The most common symptom among cases was constipation (16/17; 94%). Most patients (16/17; 94%) had at least one cranial nerve palsy, manifested as decreased head control, ptosis, weak cry, or poor suck. Outpatient IB occurs nationwide, although clinical diagnosis may be difficult because the severity of symptoms do not necessitate hospitalization or more comprehensive clinical intervention. Identification of outpatient cases requires an astute clinician and a capable, willing diagnostic testing laboratory. It is likely that more outpatient cases of IB are occurring than are presently recognized in infants mildly affected by this disease. Healthcare providers should consider the possibility of IB when presented with a previously well infant with failure to thrive, poor feeding, constipation, mild hypotonia, or cranial nerve palsy.

Synergic actions of botulinum neurotoxin A and oxaliplatin on colorectal tumour cell death through the upregulation of TRPM2 channel-mediated oxidative stress.

Botulinum neurotoxin A (BoNT) is being shown to have anticancer action as a potential adjuvant treatment. The transient receptor potential (TRP) melastatin 2 (TRPM2) stimulator action of BoNT was reported in glioblastoma cells, but not in colorectal cancer (HT29) cells. By activating TRPM2, we evaluated the impacts of BoNT and oxaliplatin (OXA) incubations on oxidant and apoptotic values within the HT29 cells. Control, BoNT (5 IU for 24 h), OXA (50 μM for 24 h) and their combinations were induced. We found that TRPM2 protein is upregulated and mediates enhanced BoNT and OXA-induced Ca2+ entry in cells as compared to control cells. The increase of free reactive oxygen species (ROS), but the decrease of glutathione is the main ROS responsible for TRPM2 activation on H29 exposure to oxidative stress. BoNT and OXA-mediated Ca2+ entry through TRPM2 stimulation in response to H2 O2 results in mitochondrial Ca2+ overload, followed by mitochondrial membrane depolarization, apoptosis and caspase-3/-8/-9, although they were diminished in the TRPM2 antagonist groups (N-(p-amylcinnamoyl)anthranilic acid and carvacrol). In conclusion, by increasing the susceptibility of HT29 tumour cells to oxidative stress and apoptosis, the combined administration of BoNT and OXA via the targeting of TRPM2 may offer a different approach to kill the tumour cells.

Botulinum neurotoxin is both poison and medicine: botulinum therapy and iatrogenic botulism

Botulinum neurotoxin is the most potent poison known worldwide. Over the past two centuries, its structure and functions have been studied in detail, and obtained in its pure form. Knowledge of the mechanisms of action of botulinum neurotoxin has determined two directions of its possible use: negative, as a biological (toxin) weapon, and positive, as an agent that eliminates pathological muscle hypertonicity and neuropathic pain and can be used for aesthetic purposes (beauty injections).
 The lecture presents the chronological development of both directions and provides a detailed analysis of the various uses of botulinum neurotoxin as a medical and cosmetic treatment. Particular attention is paid to the side effects of botulinum therapy and ways to overcome them.

Exposure of waterfowl to Clostridium botulinum in France

Botulism in wild birds is a widespread and potentially lethal disease raising major conservation issues. Botulism is also of public health concern. Due to the action of botulinum neurotoxins, mostly produced by Clostridium botulinum, botulism can affect wild birds, livestock, and humans. This study is part of a project aimed at improving our understanding of the pathogenesis of botulism in wild avifauna, which is still poorly understood. Indeed, the prevalence and dynamics of C. botulinum in the digestive tract or in bird tissue, whether as intermittent carriage related to environmental contamination or as part of the normal avian microbiota, is still unknown. In this study, we specifically addressed the presence of a healthy carrier status of wild birds, and its role in outbreaks. To answer this question, we monitored the estimated prevalence of C. botulinum in wild birds through samples from banded and swabbed birds as well as from hunted bird organs. Our results do not support the hypothesis of a healthy carriage outside of outbreaks, which raises the question of the bioavailability of the bacterium and toxin in the environment. Finally, the gene encoding botulinum neurotoxin type E was detected in keel muscle from a hunted bird, showing that recommendations on the consumption of wild bird meat are needed following a botulism outbreak.

How Does Botulinum Toxin Inhibit Itch?

