Intramuscular Botulinum Neurotoxin Serotypes E and A Elicit Distinct Effects on SNAP25 Protein Fragments, Muscular Histology, Spread and Neuronal Transport: An Integrated Histology-Based Study in the Rat.

Botulinum neurotoxins E (BoNT/E) and A (BoNT/A) act by cleaving Synaptosome-Associated Protein 25 (SNAP25) at two different C-terminal sites, but they display very distinct durations of action, BoNT/E being short acting and BoNT/A long acting. We investigated the duration of action, spread and neuronal transport of BoNT/E (6.5 ng/kg) and BoNT/A (125 pg/kg) after single intramuscular administrations of high equivalent efficacious doses, in rats, over a 30- or 75-day periods, respectively. To achieve this, we used (i) digit abduction score assay, (ii) immunohistochemistry for SNAP25 (N-ter part; SNAP25N-ter and C-ter part; SNAP25C-ter) and its cleavage sites (cleaved SNAP25; c-SNAP25E and c-SNAP25A) and (iii) muscular changes in histopathology evaluation. Combined in vivo observation and immunohistochemistry analysis revealed that, compared to BoNT/A, BoNT/E induces minimal muscular changes, possesses a lower duration of action, a reduced ability to spread and a decreased capacity to be transported to the lumbar spinal cord. Interestingly, SNAP25C-ter completely disappeared for both toxins during the peak of efficacy, suggesting that the persistence of toxin effects is driven by the persistence of proteases in tissues. These data unveil some new molecular mechanisms of action of the short-acting BoNT/E and long-acting BoNT/A, and reinforce their overall safety profiles.

Domperidone Inhibits Clostridium botulinum C2 Toxin and Bordetella pertussis Toxin.

Bordetella pertussis toxin (PT) and Clostridium botulinum C2 toxin are ADP-ribosylating toxins causing severe diseases in humans and animals. They share a common translocation mechanism requiring the cellular chaperones Hsp90 and Hsp70, cyclophilins, and FK506-binding proteins to transport the toxins' enzyme subunits into the cytosol. Inhibitors of chaperone activities have been shown to reduce the amount of transported enzyme subunits into the cytosol of cells, thus protecting cells from intoxication by these toxins. Recently, domperidone, an approved dopamine receptor antagonist drug, was found to inhibit Hsp70 activity. Since Hsp70 is required for cellular toxin uptake, we hypothesized that domperidone also protects cells from intoxication with PT and C2. The inhibition of intoxication by domperidone was demonstrated by analyzing the ADP-ribosylation status of the toxins' specific substrates. Domperidone had no inhibitory effect on the receptor-binding or enzyme activity of the toxins, but it inhibited the pH-driven membrane translocation of the enzyme subunit of the C2 toxin and reduced the amount of PTS1 in cells. Taken together, our results indicate that domperidone is a potent inhibitor of PT and C2 toxins in cells and therefore might have therapeutic potential by repurposing domperidone to treat diseases caused by bacterial toxins that require Hsp70 for their cellular uptake.

Advances in Clostridial and Related Neurotoxins.

The huge advances in genomics and molecular biology in the past two decades have made now an exciting time to study bacterial toxins, in particular, the most potent bacterial toxin known to humankind, botulinum neurotoxins (BoNTs) [...].

The Light Chain Domain and Especially the C-Terminus of Receptor-Binding Domain of the Botulinum Neurotoxin (BoNT) Are the Hotspots for Amino Acid Variability and Toxin Type Diversity.

