- Email: [email protected]
Architecture of the botulinum neurotoxin complex
S44
Abstracts / Toxicon 123 (2016) S2eS90
114. PREDICTORS OF ONABOTULINUMTOXINA RESPONSE IN PATIENTS WITH CERVICAL DYSTONIA
Table Predicted Response to Treatment With OnabotulinumtoxinA in Patients With Cervical Dystonia. Covariate
Odds ratio
95% CI
TWSTRS Total-1stdfa TWSTRS Total-2nd dfa TWSTRS Total-3rddfa CD subtype: anterocollis CD subtype: laterocollis CD subtype: retrocollis CD subtype: other Longissimus Splenius capitis Sternocleidomastoid
3.5926 16.9593 4.3805 0.3035 1.0806 1.3984 0.3544 0.4685 0.7231 1.6989
1.3285, 0.8711, 0.6063, 0.0979, 0.6885, 0.5420, 0.1005, 0.2684, 0.3656, 1.0122,
P Value 9.8110 385.6461 35.6157 0.8522 1.7020 3.8405 1.1235 0.8096 1.3957 2.8705
0.0120 0.0665 0.1507 0.0283 0.7365 0.4973 0.0858 0.0070 0.3406 0.0457
CD¼cervical dystonia; df¼degrees of freedom; TWSTRS¼Toronto Western Spasmodic Torticollis Rating Scale. a TWSTRS Total score at baseline transformed by cubic regression spline with 3 df.
Joseph Jankovic a, *, Marc Schwartz b, Aubrey Manack Adams c. a Baylor College of Medicine, Houston, TX, USA; b MedNet Solutions, Inc, Minnetonka, MN, USA; c Allergan plc, Irvine, CA, USA * Corresponding author: Neuroinfectiology Laboratory, Institute for Infectious Diseases (IFIK), University of Bern, PO Box, Friedbühlstrasse 51, CH-3001 Bern, Switzerland. E-mail address:[email protected].
Introduction and objectives: Data from the Cervical Dystonia Patient Registry for Observation of OnabotulinumtoxinA Efficacy (CD PROBE) study were analyzed to identify characteristics and treatment practices that predict response to onabotulinumtoxinA. Methods: CD PROBE is a multicenter, observational, prospective clinical registry, recruiting from 88 US sites (Janovic 2011; Janovic 2015). Patients with cervical dystonia (CD) who were new to botulinum toxin therapy or had not received it for 16 weeks and were deemed suitable for onabotulinumtoxinA treatment received 3 treatment cycles, as per their physician’s usual care. The Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) was used to assess effectiveness, defined as a 30% and/or 10point reduction in TWSTRS total score from baseline to final visit. Relationships between doses, treatment interval, and response were analyzed by linear regression. Disease and treatment characteristics as predictors of response were evaluated using logistic regression. Results: This exploratory analysis included 441 patients. There were no differences in baseline characteristics and no clinically significant differences in doses or in treatment intervals between responders (n¼254) and nonresponders (n¼187). Doses at first treatment were initially greater for responders (188 U vs 168 U; P¼0.013), but by cycle 3, doses were the same in both groups, due to dose escalation in nonresponders. Patients with a higher TWSTRS total score at baseline or injection in the sternocleidomastoid had a higher probability of response; those with anterocollis or injection in the longissimus were less likely to respond (Table). Conclusions: Consistent with clinical experience, patients with higher baseline TWSTRS total score were more likely to respond to treatment with onabotulinumtoxinA, whereas those with anterocollis were less likely to respond. Treatment of CD with onabotulinumtoxinA must be tailored to the patient’s specific needs. Funding: Allergan plc Keywords: Cervical dystonia; OnabotulinumtoxinA; Predictors; Response References Jankovic J, Adler CH, Charles PD, et al. Rationale and design of a prospective study: Cervical Dystonia Patient Registry for Observation of OnaBotulinumtoxinA Efficacy (CD PROBE). BMC Neurol. 2011;11:140. Jankovic J, Adler CH, Charles D, et al. Primary results from the cervical
dystonia patient registry for observation of onabotulinumtoxina efficacy (CD PROBE). J Neurol Sci 2015;349:84-93. 115. DEVELOPMENT OF AN IN VITRO CELL-BASED BIOASSAY FOR THE DETECTION OF CLOSTRIDIUM BOTULINUM NEUROTOXINS BY USING MESC-DERIVED NEURONS GROWN ON MULTI ELECTRODE ARRAYS Stephen P. Jenkinson a, b, c,d, *, Denis Grandgirard a, c, Martina Avondet b, Stephen L. Leib a, c. Heidemann e, Anne Tscherter e, Marc-Andre a Neuroinfectiology Laboratory, Institute for Infectious Diseases (IFIK), University of Bern, Switzerland; b Biology Division, The Spiez Laboratory, Swiss Federal Office for Civil Protection, Spiez, Switzerland; c Cluster for Regenerative Neuroscience, DCR, University of Bern, Bern, Switzerland; d Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; e Department of Physiology, University of Bern, Bern, Switzerland * Corresponding author: Neuroinfectiology Laboratory, Institute for Infectious Diseases (IFIK), University of Bern, PO Box, Friedbühlstrasse 51, CH-3001 Bern, Switzerland. E-mail address:stephen.jenkinson@ifik.unibe.ch.
