Botulinum Neurotoxin Type A Research Articles

A Highly Specific Monoclonal Antibody for Botulinum Neurotoxin Type A-Cleaved SNAP25.

Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A’s location, distribution, and movement within a cell. Due to BoNT/A’s high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25197) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25197, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25197 rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25197 in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25197-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.

Multimodal Physiotherapy as an Add-On Treatment to Botulinum Neurotoxin A-Therapy in Patients with Cervical Dystonia

<b>Background:</b> The treatment of choice for cervical dystonia comprises a local injection of botulinum neurotoxin type A (BoNT-A). Whether additional physiotherapy can achieve better results is not clearly documented in the literature. The present study aims to show possible effects of physiotherapy as an add-on treatment to therapy with botulinum neurotoxin type A in terms of pain, severity of dystonia, statics and function of the cervical spine as well as the quality of life in order to optimize the treatment of cervical dystonia. <b>Design/Methods:</b> This is a prospective, single-center, interventional study of a target patient population comprising 20 participants. The study is divided into 2 phases, with each phase lasting for 3 months. During the first phase, the usual BoNT-A will be applied. In addition, the baseline values will be collected and these assessments will be repeated at an interval of 6 weeks. In the second phase, participants will undergo a physiotherapy program consisting of two 45 min sessions per week in addition to the BoNT-A treatment. The assessments will be undertaken at the same intervals as in the first phase. <b>Analysis:</b> In order to determine the effect of the add-on physiotherapy program, the following parameters will be measured for each assessment: quality of life using the SF- 36 score and the range of motion determined by the ZEBRIS measurement system which will comprise the primary endpoints. Furthermore, for each assessment the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) and electrical activity from the trapezius and the sternocleidomastoid muscles measured by a surface electromyography will be recorded. Descriptive statistical analysis will be performed for all of the collected data. Possible changes in the measured values will be compared to the baseline values over the 2 periods by means of the Wilcoxon-test at a significance level of p<0.05. For the statistical analysis the software SPSS 22.0 will be used. <b>Trial registration:</b> Deutsches Register Klinischer Studien: DRKS00006562

IncobotulinumtoxinA in aesthetics: Russian multidisciplinary expert consensus recommendations.

BackgroundAlthough there are various international consensus recommendations on the use of botulinum neurotoxin type A (BoNT/A) in facial aesthetics, there are no global or Russian guidelines on the optimal dose of incobotulinumtoxinA, free from complexing proteins, within specific aesthetic indications. This article reports the outcomes of two expert consensus meetings, conducted to review and analyze efficacy and tolerability data for incobotulinumtoxinA in various facial aesthetic indications and to give expert consensus recommendations to ensure best clinical practice among Russian clinicians.MethodsThirteen dermatology and/or plastic surgery experts attended meetings held in Paris, France (November 2013), and Moscow, Russia (March 2014). The expert group reviewed and analyzed the existing evidence, consensus recommendations, and Russian experts’ extensive practical experience of incobotulinumtoxinA in aesthetics to reach consensus on optimal doses, potential dose adjustments, and injection sites of incobotulinumtoxinA for facial aesthetics.ResultsAll experts developed guidance on the optimal doses for incobotulinumtoxinA treatment of different regions of the upper and lower face. The expert panel agreed that there are no differences in the efficacy and duration of the effect between the four BoNT/As that are commercially available for facial aesthetic indications in Russia and that, when administered correctly, all BoNT/As can achieve optimal results. Experts also agreed that nonresponse to BoNT/A can be caused by neutralizing antibodies.ConclusionOn the basis of the scientific and clinical evidence available for incobotulinumtoxinA, coupled with the extensive clinical experience of the consensus group, experts recommended the optimal doses of incobotulinumtoxinA effective for treatment of wrinkles of the upper and lower face to achieve the expected aesthetic outcome. These first Russian guidelines on the optimal use of incobotulinumtoxinA for augmentation of glabellar lines, periorbital wrinkles, forehead lines, bunny lines, perioral wrinkles, depressor anguli oris, mentalis, masseters and platysmal bands, and performing the Nefertiti lift, are presented here.

Transurethral intraprostatic injection of botulinum neurotoxin type A for the treatment of chronic prostatitis/chronic pelvic pain syndrome: results of a prospective pilot double-blind and randomized placebo-controlled study.