Botulinum neurotoxins (BoNT) are the most potent toxins in the world. They are produced by a few dozens of strains within several clostridial species. The toxin that they produce can cause botulism, a flaccid paralysis in humans and other animals. With seven established serologically different types and over 40 subtypes, BoNTs are among the most diverse known toxins. The toxin, its structure, its function and its physiological effects on the neural cell and animal hosts along with its diversity have been the subjects of numerous studies. However, many gaps remain in our knowledge about the BoNT toxin and the species that produce them. One of these gaps involves the distribution and extent of variability along the full length of the gene and the protein as well as its domains and subdomains. In this study, we performed an extensive analysis of all of the available 143 unique BoNT-encoding genes and their products, and we investigated their diversity and evolution. Our results indicate that while the nucleotide variability is almost uniformly distributed along the entire length of the gene, the amino acid variability is not. We found that most of the differences were concentrated along the protein's light chain (LC) domain and especially, the C-terminus of the receptor-binding domain (HCC). These two regions of the protein are thus identified as the main source of the toxin type differentiation, and consequently, this toxin's versatility to bind different receptors and their isoforms and act upon different substrates, thus infecting different hosts.

Botulinum Neurotoxins in Central Nervous System: An Overview from Animal Models to Human Therapy.

Botulinum neurotoxins (BoNTs) are potent inhibitors of synaptic vesicle fusion and transmitter release. The natural target of BoNTs is the peripheral neuromuscular junction (NMJ) where, by blocking the release of acetylcholine (ACh), they functionally denervate muscles and alter muscle tone. This leads them to be an excellent drug for the therapy of muscle hyperactivity disorders, such as dystonia, spasticity, and many other movement disorders. BoNTs are also effective in inhibiting both the release of ACh at sites other than NMJ and the release of neurotransmitters other than ACh. Furthermore, much evidence shows that BoNTs can act not only on the peripheral nervous system (PNS), but also on the central nervous system (CNS). Under this view, central changes may result either from sensory input from the PNS, from retrograde transport of BoNTs, or from direct injection of BoNTs into the CNS. The aim of this review is to give an update on available data, both from animal models or human studies, which suggest or confirm central alterations induced by peripheral or central BoNTs treatment. The data will be discussed with particular attention to the possible therapeutic applications to pathological conditions and degenerative diseases of the CNS.

Neutralizing Concentrations of Anti-Botulinum Toxin Antibodies Positively Correlate with Mouse Neutralization Assay Results in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known and can induce respiratory failure requiring long-term intensive care. Treatment of botulism includes the administration of antitoxins. Monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics, due to their potency and safety. A three-mAb combination has been developed that specifically neutralizes BoNT serotype A (BoNT/A), and a separate three mAb combination has been developed that specifically neutralizes BoNT serotype B (BoNT/B). A six mAb cocktail, designated G03-52-01, has been developed that combines the anti-BoNT/A and anti-BoNT/B mAbs. The pharmacokinetics and neutralizing antibody concentration (NAC) of G03-52-01 has been determined in guinea pigs, and these parameters were correlated with protection against an inhalation challenge of BoNT/A1 or BoNT/B1. Previously, it was shown that each antibody demonstrated a dose-dependent mAb serum concentration and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intraperitoneal (IP) injection and that a single IM injection of G03-52-01 administered 48 h pre-exposure protected guinea pigs against an inhalation challenge of up to 93 LD50s of BoNT/A1 and 116 LD50s of BoNT/B1. The data presented here advance our understanding of the relationship of the neutralizing NAC to the measured circulating antibody concentration and provide additional support that a single IM or intravenous (IV) administration of G03-52-01 will provide pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A and BoNT/B.

Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E.

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

Botulinum Toxin in WW2 German and Allied Armies: Failures and Myths of Weaponization.

Botulinum toxin is nowadays approved as an effective medication for various neurological disorders. The extreme toxicity of this toxin-inducing botulism, a severe lethal muscle-paralyzing illness, has been well known since the seminal works of the end of the 19th century. Because of this toxicity, botulinum toxin was one of the first agents to be considered for use as a biological weapon. The Second World War was a crucial period for the first attempts to weaponize this toxin even if many unknown factors about botulinum toxin still existed at the outbreak of the war. Using documents from the British National Archives and other published sources, we discuss the main points of the attempts to weaponize this toxin in German and Allied armies. During WW2, Allied intelligence services regularly reported a major German threat related to the potential use of botulinum toxin as a biological weapon, especially during the preparation of Operation Overlord, the Allied invasion to liberate Europe. All these reports would ultimately prove to be inaccurate: botulinum toxin was not part of the German military arsenal even if some German scientists tried to use the results of the French pre-war military research. Misinformation spread by intelligence services stimulated military research at Porton Down facilities in England and at Camp Detrick in the USA. These studies led to a succession of failures and myths about the weaponization of botulinum toxin. Nevertheless, major progress (purification, toxoid) arose from the military research, providing useful data for the first steps of the therapeutic use of botulinum toxin in the post-war years.