Two decades after reports of the anti-pruritic effects of botulinum neurotoxins (BoNTs), there is still no approved product for the anti-itch indication of BoNTs, and most clinical case reports still focus on the off-label use of BoNTs for various itchy conditions. Few randomized clinical trials have been conducted with controversial results, and the beneficial effects of BoNTs against itch are mainly based on case studies and case series. These studies are valuable in presenting the potential application of BoNTs in chronic pruritic conditions, but due to the nature of these studies, they are categorized as providing lower levels of evidence or lower grades of recommendation. To obtain approval for the anti-pruritic indication of BoNTs, higher levels of evidence are required, which can be achieved through conducting large-scale and well-designed studies with proper control groups and established careful and reliable primary and secondary outcomes. In addition to clinical evidence, presenting the mechanism-based antipruritic action of BoNTs can potentially strengthen, accelerate, and facilitate the current efforts towards further investments in accelerating the field towards the potential approval of BoNTs for itchy conditions. This review, therefore, aimed to provide the state-of-the-art mechanisms underlying the anti-itch effect of BoNTs from basic studies that resemble various clinical conditions with itch as a hallmark. Evidence of the neuronal, glial, and immune modulatory actions of BoNTs in reducing the transmission of itch are presented, and future potential directions are outlined.

Ability of human SNAP-23 to generate high molecular weight SDS-resistant ternary SNARE complexes is influenced by C-terminal coil content

Using in vitro protein complex formation assay, ability of SNAP-25 isoforms to generate SDS-resistant ternary SNARE complexes with Syntaxin-1 and VAMP-2 was investigated. Major SNAP-25 family proteins were found to generate heat-resistant ternary complexes with varying efficiency. Compared to human SNAP-25, its non-neuronal counterparts SNAP-23 and SNAP-29 formed lower amounts of ternary complexes. Changing Pro182 in human SNAP-23 to Arg182 (SNAP-23 P182R) improved its ability to bind partners and form complexes. In silico analysis of C-terminal helical content in various SNAP-25 family members showed that except human SNAP-23, all others displayed secondary α-helical conformation. We also report that human SNAP-29 is resistant to the proteolytic action of botulinum neurotoxin A even when applied at large concentration.

Current trends in the rehabilitation of patients with spastic paresis with focal brain damage

Spasticity is one of the most frequent movement disorders and its development is associated with such CNS lesions as stroke, multiple sclerosis, cerebral palsy, craniocerebral and spinal injuries, CNS tumors, neurodegenerative diseases. Post-stroke spasticity develops in about 40% of patients, and about 15% of patients have severe and disabling spasticity. According to statistics, after TBI, about 75% of patients develop spasticity, and half of them require treatment.Spasticity worsens walking, complicates hygiene, dressing, complicates rehabilitation measures, and reduces the quality of life of patients and his family members. In recent years, this movement disorder has been actively studied, new data have appeared on the pathophysiology of spastic paresis and encouraging data on improving function in patients receiving botulinum toxin injections as part of multidisciplinary rehabilitation.The article presents data from the latest systematic reviews on the effectiveness of various rehabilitation technologies for the treatment of spasticity.The use of botulinum neurotoxin for the treatment of spastic paresis of the upper and lower extremities is the preferred method of complex multidisciplinary rehabilitation of patients with spasticity and has the highest level of evidence.Currently, there is no doubt that botulinum toxin should be used as early as possible in patients with an emerging pathological movement pattern, which can contribute to pattern change and muscle length maintenance. But the question remains: which rehabilitation technology is most effective for enhancing and prolonging the action of botulinum neurotoxin. To date, there is no clear answer to this question.

Botulinum toxin in neurological clinical practice

Botulinum toxin is produced by Clostridium botulini bacteria. It has been increasingly used as a boon in medical practice for chemodenervation in conditions like skeletal muscle spasticity, glandular hypersecretion, neuropathic pain and smooth muscle hyperactivity. The action of Botulinum neurotoxin (BoNT) is by blocking the neurotransmission at the cholinergic nerve endings, by inhibiting the docking and fusion of Acetyl Choline (Ach) vesicles for exocytosis into the synaptic area.Botulinum toxin has been used for management of neurological disorders with great patient satisfaction and appreciation. The effect of botulinum toxin wears off in 3-6 months and may require another dose. Frequent dosing may lead to production of antibodies against BoNT with consequent irresponsiveness to therapy in a few cases. Scrupulous use of botulinum toxin in the hands of experts may help a long way in giving much needed relief and respite to neurological patients and increase their quality of life. Its use in cosmetic dermatology in reducing wrinkles, by relaxing the facial muscles is very popular

Botulinum Toxin and Neuronal Regeneration after Traumatic Injury of Central and Peripheral Nervous System.