To evaluate the effect of botulinum neurotoxin type-A (BoNT-A) on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) refractory to medical therapy. Between November 2011 and January 2013, 60 men aged ≥18 years with CP/CPPS, and with National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) scores ≥10 and pain subscale scores ≥8, who were refractory to 4-6 weeks' medical therapy, underwent transurethral intraprostatic injection of BoNT-A or normal saline in a prospective pilot double-blind randomized study. The patients' NIH-CPSI total and subscale scores, American Urological Association (AUA)-symptom score (SS), visual analogue scale (VAS) and quality of life (QoL) scores and frequencies of diurnal and nocturnal urination were evaluated and compared at baseline and at 1, 3 and 6 months after injection and also were compared between the two groups. A total of 60 consecutive patients were randomized to a BoNT-A (treatment) or normal saline (placebo) group. In the treatment group at the 1-, 3- and 6-month evaluation the NIH-CPSI total and subscale scores, and the AUA-SS, VAS and QoL scores, along with frequencies of diurnal and nocturnal urinations, had significantly improved compared with baseline values (P < 0.05). By contrast, in the placebo group, none of these values showed improvement and the values were significantly different from those in the treatment group. Although the differences between the two groups in AUA-SS and frequencies of nocturnal urination were not significant at 1-month follow-up, repeated-measure analysis showed significant improvement in each of these values over the entire follow-up period in the treatment group. The most prominent improvement was related to the pain subscale score, which decreased by 64.76, 75.63 and 79.97% at 1, 3 and 6 months after treatment compared with baseline, followed by the VAS score, which decreased by 62.3, 72.4 and 82.1% at each follow-up, respectively. Only two patients developed mild transient gross haematuria, which was managed conservatively. Transurethral intraprostatic BoNT-A injection maybe an effective therapeutic option in patients with CP/CPPS as it reduces pain and improves QoL.

Heat shock protein 70 overexpression does not attenuate atrophy in botulinum neurotoxin type A-treated skeletal muscle

Botulinum neurotoxin type A (BoNT/A) is used clinically to induce therapeutic chemical denervation of spastically contracted skeletal muscles. However, BoNT/A administration can also cause atrophy. We sought to determine whether a major proteolytic pathway contributing to atrophy in multiple models of muscle wasting, the ubiquitin proteasome system (UPS), is involved in BoNT/A-induced atrophy. Three and ten days following BoNT/A injection of rat hindlimb, soleus muscle fiber cross-sectional area was reduced 25 and 65%, respectively. The transcriptional activity of NF-κB and Foxo was significantly elevated at 3 days (2- to 4-fold) and 10 days (5- to 6-fold). Muscle RING-finger protein-1 (MuRF1) activity was elevated (2-fold) after 3 days but not 10 days, while atrogin-1 activity was not elevated at any time point. BoNT/A-induced polyubiquitination occurred after 3 days (3-fold increase) but was totally absent after 10 days. Proteasome activity was elevated (1.5- to 2-fold) after 3 and 10 days. We employed the use of heat shock protein 70 (Hsp70) to inhibit NF-κB and Foxo transcriptional activity. Electrotransfer of Hsp70 into rat soleus, before BoNT/A administration, was insufficient to attenuate atrophy. It was also insufficient to decrease BoNT/A-induced Foxo activity at 3 days, although NF-κB activity was abolished. By 10 days both NF-κB and Foxo activation were abolished by Hsp70. Hsp70-overexpression was unable to alter the levels of BoNT/A-induced effects on MuRF1/atrogin-1, polyubiquitination, or proteasome activity. In conclusion, Hsp70 overexpression is insufficient to attenuate BoNT/A-induced atrophy. It remains unclear what proteolytic mechanism/s are contributing to BoNT/A-induced atrophy, although a Foxo-MuRF1-ubiquitin-proteasome contribution may exist, at least in early BoNT/A-induced atrophy. Further clarification of UPS involvement in BoNT/A-induced atrophy is warranted.

Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a.

Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity.

Botulinum neurotoxin type A induces TLR2-mediated inflammatory responses in macrophages.

Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal–regulated kinase (ERK), and p38 mitogen–activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

Effects of Botulinum Toxin A Injection on Healing and Tensile Strength of Ruptured Rabbit Achilles Tendons

Tendon lacerations are most commonly managed with surgical repair. Postoperative complications such as adhesions and ruptures often occur with immobilization. Early postoperative mobilization is therefore advised to minimize complications and time required to return to daily life. The aim of this study was to evaluate whether botulinum neurotoxin type-A (BoNT-A) can be used to enhance healing and prevent rupture in mobilized animals with Achilles tenotomy. Twenty-seven rabbits were divided into 3 groups, namely, I, II, and III, after surgical 1-sided Achilles tenotomy and end-to-end repair. The control group for biomechanical comparisons consisted of randomly selected contralateral (unoperated) healthy Achilles tendons. Group I received BoNT-A (4 U/kg) injection into the calf muscles. One week later, electromyographical confirmation was performed to establish the effects of injection. Surgery was then performed. Animals in the second group (n = 9, group II) were immobilized with a cast postoperatively. The third group (n = 9, group III) was mobilized immediately with no cast or BoNT-A. Tendons were harvested and gap formation or ruptures as well as strength of the repaired tendon were assessed 6 weeks after surgery. Achilles tendons healed in all animals injected with BoNT-A, whereas all were ruptured in group III. All Achilles tendons of animals in groups I and II healed. However, group I repaired tendons were biomechanically equivalent to healthy tendons, whereas group II repaired tendons demonstrated significantly decreased tensile strength (P = 0.009). The present study suggests that local injection of BoNT-A can be used for treatment of tendon rupture and may replace the use of cast for immobilization. However, further studies are needed to determine whether BoNT-A injection can have a beneficial effect on the healing of tendon repairs in humans.

An Animal Model for Inducing Deviation of the Mandible

Altering the occlusal surface is still a common choice for inducing a deviated mandible in an animal model. Botulinum neurotoxin type A (BoTx/A) can block the action potential transmission in neuromuscular junctions by inhibiting acetylcholine release without damaging the nerves and muscle structures. Our present study was aimed at developing an easy-to-reproduce animal model of asymmetric mandibles in which injection of BoTx/A was applied. A total of 96 healthy 4-week-old male Sprague-Dawley rats were divided into 2 groups: an experimental group (n= 48) with BoTx/A injection and a control group (n= 48) with sterile saline injection at 4 sites of the right masseter muscle. Twelve rats from each group were humanely euthanized at weeks 1, 2, 3, and 4 for morphometric analysis using the micro-computed tomography (CT) findings. The micro-CT scans revealed facial asymmetry in the experimental group, with no facial asymmetry in the control group after injection. Significant differences were found between the experimental and control groups regarding the indexes containing the mandibular length (length from condyle to menton, length from coronoid to menton, and length of mandibular corpus from gonion to menton) and ramus height (posterior border and middle region near coronoid, and height of anterior mandible at vertical distance from menton). Our data have indicated that this deviated mandible animal model induced by injection of BoTx/A is highly reproducible and might be proved suitable for future studies of the asymmetric mandible.

786 Retrograde transport of botulinum neurotoxin type A (BoNT/A) to the central nervous system (CNS) and central changes in markers related to lower urinary tract control after bladder injections in rats

786 Retrograde transport of botulinum neurotoxin type A (BoNT/A) to the central nervous system (CNS) and central changes in markers related to lower urinary tract control after bladder injections in rats

Botulinum neurotoxin type A modulates vesicular release of glutamate from satellite glial cells.

This study investigated the presence of cell membrane docking proteins synaptosomal-associated protein, 25 and 23 kD (SNAP-25 and SNAP-23) in satellite glial cells (SGCs) of rat trigeminal ganglion; whether cultured SGCs would release glutamate in a time- and calcium-dependent manner following calcium-ionophore ionomycin stimulation; and if botulinum neurotoxin type A (BoNTA), in a dose-dependent manner, could block or decrease vesicular release of glutamate. SGCs were isolated from the trigeminal ganglia (TG) of adult Wistar rats and cultured for 7 days. The presence of SNAPs in TG sections and isolated SGCs were investigated using immunohistochemistry and immunocytochemistry, respectively. SGCs were stimulated with ionomycin (5 μM for 4, 8, 12 and 30 min.) to release glutamate. SGCs were then pre-incubated with BoNTA (24 hrs with 0.1, 1, 10 and 100 pM) to investigate if BoNTA could potentially block ionomycin-stimulated glutamate release. Glutamate concentrations were measured by ELISA. SNAP-25 and SNAP-23 were present in SGCs in TG sections and in cultured SGCs. Ionomycin significantly increased glutamate release from cultured SGCs 30 min. following the treatment (P < 0.001). BoNTA (100 pM) significantly decreased glutamate release (P < 0.01). Results from this study demonstrated that SGCs, when stimulated with ionomycin, released glutamate that was inhibited by BoNTA, possibly through cleavage of SNAP-25 and/or SNAP-23. These novel findings demonstrate the existence of vesicular glutamate release from SGCs, which could potentially play a role in the trigeminal sensory transmission. In addition, interaction of BoNTA with non-neuronal cells at the level of TG suggests a potential analgesic mechanism of action of BoNTA.