Intramuscular injection of Botox causes tendon atrophy by induction of senescence of tendon-derived stem cells.

BACKGROUND: Botulinum toxin (Botox) injection is in widespread clinical use for the treatment of muscle spasms and tendinopathy but the mechanism of action is poorly understood. HYPOTHESIS: We hypothesised that the reduction of patellar-tendon mechanical-loading following intra-muscular injection of Botox results in tendon atrophy that is at least in part mediated by the induction of senescence of tendon-derived stem cells (TDSCs). STUDY DESIGN: Controlled laboratory study METHODS: A total of 36 mice were randomly divided into 2 groups (18 Botox-injected and 18 vehicle-only control). Mice were injected into the right vastus lateralis of quadriceps muscles either with Botox (to induce mechanical stress deprivation of the patellar tendon) or with normal saline as a control. At 2 weeks post-injection, animals were euthanized prior to tissues being harvested for either evaluation of tendon morphology or in vitro studies. TDSCs were isolated by cell-sorting prior to determination of viability, differentiation capacity or the presence of senescence markers, as well as assessing their response to mechanical loading in a bioreactor. Finally, to examine the mechanism of tendon atrophy in vitro, the PTEN/AKT-mediated cell senescence pathway was evaluated in TDSCs from both groups. RESULTS: Two weeks after Botox injection, patellar tendons displayed several atrophic features including tissue volume reduction, collagen fibre misalignment and increased degradation. A colony formation assay revealed a significantly reduced number of colony forming units of TDSCs in the Botox-injected group compared to controls. Multipotent differentiation capacities of TDSCs were also diminished after Botox injection. To examine if mechanically deprived TDSC are capable of forming tendon tissue, we used an isolated bioreactor system to culture tendon constructs using TDSC. These results showed that TDSCs from the Botox-treated group failed to restore tenogenic differentiation after appropriate mechanical loading. Examination of the signalling pathway revealed that injection of Botox into quadriceps muscles causes PTEN/AKT-mediated cell senescence of TDSCs. CONCLUSION: Intramuscular injection of Botox interferes with tendon homeostasis by inducing tendon atrophy and senescence of TDSCs. Botox injection may have long-term adverse consequences for the treatment of tendinopathy. CLINICAL RELEVANCE: Intramuscular Botox injection for tendinopathy or tendon injury could result in adverse effects in human tendons and evaluation of its long-term efficacy is warranted.

Influence of aerodynamic particle size on botulinum neurotoxin potency in mice.

OBJECTIVE: For many agents, the aerodynamic particle size can affect both the virulence and disease course in animal models. Botulinum neurotoxins (BoNTs), which are widely known as potential bioterrorism agents, have been shown to be toxic via multiple routes of exposure, including small particle inhalation (1-3 µm MMAD). However, the impact of larger particle sizes on the potency of BoNT has not been previously reported. In this study, we compared the potency of BoNT in small and large particle aerosols. MATERIALS AND METHODS: Outbred mice (ICR (CD-1®)) were exposed to BoNT-containing aerosols with differing mass median aerodynamic diameters (MMADs) of 1.1, 4.9, and 7.6 microns. The effects of bioaerosol sampler and inhalation exposure modality were studied. RESULTS AND DISCUSSION: Collecting aerosolized BoNT onto gelatin filters or into liquid impingers resulted in equivalent estimates of aerosol concentration. Nose-only and whole-body inhalation exposure resulted in nearly identical estimates of the median lethal dose (LD50). The LD50 for inhaled BoNT increased approximately 50-fold when the median aerodynamic particle size was increased from 1.1 to 4.9 µm, from 139 (95% CI: 111-185) to 7324 (95% CI: 4287-10 891) mouse intraperitoneal median lethal doses (MIPLD50). These results demonstrate the importance of aerodynamic particle size and regional deposition patterns with regards to BoNT inhalational toxicity. CONCLUSIONS: These data will be useful for medical countermeasure development, as well as biodefense preparedness modeling by demonstrating that the estimates of dose and toxicity of an inhaled aerosol containing BoNT can be significantly affected by a range of factors.