Botulinum neurotoxins (BoNTs) are toxins produced by the bacteria Clostridium botulinum, the causing agent for botulism, in different serotypes, seven of which (A–G) are well characterized, while others, such as H or FA, are still debated. BoNTs exert their action by blocking SNARE (soluble N-ethylmale-imide-sensitive factor-attachment protein receptors) complex formation and vesicle release from the neuronal terminal through the specific cleavage of SNARE proteins. The action of BoNTs at the neuromuscular junction has been extensively investigated and knowledge gained in this field has set the foundation for the use of these toxins in a variety of human pathologies characterized by excessive muscle contractions. In parallel, BoNTs became a cosmetic drug due to its power to ward off facial wrinkles following the activity of the mimic muscles. Successively, BoNTs became therapeutic agents that have proven to be successful in the treatment of different neurological disorders, with new indications emerging or being approved each year. In particular, BoNT/A became the treatment of excellence not only for muscle hyperactivity conditions, such as dystonia and spasticity, but also to reduce pain in a series of painful states, such as neuropathic pain, lumbar and myofascial pain, and to treat various dysfunctions of the urinary bladder. This review summarizes recent experimental findings on the potential efficacy of BoNTs in favoring nerve regeneration after traumatic injury in the peripheral nervous system, such as the injury of peripheral nerves, like sciatic nerve, and in the central nervous system, such as spinal cord injury.

Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties.

SummaryThe fusion pore is the first crucial intermediate formed during exocytosis, yet little is known regarding the mechanisms that determine the size and kinetic properties of these transient structures1. Here, we reduced the number of available SNAREs in neurons and observed changes in transmitter release suggestive of alterations in fusion pores. To address this, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores, and enabled the escape of larger cargos. To determine whether this was due to changes in nascent pore size versus stability, we developed a novel approach, based on nanodiscs and planar lipid bilayer electrophysiology, that affords μsec time resolution at the single event level. Remarkably, both parameters were affected by SNARE copy number. Increasing the number of v-SNAREs per nanodisc from three to five caused a two-fold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, trans-SNARE pairing was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable, SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, that mimics the action of botulinum neurotoxin A, dramatically reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determine fundamental properties of individual pores.

Substrate cleavage and duration of action of botulinum neurotoxin type FA (“H, HA”)

Botulinum neurotoxin (BoNT) type FA is the only known naturally occurring chimeric BoNT of domains of BoNT/A and BoNT/F. BoNT/FA consists of an F5-like light chain (LC), a unique heavy chain (HC) translocation domain, and a HC receptor binding domain similar to BoNT/A1. Previous analyses of purified BoNT/FA have indicated a 5–10-fold greater potency in cultured human or rat neurons as compared to BoNT/A1 and a 400–500-fold greater potency compared to BoNT/B1. However, in vivo potency in mice was about 5-fold lower than BoNT/A1 or/B1. In this report, species specificity was examined by cell-based assays using primary neurons from mice and examining VAMP1 and 2 cleavage. The data indicated similar potency of BoNT/FA in primary mouse spinal cord neurons as previously observed in primary rat and human induced pluripotent stem cell (hiPSC) derived neuronal cell models, and equal enzymatic cleavage of mouse VAMP1 and 2 isoforms. Since the duration of action of BoNTs is due to continuous enzymatic activity of the LC in the neuronal cytosol, BoNT/FA was expected to have a short duration of action due to its F-type LC. In this report the duration of action of BoNT/FA was compared to that of BoNT/F1,/F5, and/B1 in both hiPSC derived neurons and in the in vivo mouse model. The data indicate a duration of action of BoNT/FA similar to BoNT/B1, while BoNT/F5 had a short duration of action similar to BoNT/F1.

The binding of botulinum neurotoxins to different peripheral neurons

Botulinum neurotoxins are the most potent toxins known. The double receptor binding modality represents one of the most significant properties of botulinum neurotoxins and largely accounts for their incredible potency and lethality. Despite the high affinity and the very specific binding, botulinum neurotoxins are versatile and multi-tasking toxins. Indeed they are able to act both at the somatic and at the autonomic nervous system.In spite of the preference for cholinergic nerve terminals botulinum neurotoxins have been shown to inhibit to some extent also the noradrenergic postganglionic sympathetic nerve terminals and the afferent nerve terminals of the sensory neurons inhibiting the release of neuropeptides and glutamate, which are responsible of nociception. Therefore, there is increasing evidence that the therapeutic effect in both motor and autonomic disorders is based on a complex mode of botulinum neurotoxin action modulating the activity of efferent as well as afferent nerve fibres.

Full Solution-Phase Synthesis of Acetyl Hexapeptide-3 by Fragments Coupling Strategy

Acetyl hexapeptide-3, a competitive SNAP-25 inhibitor that mimics the action of botulinum neurotoxins but is nontoxic, was successively synthesized in a solution by peptide chemistry with a “3+2+1” fragment coupling strategy. The difficulties encountered in the convergent route, an optimization on scalable process, and controls on potential epimerization are systematically discussed. Final purification led to the target product’s overall 36.5% yield with 98.3% purity and excellent optical purity.

Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology.