Introduction and objectives: Clostridium botulinum neurotoxins (BoNTs) are the most poisonous naturally occurring protein toxins known to man, with the toxicity range in humans starting as low as 0.3 ng/kg when administered intravenously. All BoNT serotypes (A-G) consist of 2 subunits, a heavy chain (HC) of ~100 kDa and a light chain (LC) of ~50 kDa, which are linked by a disulphide bond. Upon binding to specific membrane receptors on the plasmalemma of neurons and uptake in the synaptic vesicles, the HC translocates the LC over the membrane where it exerts its cleaving activity against SNARE proteins, thus inhibiting neurotransmitter release. Currently, the potency of biologically active BoNT is monitored using the murine LD50 assay. Using differentiated neurons from mouse embryonic stem cells (mESCs), we intend to develop an in vitro assay capable of detecting BoNT activity using electrophysiological recording techniques. Method: mESCs were differentiated towards neurons by embryoid body (EB) formation. After 7 days, EBs were dissociated and the cells were cultured for 3 weeks on multielectrode arrays (MEAs) allowing the extracellular recording of spontaneous neuronal activity. The cultures were then treated with varying concentrations of BoNT serotype A (BoNT/ A), and spontaneous network bursts, evoked through synaptic transmission, as well as total network activity, were recorded 6 hours after exposure to the toxin. Results: Forty-eight cultures were assessed in 6 independent experiments. Twenty-three cultures were used to quantify the effect of the toxin on synapses when treated with either 333 femtomoles (fmol) or 33 fmol BoNT/A, while 27 cultures served as controls and did not receive any treatment. Exposure to BoNT/A for 6 hours resulted in a significant decrease of the burst rate for toxin concentrations of 333 fmol (49.5 ± 27.2 %; P<0.0005; n¼7) and 33 fmol (66.7 ± 42.5 %; P<0.005; n¼16) compared with untreated cultures (100 ± 19.7 %; n¼27). A significant decrease in the total network activity was observed for toxin concentrations of 333 fmol (45.5 ± 48.0 %; P<0.005; n¼7) and 33 fmol (78.0 ± 70.9 %; P<0.05; n¼16) compared with untreated cultures (100 ± 31.9 %; n¼27). Conclusion: The present assay detects toxin activity of BoNT/A. Thus proof of principle has been achieved. Given sufficient robustness and a further increase in sensitivity to detect BoNT/A, this assay may replace the murine LD50 assay in the batch control of BoNT products. Keywords: Bioassay; BoNT; Clostridium botulinum; Embryonic stem cells; Neuronal cultures; MEAs 116. ARCHITECTURE OF THE BOTULINUM NEUROTOXIN COMPLEX Rongsheng Jin. University of California Irvine, Department of Physiology & Biophysics, Medical Sciences I, Room C-333, Irvine 92697, CA, USA. E-mail address:r.jin@ uci.edu
Abstracts / Toxicon 123 (2016) S2eS90
Introduction and objectives: Botulinum neurotoxin serotype A (BoNT/A) is naturally produced in bacteria with the nontoxic nonhemagglutinin (NTNHA) protein and 3 hemagglutinins (HA70, HA33, and HA17). Together they form a progenitor toxin complex (PTC), the largest bacterial toxin complex known to date. In food-borne botulism, the PTC functions as a molecular machine that protects BoNT/A and helps it breach the host defense in the gastrointestinal (GI) tract. However, the molecular mechanism underlying the PTC function remains poorly defined. Methods: We prepared the highly homogeneous recombinant forms of all PTC components (BoNT/A, NTNHA, HA17, HA33, and HA70) using Escherichia coli. We determined numerous 3-dimensional (3D) structures for individual PTC components and various subcomplexes using x-ray crystallography. We then determined the first pseudo-atomic structure of a 760-kDaelarge PTC (L-PTC) of BoNT/A using a combination of x-ray crystallography, single-particle electron microscopy, and 3D reconstruction (3D-EM). Furthermore, we systematically examined the interactions between the HA complex and an important cell adhesion protein, E-cadherin, and determined the first crystal structure of an HA complex bound to Ecadherin. Results: We demonstrated that the overall architecture of the L-PTC of BoNT/A is composed of 2 structurally and functionally independent entities, reminiscent of an Apollo lunar landing vessel. Toxin’s “ascent stage” (~300 kDa) is formed by BoNT/A and NTNHA that protects BoNT/A from destruction in the GI tract. The “descent stage” (~470 kDa, termed