Intoxication of mammalian cells with binary clostridial enterotoxins is inhibited by the combination of pharmacological chaperone inhibitors.

Binary enterotoxins Clostridioides difficile CDT toxin, Clostridium botulinum C2 toxin, and Clostridium perfringens iota toxin consist of two separate protein components. The B-components facilitate receptor-mediated uptake into mammalian cells and form pores into endosomal membranes through which the enzymatic active A-components translocate into the cytosol. Here, the A-components ADP-ribosylate G-actin which leads to F-actin depolymerization followed by rounding of cells which causes clinical symptoms. The protein folding helper enzymes Hsp90, Hsp70, and peptidyl-prolyl cis/trans isomerases of the cyclophilin (Cyp) and FK506 binding protein (FKBP) families are required for translocation of A-components of CDT, C2, and iota toxins from endosomes to the cytosol. Here, we demonstrated that simultaneous inhibition of these folding helpers by specific pharmacological inhibitors protects mammalian, including human, cells from intoxication with CDT, C2, and iota toxins, and that the inhibitor combination displayed an enhanced effect compared to application of the individual inhibitors. Moreover, combination of inhibitors allowed a concentration reduction of the individual compounds as well as decreasing of the incubation time with inhibitors to achieve a protective effect. These results potentially have implications for possible future therapeutic applications to relieve clinical symptoms caused by bacterial toxins that depend on Hsp90, Hsp70, Cyps, and FKBPs for their membrane translocation into the cytosol of target cells.

Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay.

Botulinum neurotoxins (BoNTs) are among the most poisonous known biological substances, and therefore the availability of reliable, easy-to use tools for BoNT detection are important goals for food safety and human and animal health. The reference method for toxin detection and identification is the mouse bioassay (MBA). An EndoPep-MS method for BoNT differentiation has been developed based on mass spectrometry. We have validated and implemented the EndoPep-MS method on a Bruker MALDI Biotyper for the detection of BoNT/C and D serotypes. The method was extensively validated using experimentally and naturally contaminated samples comparing the results with those obtained with the MBA. Overall, the limit of detection (LoD) for both C and D toxins were less than or equal to two mouse lethal dose 50 (mLD50) per 500 µL for all tested matrices with the exception of feces spiked with BoNT/C which showed signals not-related to specific peptide fragments. Diagnostic sensitivity, specificity and positive predictive value were 100% (95% CI: 87.66-100%), 96.08% (95% CI: 86.54-99.52%), and 93.33% (95% CI: 78.25-98.20%), respectively, and accuracy was 97.47% (95% CI: 91.15-99.69%). In conclusion, the tests carried out showed that the EndoPep-MS method, initially developed using more powerful mass spectrometers, can be applied to the Bruker MALDI Biotyper instrument with excellent results including for detection of the proteolytic activity of BoNT/C, BoNT/D, BoNT/CD, and BoNT/DC toxins.

Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells.