Microfluidic Detection of Botulinum Neurotoxin Type A Utilizing Polyacrylamide Hydrogels With SNAP-25 Peptide Crosslinker

We synthesized a new botulinum neurotoxin type A (BoNT/A) sensing material consisting of a photopolymerizable polyacrylamide hydrogel incorporating a 25-mer BoNT/A recognizable peptide substrate coupled to an acryloyl-PEG-NHS crosslinker that readily reacts with free amines at pH 8-9 via the NHS moiety. The peptide substrate residues are slightly modified from synaptosomal-associated protein SNAP-25 between amino acid residues 187-203 with an additional three glycine spacers and a lysine on each end for coupling to the crosslinker via NHS-ester conjugation reactions. The alpha and epsilon amines of the N-terminal lysine and epsilon amine of the C-terminal lysine were used to couple the peptide to the crosslinker instead of previously used cysteines to avoid having to remove dithiothreitol (DTT) after sample preparation or potential false positive substrate cleavage by DTT if not removed after sample preparation. By integrating this hydrogel into a microchannel, we developed a BoNT sensor based on the morphology change of this hydrogel. The hydrogel in the microchannel can be degraded in 22 h at 45 μg/mL of light chain (LC) of BoNT/A and 90 h at the lowest concentration of 4.5 μg/mL of LC, but remains intact in Hepes buffer and DTT solutions. The results indicate that the hydrogel-based device can potentially be a portable sensor for the detection of active BoNT/A with high accuracy and specificity.

Antinociceptive Effects of Transcytosed Botulinum Neurotoxin Type A on Trigeminal Nociception in Rats.

We examined the effects of peripherally or centrally administered botulinum neurotoxin type A (BoNT-A) on orofacial inflammatory pain to evaluate the antinociceptive effect of BoNT-A and its underlying mechanisms. The experiments were carried out on male Sprague-Dawley rats. Subcutaneous (3 U/kg) or intracisternal (0.3 or 1 U/kg) administration of BoNT-A significantly inhibited the formalin-induced nociceptive response in the second phase. Both subcutaneous (1 or 3 U/kg) and intracisternal (0.3 or 1 U/kg) injection of BoNT-A increased the latency of head withdrawal response in the complete Freund's adjuvant (CFA)-treated rats. Intracisternal administration of N-methyl-D-aspartate (NMDA) evoked nociceptive behavior via the activation of trigeminal neurons, which was attenuated by the subcutaneous or intracisternal injection of BoNT-A. Intracisternal injection of NMDA up-regulated c-Fos expression in the trigeminal neurons of the medullary dorsal horn. Subcutaneous (3 U/kg) or intracisternal (1 U/kg) administration of BoNT-A significantly reduced the number of c-Fos immunoreactive neurons in the NMDA-treated rats. These results suggest that the central antinociceptive effects the peripherally or centrally administered BoNT-A are mediated by transcytosed BoNT-A or direct inhibition of trigeminal neurons. Our data suggest that central targets of BoNT-A might provide a new therapeutic tool for the treatment of orofacial chronic pain conditions.

RNA aptamer as potential antidote against botulism: an in vivo report

Botulinum neurotoxins (BoNTs) are the most toxic substances to mankind, and yet, there are no therapeutics available to reverse the paralysis, the deadly symptom of botulism caused by BoNTs. Here we report that RNA aptamer, selected through in vitro process against light chain of botulinum neurotoxin type A (BoNT/A) showed in vivo protection efficacy against botulism. Our results showed that RNA aptamer not only accelerated the recovery of local paralysis induced by BoNT/A through mouse exercise wheel assay, but also significantly delayed the time-to-death through mouse protection assay, and rescued one mouse (out of six) completely intoxicated by BoNT/A. Our results demonstrated the potential using RNA aptamer as antidote against deadly botulism.

Clinical outcomes of individualized botulinum neurotoxin type A injection techniques in patients with essential blepharospasm.