HA complex) consists of 3 hemagglutinins, which assemble into a 3-fold symmetric hetero-dodecameric complex. The HA complex has 9 carbohydrate-binding sites, which allow BoNT/A to land on the cell surface in the small-intestine lumen by docking onto host carbohydrates. Furthermore, we found that the HA complex recognizes E-cadherin with high specificity involving extensive intermolecular interactions. Binding of the HA complex sequesters E-cadherin in the monomeric state, thereby compromising the E-cadherinemediated intercellular barrier in the intestines and facilitating paracellular absorption of the BoNT/A complex. We successfully reconstituted the complete 14-subunit BoNT/A L-PTC in vitro, using all recombinantly produced PTC components. We found that abolishing either E-cadherin or carbohydrate binding of the HA complex using structure-based mutagenesis drastically reduced oral toxicity of the BoNT/A complex in vivo. Conclusions: We revealed the first atomic architecture of a gigantic multiprotein BoNT/A complex. This structure combined with the complementary functional studies established the molecular mechanism of how PTC contributes to the oral toxicity of BoNT/A. These new findings will promote the development of novel chemical inhibitors or antibody/peptide inhibitors as countermeasures against BoNTs and also open the door for alternative applications of this sophisticated bacterial machinery. Keywords: Botulinum neurotoxin; Crystal structure; E-cadherin; HA complex; Host receptors; Progenitor toxin complex
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Fig. 1. The molecular architecture of the large PTC (L-PTC) of BoNT/A. The minimally functional PTC (M-PTC) is composed of BoNT/A (purple) and NTNHA (salmon), and the HA complex is composed of HA70 (blue), HA17 (yellow), and HA33 (orange).
117. LONGITUDINAL STUDIES OF CERVICAL DYSTONIA H.A. Jinnah. for the Dystonia Coalition Investigators Emory University, 100 Woodruff Circle, 6300 Woodruff Memorial Building, Atlanta, GA 30322E-mail address:[email protected] Cervical dystonia is defined by excessive contraction of the neck muscles. It is the most common of all adult-onset focal dystonias. Numerous crosssectional studies have described its cardinal features, which include abnormal head positions, repetitive phasic head movements, neck and shoulder muscle spasms, neck and shoulder pain, and other problems. Although the clinical features of cervical dystonia are well described, few studies have documented the evolution of these features in longitudinal study designs. Early studies were relatively small and limited to experience from individual expert centers. These studies often implied worsening over a period of a few months to a year, followed by relatively static abnormalities that only rarely remitted. This worsening could take the form of increased severity of neck muscle contractions, or spread of contractions to muscles outside of the neck region. Later studies with longer periods of follow up suggested that some cases might continue to worsen for at least 5-10 years after onset. One of the goals of the Dystonia Coalition has been to evaluate the evolution of the clinical features of cervical dystonia using a prospectively designed standard rating protocol in a multicenter setting. An interim analysis of results for the period of recruitment from 5 January 2011, through 30 September 2015, was conducted. In this cohort, 37 sites had enrolled 2257 subjects with various types of isolated dystonia, including 1582 with focal or segmental cervical dystonia. Among these cases, 22.8% had evidence of spread outside of the neck region. Identification of patients with cervical dystonia who are at risk for worsening is vital for counseling them regarding their future. It is also important for symptomatic treatment, as the dose and pattern of botulinum toxin may change over time. Longitudinal data is vital for the design of any future disease-modifying treatments aimed at slowing or stopping progression of the disorder. Keywords: Cervical dystonia; Natural history study; Progression; Torticollis 118. PRACTICAL MANAGEMENT OF FLU-LIKE SYMPTOMS AMONG PATIENTS RECEIVING BOTULINUM TOXIN INJECTIONS H.A. Jinnah*, Adam Kassem. Emory University, Atlanta, GA, USA * Corresponding author: Emory University, 100 Woodruff Circle, 6300 Woodruff Memorial Building, Atlanta, GA 30322, USA. E-mail address:[email protected].
Introduction and objectives: Some patients experience a flu-like (FL) syndrome following botulinum toxin (BoNT) injections. These symptoms suggest an immunological response (IR) to the injections, even though the clinical outcome from treatment remains good. These observations, together with evidence that BoNT itself has low immunogenic potential, suggest that the IR in some cases may be a reaction to accompanying ingredients. The purpose of this study was to determine whether switching to a different BoNT product, which contains different types or amounts of accompanying ingredients, reduces the development of the FL symptoms. Methods: The study was conducted with 5 subjects who regularly complained of severe FL reactions following BoNT injections for dystonia. We methodically collected details of the treatment associated with FL symptoms, before and after switching to an alternative brand. Results: All 5 subjects who reported FL symptoms experienced either a reduction or elimination of FL symptoms after switching from the first product to a second product. All 5 patients reported maintenance of clinical benefits. The clinical benefits and lessening of FL symptoms were maintained for at least 3 injection cycles with the second brand, making chance occurrence unlikely.
Abstracts / Toxicon 123 (2016) S2eS90
114. PREDICTORS OF ONABOTULINUMTOXINA RESPONSE IN PATIENTS WITH CERVICAL DYSTONIA
Table Predicted Response to Treatment With OnabotulinumtoxinA in Patients With Cervical Dystonia. Covariate
Odds ratio
95% CI
TWSTRS Total-1stdfa TWSTRS Total-2nd dfa TWSTRS Total-3rddfa CD subtype: anterocollis CD subtype: laterocollis CD subtype: retrocollis CD subtype: other Longissimus Splenius capitis Sternocleidomastoid
3.5926 16.9593 4.3805 0.3035 1.0806 1.3984 0.3544 0.4685 0.7231 1.6989
1.3285, 0.8711, 0.6063, 0.0979, 0.6885, 0.5420, 0.1005, 0.2684, 0.3656, 1.0122,
P Value 9.8110 385.6461 35.6157 0.8522 1.7020 3.8405 1.1235 0.8096 1.3957 2.8705
0.0120 0.0665 0.1507 0.0283 0.7365 0.4973 0.0858 0.0070 0.3406 0.0457
CD¼cervical dystonia; df¼degrees of freedom; TWSTRS¼Toronto Western Spasmodic Torticollis Rating Scale. a TWSTRS Total score at baseline transformed by cubic regression spline with 3 df.
Joseph Jankovic a, *, Marc Schwartz b, Aubrey Manack Adams c. a Baylor College of Medicine, Houston, TX, USA; b MedNet Solutions, Inc, Minnetonka, MN, USA; c Allergan plc, Irvine, CA, USA * Corresponding author: Neuroinfectiology Laboratory, Institute for Infectious Diseases (IFIK), University of Bern, PO Box, Friedbühlstrasse 51, CH-3001 Bern, Switzerland. E-mail address:[email protected].
Introduction and objectives: Data from the Cervical Dystonia Patient Registry for Observation of OnabotulinumtoxinA Efficacy (CD PROBE) study were analyzed to identify characteristics and treatment practices that predict response to onabotulinumtoxinA. Methods: CD PROBE is a multicenter, observational, prospective clinical registry, recruiting from 88 US sites (Janovic 2011; Janovic 2015). Patients with cervical dystonia (CD) who were new to botulinum toxin therapy or had not received it for 16 weeks and were deemed suitable for onabotulinumtoxinA treatment received 3 treatment cycles, as per their physician’s usual care. The Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) was used to assess effectiveness, defined as a 30% and/or 10point reduction in TWSTRS total score from baseline to final visit. Relationships between doses, treatment interval, and response were analyzed by linear regression. Disease and treatment characteristics as predictors of response were evaluated using logistic regression. Results: This exploratory analysis included 441 patients. There were no differences in baseline characteristics and no clinically significant differences in doses or in treatment intervals between responders (n¼254) and nonresponders (n¼187). Doses at first treatment were initially greater for responders (188 U vs 168 U; P¼0.013), but by cycle 3, doses were the same in both groups, due to dose escalation in nonresponders. Patients with a higher TWSTRS total score at baseline or injection in the sternocleidomastoid had a higher probability of response; those with anterocollis or injection in the longissimus were less likely to respond (Table). Conclusions: Consistent with clinical experience, patients with higher baseline TWSTRS total score were more likely to respond to treatment with onabotulinumtoxinA, whereas those with anterocollis were less likely to respond. Treatment of CD with onabotulinumtoxinA must be tailored to the patient’s specific needs. Funding: Allergan plc Keywords: Cervical dystonia; OnabotulinumtoxinA; Predictors; Response References Jankovic J, Adler CH, Charles PD, et al. Rationale and design of a prospective study: Cervical Dystonia Patient Registry for Observation of OnaBotulinumtoxinA Efficacy (CD PROBE). BMC Neurol. 2011;11:140. Jankovic J, Adler CH, Charles D, et al. Primary results from the cervical
dystonia patient registry for observation of onabotulinumtoxina efficacy (CD PROBE). J Neurol Sci 2015;349:84-93. 115. DEVELOPMENT OF AN IN VITRO CELL-BASED BIOASSAY FOR THE DETECTION OF CLOSTRIDIUM BOTULINUM NEUROTOXINS BY USING MESC-DERIVED NEURONS GROWN ON MULTI ELECTRODE ARRAYS Stephen P. Jenkinson a, b, c,d, *, Denis Grandgirard a, c, Martina Avondet b, Stephen L. Leib a, c. Heidemann e, Anne Tscherter e, Marc-Andre a Neuroinfectiology Laboratory, Institute for Infectious Diseases (IFIK), University of Bern, Switzerland; b Biology Division, The Spiez Laboratory, Swiss Federal Office for Civil Protection, Spiez, Switzerland; c Cluster for Regenerative Neuroscience, DCR, University of Bern, Bern, Switzerland; d Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; e Department of Physiology, University of Bern, Bern, Switzerland * Corresponding author: Neuroinfectiology Laboratory, Institute for Infectious Diseases (IFIK), University of Bern, PO Box, Friedbühlstrasse 51, CH-3001 Bern, Switzerland. E-mail address:stephen.jenkinson@ifik.unibe.ch.
Introduction and objectives: Clostridium botulinum neurotoxins (BoNTs) are the most poisonous naturally occurring protein toxins known to man, with the toxicity range in humans starting as low as 0.3 ng/kg when administered intravenously. All BoNT serotypes (A-G) consist of 2 subunits, a heavy chain (HC) of ~100 kDa and a light chain (LC) of ~50 kDa, which are linked by a disulphide bond. Upon binding to specific membrane receptors on the plasmalemma of neurons and uptake in the synaptic vesicles, the HC translocates the LC over the membrane where it exerts its cleaving activity against SNARE proteins, thus inhibiting neurotransmitter release. Currently, the potency of biologically active BoNT is monitored using the murine LD50 assay. Using differentiated neurons from mouse embryonic stem cells (mESCs), we intend to develop an in vitro assay capable of detecting BoNT activity using electrophysiological recording techniques. Method: mESCs were differentiated towards neurons by embryoid body (EB) formation. After 7 days, EBs were dissociated and the cells were cultured for 3 weeks on multielectrode arrays (MEAs) allowing the extracellular recording of spontaneous neuronal activity. The cultures were then treated with varying concentrations of BoNT serotype A (BoNT/ A), and spontaneous network bursts, evoked through synaptic transmission, as well as total network activity, were recorded 6 hours after exposure to the toxin. Results: Forty-eight cultures were assessed in 6 independent experiments. Twenty-three cultures were used to quantify the effect of the toxin on synapses when treated with either 333 femtomoles (fmol) or 33 fmol BoNT/A, while 27 cultures served as controls and did not receive any treatment. Exposure to BoNT/A for 6 hours resulted in a significant decrease of the burst rate for toxin concentrations of 333 fmol (49.5 ± 27.2 %; P<0.0005; n¼7) and 33 fmol (66.7 ± 42.5 %; P<0.005; n¼16) compared with untreated cultures (100 ± 19.7 %; n¼27). A significant decrease in the total network activity was observed for toxin concentrations of 333 fmol (45.5 ± 48.0 %; P<0.005; n¼7) and 33 fmol (78.0 ± 70.9 %; P<0.05; n¼16) compared with untreated cultures (100 ± 31.9 %; n¼27). Conclusion: The present assay detects toxin activity of BoNT/A. Thus proof of principle has been achieved. Given sufficient robustness and a further increase in sensitivity to detect BoNT/A, this assay may replace the murine LD50 assay in the batch control of BoNT products. Keywords: Bioassay; BoNT; Clostridium botulinum; Embryonic stem cells; Neuronal cultures; MEAs 116. ARCHITECTURE OF THE BOTULINUM NEUROTOXIN COMPLEX Rongsheng Jin. University of California Irvine, Department of Physiology & Biophysics, Medical Sciences I, Room C-333, Irvine 92697, CA, USA. E-mail address:r.jin@ uci.edu
Abstracts / Toxicon 123 (2016) S2eS90
Introduction and objectives: Botulinum neurotoxin serotype A (BoNT/A) is naturally produced in bacteria with the nontoxic nonhemagglutinin (NTNHA) protein and 3 hemagglutinins (HA70, HA33, and HA17). Together they form a progenitor toxin complex (PTC), the largest bacterial toxin complex known to date. In food-borne botulism, the PTC functions as a molecular machine that protects BoNT/A and helps it breach the host defense in the gastrointestinal (GI) tract. However, the molecular mechanism underlying the PTC function remains poorly defined. Methods: We prepared the highly homogeneous recombinant forms of all PTC components (BoNT/A, NTNHA, HA17, HA33, and HA70) using Escherichia coli. We determined numerous 3-dimensional (3D) structures for individual PTC components and various subcomplexes using x-ray crystallography. We then determined the first pseudo-atomic structure of a 760-kDaelarge PTC (L-PTC) of BoNT/A using a combination of x-ray crystallography, single-particle electron microscopy, and 3D reconstruction (3D-EM). Furthermore, we systematically examined the interactions between the HA complex and an important cell adhesion protein, E-cadherin, and determined the first crystal structure of an HA complex bound to Ecadherin. Results: We demonstrated that the overall architecture of the L-PTC of BoNT/A is composed of 2 structurally and functionally independent entities, reminiscent of an Apollo lunar landing vessel. Toxin’s “ascent stage” (~300 kDa) is formed by BoNT/A and NTNHA that protects BoNT/A from destruction in the GI tract. The “descent stage” (~470 kDa, termed HA complex) consists of 3 hemagglutinins, which assemble into a 3-fold symmetric hetero-dodecameric complex. The HA complex has 9 carbohydrate-binding sites, which allow BoNT/A to land on the cell surface in the small-intestine lumen by docking onto host carbohydrates. Furthermore, we found that the HA complex recognizes E-cadherin with high specificity involving extensive intermolecular interactions. Binding of the HA complex sequesters E-cadherin in the monomeric state, thereby compromising the E-cadherinemediated intercellular barrier in the intestines and facilitating paracellular absorption of the BoNT/A complex. We successfully reconstituted the complete 14-subunit BoNT/A L-PTC in vitro, using all recombinantly produced PTC components. We found that abolishing either E-cadherin or carbohydrate binding of the HA complex using structure-based mutagenesis drastically reduced oral toxicity of the BoNT/A complex in vivo. Conclusions: We revealed the first atomic architecture of a gigantic multiprotein BoNT/A complex. This structure combined with the complementary functional studies established the molecular mechanism of how PTC contributes to the oral toxicity of BoNT/A. These new findings will promote the development of novel chemical inhibitors or antibody/peptide inhibitors as countermeasures against BoNTs and also open the door for alternative applications of this sophisticated bacterial machinery. Keywords: Botulinum neurotoxin; Crystal structure; E-cadherin; HA complex; Host receptors; Progenitor toxin complex
S45
Fig. 1. The molecular architecture of the large PTC (L-PTC) of BoNT/A. The minimally functional PTC (M-PTC) is composed of BoNT/A (purple) and NTNHA (salmon), and the HA complex is composed of HA70 (blue), HA17 (yellow), and HA33 (orange).
117. LONGITUDINAL STUDIES OF CERVICAL DYSTONIA H.A. Jinnah. for the Dystonia Coalition Investigators Emory University, 100 Woodruff Circle, 6300 Woodruff Memorial Building, Atlanta, GA 30322E-mail address:[email protected] Cervical dystonia is defined by excessive contraction of the neck muscles. It is the most common of all adult-onset focal dystonias. Numerous crosssectional studies have described its cardinal features, which include abnormal head positions, repetitive phasic head movements, neck and shoulder muscle spasms, neck and shoulder pain, and other problems. Although the clinical features of cervical dystonia are well described, few studies have documented the evolution of these features in longitudinal study designs. Early studies were relatively small and limited to experience from individual expert centers. These studies often implied worsening over a period of a few months to a year, followed by relatively static abnormalities that only rarely remitted. This worsening could take the form of increased severity of neck muscle contractions, or spread of contractions to muscles outside of the neck region. Later studies with longer periods of follow up suggested that some cases might continue to worsen for at least 5-10 years after onset. One of the goals of the Dystonia Coalition has been to evaluate the evolution of the clinical features of cervical dystonia using a prospectively designed standard rating protocol in a multicenter setting. An interim analysis of results for the period of recruitment from 5 January 2011, through 30 September 2015, was conducted. In this cohort, 37 sites had enrolled 2257 subjects with various types of isolated dystonia, including 1582 with focal or segmental cervical dystonia. Among these cases, 22.8% had evidence of spread outside of the neck region. Identification of patients with cervical dystonia who are at risk for worsening is vital for counseling them regarding their future. It is also important for symptomatic treatment, as the dose and pattern of botulinum toxin may change over time. Longitudinal data is vital for the design of any future disease-modifying treatments aimed at slowing or stopping progression of the disorder. Keywords: Cervical dystonia; Natural history study; Progression; Torticollis 118. PRACTICAL MANAGEMENT OF FLU-LIKE SYMPTOMS AMONG PATIENTS RECEIVING BOTULINUM TOXIN INJECTIONS H.A. Jinnah*, Adam Kassem. Emory University, Atlanta, GA, USA * Corresponding author: Emory University, 100 Woodruff Circle, 6300 Woodruff Memorial Building, Atlanta, GA 30322, USA. E-mail address:[email protected].
Introduction and objectives: Some patients experience a flu-like (FL) syndrome following botulinum toxin (BoNT) injections. These symptoms suggest an immunological response (IR) to the injections, even though the clinical outcome from treatment remains good. These observations, together with evidence that BoNT itself has low immunogenic potential, suggest that the IR in some cases may be a reaction to accompanying ingredients. The purpose of this study was to determine whether switching to a different BoNT product, which contains different types or amounts of accompanying ingredients, reduces the development of the FL symptoms. Methods: The study was conducted with 5 subjects who regularly complained of severe FL reactions following BoNT injections for dystonia. We methodically collected details of the treatment associated with FL symptoms, before and after switching to an alternative brand. Results: All 5 subjects who reported FL symptoms experienced either a reduction or elimination of FL symptoms after switching from the first product to a second product. All 5 patients reported maintenance of clinical benefits. The clinical benefits and lessening of FL symptoms were maintained for at least 3 injection cycles with the second brand, making chance occurrence unlikely.