C3 protein toxins produced by Clostridium (C.) botulinum and C. limosum are mono-ADP-ribosyltransferases, which specifically modify the GTPases Rho A/B/C in the cytosol of monocytic cells, thereby inhibiting Rho-mediated signal transduction in monocytes, macrophages, and osteoclasts. C3 toxins are selectively taken up into the cytosol of monocytic cells by endocytosis and translocate from acidic endosomes into the cytosol. The C3-catalyzed ADP-ribosylation of Rho proteins inhibits essential functions of these immune cells, such as migration and phagocytosis. Here, we demonstrate that C3 toxins enter and intoxicate dendritic cells in a time- and concentration-dependent manner. Both immature and mature human dendritic cells efficiently internalize C3 exoenzymes. These findings could also be extended to the chimeric fusion toxin C2IN-C3lim. Moreover, stimulated emission depletion (STED) microscopy revealed the localization of the internalized C3 protein in endosomes and emphasized its potential use as a carrier to deliver foreign proteins into dendritic cells. In contrast, the enzyme C2I from the binary C. botulinum C2 toxin was not taken up into dendritic cells, indicating the specific uptake of C3 toxins. Taken together, we identified human dendritic cells as novel target cells for clostridial C3 toxins and demonstrated the specific uptake of these toxins via endosomal vesicles.

Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays.

Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.

Potency and stability of a trivalent, catalytically inactive vaccine against botulinum neurotoxin serotypes C, E and F (triCEF).

Botulism is an acute neuroparalytic affliction of the motor and autonomic neurons caused by the toxins produced from Clostridium botulinum and related bacterial strains. The botulinum neurotoxins, or BoNTs, consist of a phylogenetically diverse group of highly potent protein toxins. Current medical interventions for confirmed cases of botulism are limited to immediate administration of antitoxins and respiratory support. There is currently no licensed vaccine against botulism in the United States. The most widely distributed botulism vaccine was a pentavalent BoNT toxoid (PBT) against serotypes A-E administered until 2011 under an investigational new drug license. A binary vaccine composed of the recombinant, non-toxic, receptor binding domains (RBD) of serotypes/A1 and/B1 has completed a phase II clinical trial, but has yet to attain full licensure. We have previously published data demonstrating catalytically inactive, full length botulinum neurotoxin holoproteins (ciBoNT HPs) against serotypes/A1,/B1,/C1,/E1 and/F1 provide equivalent or superior potency against parental and dissimilar subtype toxins as compared the RBD vaccines. Here we describe the consistent potencies of the three independent lots each of ciBoNT/C1,/E1, and/F1 HPs against substantial monovalent challenges of the parental toxins. We also present data that a trivalent formulation of ciBoNT/C1,/E1 and/F1 (triCEF) maintains potency against both monovalent and polyvalent toxin challenges when stored as an adjuvanted vaccine at 4-8 °C for up to 2 years.

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins.

Botulinum Neurotoxins (BoNTs) are a large protein family that includes the most potent neurotoxins known to humankind. BoNTs delivered locally in humans at low doses are widely used pharmaceuticals. Reliable and quantitative detection of BoNTs is of paramount importance for the clinical diagnosis of botulism, basic research, drug development, potency determination, and detection in clinical, environmental, and food samples. Ideally, a definitive assay for BoNT should reflect the activity of each of the four steps in nerve intoxication. The in vivo mouse bioassay (MBA) is the 'gold standard' for the detection of BoNTs. The MBA is sensitive, robust, semi-quantitative, and reliable within its sensitivity limits. Potential drawbacks with the MBA include assay-to-assay potency variations, especially between laboratories, and false positives or negatives. These limitations can be largely avoided by careful planning and performance. Another detection method that has gained importance in recent years for research and potency determination of pharmaceutical BoNTs is cell-based assays, as these assays can be highly sensitive, quantitative, human-specific, and detect fully functional holotoxins at physiologically relevant concentrations. A myriad of other in vitro BoNT detection methods exist. This review focuses on critical factors and assay limitations of the mouse bioassay and cell-based assays for BoNT detection.

The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA.

BACKGROUND: Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. METHODS: monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. RESULTS: 21 unique BoNT/H LC-HN mAbs were obtained; 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. CONCLUSIONS: The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.

Tables of Toxicity of Botulinum and Tetanus Neurotoxins.

Tetanus and botulinum neurotoxins are the most poisonous substances known, so much so as to be considered for a possible terrorist use. At the same time, botulinum neurotoxin type A1 is successfully used to treat a variety of human syndromes characterized by hyperactive cholinergic nerve terminals. The extreme toxicity of these neurotoxins is due to their neurospecificity and to their metalloprotease activity, which results in the deadly paralysis of tetanus and botulism. Recently, many novel botulinum neurotoxins and some botulinum-like toxins have been discovered. This large number of toxins differs in terms of toxicity and biological activity, providing a potential goldmine for novel therapeutics and for new molecular tools to dissect vesicular trafficking, fusion, and exocytosis. The scattered data on toxicity present in the literature require a systematic organization to be usable by scientists and clinicians. We have assembled here the data available in the literature on the toxicity of these toxins in different animal species. The internal comparison of these data provides insights on the biological activity of these toxins.

Comparative functional analysis of mice after local injection with botulinum neurotoxin A1, A2, A6, and B1 by catwalk analysis.

Botulinum neurotoxins (BoNTs) are potent neurotoxins and are the causative agent of botulism, as well as valuable pharmaceuticals. BoNTs are divided into seven serotypes that comprise over 40 reported subtypes. BoNT/A1 and BoNT/B1 are currently the only subtypes approved for pharmaceutical use in the USA. While several other BoNT subtypes including BoNT/A2 and/A6 have been proposed as promising pharmaceuticals, detailed characterization using in vivo assays are essential to determine their pharmaceutical characteristics compared to the currently used BoNT/A1 and/B1. Several methods for studying BoNTs in mice are being used, but no objective and quantitative assay for assessment of functional outcomes after injection has been described. Here we describe the use of CatWalk XT as a new analytical tool for the objective and quantitative analysis of the paralytic effect after local intramuscular injection of BoNT subtypes A1, A2, A6, and B1. Catwalk is a sophisticated gait and locomotion analysis system that quantitatively analyzes a rodent's paw print dimensions and footfall patterns while traversing a glass plate during unforced walk. Significant changes were observed in several gait parameters in mice after local intramuscular injection of all tested BoNT subtypes, however, no changes were observed in mice injected intraperitoneally with the same BoNTs. While a clear difference in time to peak paralysis was observed between BoNT/A1 and/B1, injection of all four toxins resulted in a deficit in the injected limb with the other limbs functionally compensating and with no qualitative differences between the four BoNT subtypes. The presented data demonstrate the utility of CatWalk as a tool for functional outcomes after local BoNT injection through its ability to collect large amounts of quantitative data and objectively analyze sensitive changes in static and dynamic gait parameters.

Botulinum toxin type D blocks autonomic cholinergic synapses in humans: discussion of a potential therapeutic use.

Based on epidemiological data it was believed that botulinumtoxin type D (BT-D) may not block human cholinergic synapses. We wanted to investigate BT-D's effect on the autonomic cholinergic synapse in humans. For this, we compared in four volunteers intraindividually the hypohidrotic effect of intradermal BT-D and BT-A in Minor's iodine starch sweat test. Altogether, we studied BT-D in doses of 4, 8, 16 and 32MU and BT-A in doses of 2, 4, 8 and 16MU at weekly intervals throughout a period of 13 weeks. All BT doses were diluted in 0.2 ml 0.9% NaCl/H2O. Overall 704 data points were collected. Combined over all four subjects and all four doses BT-D's hypohidrotic effect intensity was half of BT-A's. BT-D's effect peaked around 5 weeks, BT-A's around 7 weeks. BT-D's effect duration was around 12 weeks, of BT-A's was around 14 weeks. For both BT types the hypohidrotic effect was dose dependent. BT-D, when injected intradermally, can block autonomic cholinergic synapses in humans. Compared to BT-A, BT-D's effect intensity was half and its effect duration was some 2 weeks shorter. With its weaker and shorter effect BT-D does not seem to promise therapeutic effects superior to BT-A.