PurposeTo assess the clinical outcomes following botulinum neurotoxin type A (BoNT-A) treatment with an individualized injection technique based on the types of spasms and to compare the results of the individualized injection technique with those of the conventional injection technique in the same patients.MethodsFrom November 2011 to July 2013, 77 BoNT-A injections were performed in 38 patients. Eighteen patients were treated with conventional BoNT-A injections before 2011, and 20 patients were referred to our hospital for unsatisfactory results after a conventional injection technique. We classified the patients by spasm-dominant sites: the lateral orbital area, representing the orbital orbicularis-dominant group (ODG); the glabella, representing the corrugator-dominant group (CDG); and the ptosis, representing the palpebral part of the orbicularis-dominant group (PDG). We increased the injection dose into the spasm-dominant sites of the blepharospasm groups. We assessed subjective symptom scores (functional disability score, FDS) after treatment.ResultsThis study included 38 patients (26 women, 12 men; mean age, 60.6 ± 10.9 years). There were 21 patients in the ODG, 10 patients in the CDG, and 7 patients in the PDG. Mean ages were 59.7 ± 12.6, 59.8 ± 8.5, and 66.8 ± 9.0 years, and mean BoNT-A injection dose was 38.8 ± 11.2, 38.8 ± 11.2, and 38.8 ± 10.8 U in each group, respectively (p = 0.44, 0.82 Kruskal-Wallis test). Mean FDS after injection was 1.7 ± 0.7 in the ODG, 1.4 ± 0.8 in the CDG, and 1.2 ± 0.3 in the PDG. There were significant differences in reading and job scale among the three groups. In a comparison between the conventional and individualized injection techniques, there was a significant improvement in mean FDS and in the reading scale in the PDG with the individualized injection technique. The success rate was 92.1% in the conventional injection group and 94.1% in the individualized injection group.ConclusionsThe individualized injection technique of BoNT-A according to the spasm-dominant site is an effective and safe treatment method for essential blepharospasm patients.

116. Functional connectivity between cortical speech network and primary motor cortex is abnormal in spasmodic dysphonia

In healthy humans, the execution of linguistic tasks modifies the excitability of the hand area of the dominant primary motor cortex (M1), as tested by TMS. Our aim was to investigate the effect of linguistic tasks on the excitability of the M1 in patients with adductor type-spasmodic dysphonia (ASD), before and after Botulinum Neurotoxin Type A (BoNT-A) injections. We studied 10 patients with ASD and 10 age-matched healthy subjects. All participants were right handed. The excitability of either M1 hand area was evaluated at the baseline and during different linguistic and non-linguistic tasks: (1) reading aloud of single words; (2) silent reading; (3) looking at meaningless non-letter strings; (4) oral movements without vocalization; (5) producing simple syllabic phonation. In healthy controls, the motor evoked potential (MEP) elicited by TMS of the dominant M1 were significantly larger during reading aloud. In ASD patients, MEP enhancement in the dominant hand emerged not only during reading aloud but also during syllabic phonation. BoNT-A injections restored the neurophysiologic abnormalities. We conclude that ASD is characterized by an abnormal excitability of the hand area of the dominant M1 during specific linguistic tasks. This likely reflects an altered functional connectivity between cortical speech network and M1.

Factors influencing secondary non-response to botulinum toxin type A injections in cervical dystonia.

The development of secondary non-response (SNR) to botulinum neurotoxin type-A (BoNT-A) is considered a key issue in the management of cervical dystonia (CD). This case-controlled study was performed to systematically identify factors influencing SNR during BoNT-A therapy. This was a retrospective, international, non-interventional study of CD patients. Patients with SNR were matched with up to three responder patients (control) on the basis of duration of therapy and number of injection cycles. Factors influencing the development of SNR were screened using a univariate logistic regression model and confirmed using a multivariate conditional logistic regression model. 216 patients were enrolled, and 201 (SNR = 52; responder = 149) were matched and subdivided into blocks (doublets, triplets or quadruplets). At baseline, a significantly higher proportion of SNR patients had received previous or concomitant therapies (p = 0.038) and surgery for CD (p = 0.007) compared with controls. Although disease severity at onset was similar between groups, a significantly higher proportion of SNR patients experienced severe CD at the time of SNR compared with controls at the last documented visit. Multivariate analyses identified five factors that were significantly associated in predicting SNR (odds ratio [OR] > 1 indicated higher chances for being SNR): previous surgical procedure for CD (OR 9.8, p = 0.013), previous BoNT-A related severe adverse event (AE) (OR 5.6 p = 0.027), physical therapy (OR 4.6, p = 0.028), neuroleptic use (OR 3.3, p = 0.019) and average BoNT-A dose (OR 2.7, p = 0.010). These findings suggest that SNR may not reflect true pharmacological resistance to BoNT-A therapy, but may be related to underlying disease severity.

Regions recognized on the light chain of botulinum neurotoxin type A by T lymphocytes of SJL and BALB/c mice primed with inactivated toxin

Lymph node cells (LNC) from SJL (H-2s) and BALB/c (H-2d) mice primed once with inactivated botulinum neurotoxin type A (BoNT/A) were examined for their T-cell responses to each of 32 synthetic overlapping peptides (19 residues each, L1–L32) that encompass the entire L chain (residues 1–448) of BoNT/A. LNC of SJL gave strong responses to 6 regions on, L2 (residues 15–23), L10/11/12 (127–173), L19 (253–271) and L21 (281–299), and moderate to weak responses to L9 (113–131), L14/15 (183–215) and L27 (365–383). In BALB/c, LNC gave a substantial T-cell response only against peptide L12 (residues 155–173), and responded very weakly to 9 other peptides. The results were compared with the recognition profiles determined previously in these two strains after multiple BoNT/A injections. Overall responses to the L-chain peptides of T cells in later profiles were found to be somewhat weakened in SJL and stayed essentially at a similar level in BALB/c, although responses to BoNT/A increased. In SJL, response to L10 (127–145) remained the highest in the later profile. Strong responses against L12 (155–173) observed in both strains at early stage were reduced to an insignificant level. Cross-reactivity to tetanus neurotoxin by BoNT/A-specific T cells was observed in SJL but not in BALB/c. Design of an effective synthetic peptide vaccine will require incorporation of both T cell- and Ab-recognition elements of the BoNT molecule. Significance and possible implications of these results on BoNT/A-specific T-cell responses of BoNT-treated patients are discussed.

Comparison of immunostrips with mouse bioassay and bacterial culture in detecting botulinum toxins in bottles from suspected Taiwan high-speed rail bomber

In April 2013, a bomb-threat incident occurred on the Taiwan high-speed railway. Two suspects claimed to have placed explosive devices on a high-speed train and outside a lawmaker’s office. Afterward, the Criminal Investigation Bureau also found several bottles in the main suspect’s apartment labeled “botulinum toxin.” The Institute of Preventive Medicine was entrusted by the Institute of Forensic Medicine, Ministry of Justice, to verify whether the bottles contained botulinum neurotoxin (BoNT). Three different analyses, including rapid detection strips, toxin neutralization tests, and bacterial culture, were processed. The results provided conclusive evidence that botulinum neurotoxin type A (BoNT/A) was present in the samples. This study demonstrated that the BoNT/A strip assay is an excellent method for BoNT/A detection, and has potential use as an early warning tool in screening of food products for botulinum toxins.

Chapter 3: Molecular basis for the therapeutic effectiveness of botulinum neurotoxin type A.

The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine glands is attributable to a unique conflation of properties honed to exploit and inactivate synaptic transmission. Specific, high-affinity coincident binding to gangliosides plus an intraluminal loop of synaptic vesicle protein 2 (SV2) by the heavy chain (HC) of BoNT/A confers selectivity for presynaptic nerve terminals and subsequent uptake by endocytosis. Upon vesicle acidification, the HC forms a channel for transmembrane transfer of the light chain to the cytosol, as observed by single channel recordings. The light chain is a Zn(2+) -dependent endoprotease that cleaves and inactivates SNAP-25, thereby blocking exocytotic release of transmitters, a discovery that revealed the pivotal role of the latter in synaptic vesicle fusion. A di-leucine motif in BoNT/A light chain stabilizes this protease, contributing to its longevity inside nerves. The ubiquity of SV2 and SNAP-25 has prompted re-evaluation of the nerve types susceptible to BoNT/A. In urology, there is emerging evidence that BoNT/A blocks neuropeptide release from afferent nerves, exocytosis of acetylcholine and purines from efferent nerves, and possibly ATP release from the urothelium. Suppression by BoNT/A of the surface expression of nociceptor channels on bladder afferents might also contribute to its improvement of urological sensory symptoms.