The study of botulinum neurotoxins (BoNT) is rapidly progressing in many aspects. Novel BoNTs are being discovered owing to next generation sequencing, but their biologic and pharmacological properties remain largely unknown. The molecular structure of the large protein complexes that the toxin forms with accessory proteins, which are included in some BoNT type A1 and B1 pharmacological preparations, have been determined. By far the largest effort has been dedicated to the testing and validation of BoNTs as therapeutic agents in an ever increasing number of applications, including pain therapy. BoNT type A1 has been also exploited in a variety of cosmetic treatments, alone or in combination with other agents, and this specific market has reached the size of the one dedicated to the treatment of medical syndromes. The pharmacological properties and mode of action of BoNTs have shed light on general principles of neuronal transport and protein-protein interactions and are stimulating basic science studies. Moreover, the wide array of BoNTs discovered and to be discovered and the production of recombinant BoNTs endowed with specific properties suggest novel uses in therapeutics with increasing disease/symptom specifity. These recent developments are reviewed here to provide an updated picture of the biologic mechanism of action of BoNTs, of their increasing use in pharmacology and in cosmetics, and of their toxicology.

The Botulinum Toxin as a Therapeutic Agent: Molecular Structure and Mechanism of Action in Motor and Sensory Systems.

Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is the most potent molecule known to mankind. Higher potency of BoNT is attributed to several factors, including structural and functional uniqueness, target specificity, and longevity. Although BoNT is an extremely toxic molecule, it is now increasingly used for the treatment of disorders related to muscle hyperactivity and glandular hyperactivity. Weakening of muscles due to peripheral action of BoNT produces a therapeutic effect. Depending on the target tissue, BoNT can block the cholinergic neuromuscular or cholinergic autonomic innervation of exocrine glands and smooth muscles. In recent observations of the analgesic properties of BoNT, the toxin modifies the sensory feedback loop to the central nervous system. Differential effects of BoNT in excitatory and inhibitory neurons provide a unique therapeutic tool. In this review the authors briefly summarize the structure and mechanism of actions of BoNT on motor and sensory neurons to explain its therapeutic effects and future potential.

Explanation of timing of botulinum neurotoxin effects, onset and duration, and clinical ways of influencing them

While the steps in the action of botulinum neurotoxin (BoNT) are well known, the factors underlying the timing of these steps are not fully understood. After toxin is injected into a muscle, it resides in the extracellular space and must be taken up into the nerve terminals. More toxin will be taken up if near the endplate. Toxin is distributed mainly by convection and there is likely little diffusion. Toxin that is not taken up will go into the general circulation where it may have a slight systemic effect. The uptake is activity and temperature dependent. Encouraging the unwanted muscle contractions after injection should be helpful. Cooling will decrease the uptake. The times for washout from the extracellular space and uptake of the toxin are not well established, but are likely measured in minutes. Toxin in the general circulation has a long half time. The time from injection to weakness is determined by how long it takes to get sufficient damage of the SNARE proteins to interfere with synaptic release. Toxins are zinc dependent proteases, and supplemental zinc may produce a greater effect. There will be weakness as long as there is residual toxin in the nerve ending.

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

Botulinum Toxin as a Pain Killer: Players and Actions in Antinociception.

Botulinum neurotoxins (BoNTs) have been widely used to treat a variety of clinical ailments associated with pain. The inhibitory action of BoNTs on synaptic vesicle fusion blocks the releases of various pain-modulating neurotransmitters, including glutamate, substance P (SP), and calcitonin gene-related peptide (CGRP), as well as the addition of pain-sensing transmembrane receptors such as transient receptor potential (TRP) to neuronal plasma membrane. In addition, growing evidence suggests that the analgesic and anti-inflammatory effects of BoNTs are mediated through various molecular pathways. Recent studies have revealed that the detailed structural bases of BoNTs interact with their cellular receptors and SNAREs. In this review, we discuss the molecular and cellular mechanisms related to the efficacy of BoNTs in alleviating human pain and insights on engineering the toxins to extend therapeutic interventions related to nociception.

A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin.

Botulism is a potentially fatal paralytic disease caused by the action of botulinum neurotoxin (BoNT) on nerve cells. There are 7 known serotypes (A-G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was recently reported to produce BoNT serotype B (BoNT/B) and a novel BoNT, designated as BoNT/H. The BoNT gene (bont) sequence of BoNT/H was compared to known bont sequences. Genetic analysis suggested that BoNT/H has a hybrid-like structure containing regions of similarity to the structures of BoNT/A1 and BoNT/F5. This novel BoNT was serologically characterized by the mouse neutralization assay and a neuronal cell-based assay. The toxic effects of this hybrid-like BoNT were completely eliminated by existing serotype A antitoxins, including those contained in multivalent therapeutic antitoxin products that are the mainstay of human botulism treatment.

Botulinum neurotoxin serotypes detected by electrochemical impedance spectroscopy.

Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A–E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins.