The Botulinum J. http://www.inderscience.com/browse/index.php?journalID=262&year=2011&vol=2&issue=1 Inderscience Publishers Ltd en-uk The Botulinum J. 1754-7318 1754-7326 © 2011 Inderscience Publishers Ltd editor@inderscience.com https://www.inderscience.com/images/files/coverImgs/tbj_scovertbj.jpg http://www.inderscience.com/browse/index.php?journalID=262&year=2011&vol=2&issue=1 http://www.inderscience.com/link.php?id=41834 Over the years, scientists have worked with BoNT as crude and purified complexes, both in solution or crystalline. They have also used purified neurotoxins, again in solution or crystalline, either as true crystals or so-called 'crystalline' precipitates from purifications with ammonium sulphate fractionations. They use protein concentration measured by one of several methods, LD50 units measured by one of several methods and now also concentration itself, expressed in molarity to describe the quantities they use. There are even signs that extinction coefficients are starting to appear in the literature. However, the community of BoNT scientists is not good at indicating what their measurements refer to when they cite these values. That is the dilemma briefly discussed in this commentary. Units, weights and concentrations: the botulinum toxin dilemma
Andrew Pickett
The Botulinum J., Vol. 2, No. 1 (2011) pp. 4 - 5
Over the years, scientists have worked with BoNT as crude and purified complexes, both in solution or crystalline. They have also used purified neurotoxins, again in solution or crystalline, either as true crystals or so-called 'crystalline' precipitates from purifications with ammonium sulphate fractionations. They use protein concentration measured by one of several methods, LD50 units measured by one of several methods and now also concentration itself, expressed in molarity to describe the quantities they use. There are even signs that extinction coefficients are starting to appear in the literature. However, the community of BoNT scientists is not good at indicating what their measurements refer to when they cite these values. That is the dilemma briefly discussed in this commentary.

]]> 10.1504/TBJ.2011.041834 The Botulinum J., Vol. 2, No. 1 (2011) pp. 4 - 5 Andrew Pickett Toxin Science Limited, Wrexham, UK botulinum neurotoxins BoNT units weights concentrations measurements. 2011-08-06T23:20:50-05:00 2 1 4 5 2011-08-06T23:20:50-05:00 http://www.inderscience.com/link.php?id=41812 This placebo-controlled crossover trial investigate the safety and efficacy of onabotulinumtoxinA (BOTOX, Allergan, Inc.) for the treatment of cervical myofascial pain syndrome (CMPS). A group of subjects with Cervical Dystonia (CD) served as additional controls. A change in diary pain score from baseline to week 3 in placebo vs. onabotulinumtoxinA was the primary outcome measure. Both CMPS and CD subjects showed a trend towards significant pain relief from onabotulinumtoxinA. This suggests that the study was underpowered and that a larger sample size would be necessary in future trials of onabotulinumtoxinA for CMPS. A pilot trial of onabotulinumtoxinA for the treatment of cervicothoracic myofascial pain
Thomas L. Davis, John Y. Fang, Chandler E. Gill, P. David Charles
The Botulinum J., Vol. 2, No. 1 (2011) pp. 6 - 15
This placebo-controlled crossover trial investigate the safety and efficacy of onabotulinumtoxinA (BOTOX, Allergan, Inc.) for the treatment of cervical myofascial pain syndrome (CMPS). A group of subjects with Cervical Dystonia (CD) served as additional controls. A change in diary pain score from baseline to week 3 in placebo vs. onabotulinumtoxinA was the primary outcome measure. Both CMPS and CD subjects showed a trend towards significant pain relief from onabotulinumtoxinA. This suggests that the study was underpowered and that a larger sample size would be necessary in future trials of onabotulinumtoxinA for CMPS.

]]> 10.1504/TBJ.2011.041812 The Botulinum J., Vol. 2, No. 1 (2011) pp. 6 - 15 Thomas L. Davis John Y. Fang Chandler E. Gill P. David Charles Department of Neurology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Vanderbilt Department of Neurology, Medical Center North, Nashville, TN 37232, USA. ' Department of Neurology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Department of Neurology, Vanderbilt University Medical Center, 1161 21st Ave. South, Suite A-1101 Medical Center North, Nashville, TN 37232-2551, USA. ' Stritch School of Medicine, Loyola University, Maywood, IL 60153, USA; Loyola University Stritch School of Medicine, 2160 1st Avenue, Maywood, IL 60153, USA. ' Department of Neurology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Vanderbilt University Medical Center, 1161 21st Avenue South, Suite A-1106 Medical Center North, Nashville, TN 37232-2551, USA CMPS cervicothoracic myofascial pain syndrome BOTOX safety cervical dystonia botulinum toxin cervical myofascial pain syndrome BOTOX efficacy onabotulinumtoxinA pain relief. 2011-08-06T23:20:50-05:00 2 1 6 15 2011-08-06T23:20:50-05:00 http://www.inderscience.com/link.php?id=41813 Botulinum Neurotoxins (BoNTs) are used therapeutically and in cosmetics, providing potential for bioterrorist activity, thus driving the search for small-molecule BoNT inhibitors. This report describes a 70,000-compound screen for inhibition of BoNT/A using a FRET assay to detect proteolysis of a peptide substrate. Hits were confirmed, followed by evaluation to determine compound specificity. Inhibitors fell into three main chemical classes, and on the basis of potency and specificity of inhibition, the activities of two chemotypes were examined further. Compounds exhibited specificity for BoNT/A LC inhibition with respect to other metalloproteases and displayed activity in a neuronal assay for botulinum intoxication. Novel benzimidazole inhibitors of botulinum neurotoxin/A display enzyme and cell-based potency
Steven C. Cardinale, Michelle M. Butler, Gordon Ruthel, Jonathan E. Nuss, Laura M. Wanner, Bing Li, Ramdas P. Pai, Norton P. Peet, Sina Bavari, Terry L. Bowlin
The Botulinum J., Vol. 2, No. 1 (2011) pp. 16 - 29
Botulinum Neurotoxins (BoNTs) are used therapeutically and in cosmetics, providing potential for bioterrorist activity, thus driving the search for small-molecule BoNT inhibitors. This report describes a 70,000-compound screen for inhibition of BoNT/A using a FRET assay to detect proteolysis of a peptide substrate. Hits were confirmed, followed by evaluation to determine compound specificity. Inhibitors fell into three main chemical classes, and on the basis of potency and specificity of inhibition, the activities of two chemotypes were examined further. Compounds exhibited specificity for BoNT/A LC inhibition with respect to other metalloproteases and displayed activity in a neuronal assay for botulinum intoxication.

]]> 10.1504/TBJ.2011.041813 The Botulinum J., Vol. 2, No. 1 (2011) pp. 16 - 29 Steven C. Cardinale Michelle M. Butler Gordon Ruthel Jonathan E. Nuss Laura M. Wanner Bing Li Ramdas P. Pai Norton P. Peet Sina Bavari Terry L. Bowlin Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA. ' Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA. ' US Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702, USA. ' US Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702, USA. ' US Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702, USA. ' Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA. ' Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA. ' Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA. ' US Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702, USA. ' Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA botulinum neurotoxins serotype A bioterrorism SNAP-25 high throughput screening benzimidazole acrylonitrile hydroxyquinoline small molecule inhibitors drug discovery metalloprotease BoNT inhibitors. 2011-08-06T23:20:50-05:00 2 1 16 29 2011-08-06T23:20:50-05:00 http://www.inderscience.com/link.php?id=41814 Research on more sensitive cell based assays is essential to assess neutralising activities of Botulinum neurotoxins antibodies. A reproducible and robust differentiation protocol was applied on human neuroblastoma SH-SY5Y cells generating neuronal cultures stable for 2 3 weeks. The presence of specific neuronal markers was assessed with labelled antibodies. Accurate and reproducible noradrenaline release levels were obtained which were significantly higher in differentiated cells and correlated with vesicle turnover assessments. The suitability of differentiated SH-SY5Y cells for detecting low levels of Botulinum type A and for toxin neutralisation assay was evidenced using functional end points relevant to Botulinum neurotoxicity. Enhanced sensitivity to Botulinum type A neurotoxin of human neuroblastoma SH-SY5Y cells after differentiation into mature neuronal cells
C. Rasetti-Escargueil, C.B. Machado, R. Preneta-Blanc, R.A. Fleck, D. Sesardic
The Botulinum J., Vol. 2, No. 1 (2011) pp. 30 - 48
Research on more sensitive cell based assays is essential to assess neutralising activities of Botulinum neurotoxins antibodies. A reproducible and robust differentiation protocol was applied on human neuroblastoma SH-SY5Y cells generating neuronal cultures stable for 2 3 weeks. The presence of specific neuronal markers was assessed with labelled antibodies. Accurate and reproducible noradrenaline release levels were obtained which were significantly higher in differentiated cells and correlated with vesicle turnover assessments. The suitability of differentiated SH-SY5Y cells for detecting low levels of Botulinum type A and for toxin neutralisation assay was evidenced using functional end points relevant to Botulinum neurotoxicity.

]]> 10.1504/TBJ.2011.041814 The Botulinum J., Vol. 2, No. 1 (2011) pp. 30 - 48 C. Rasetti-Escargueil C.B. Machado R. Preneta-Blanc R.A. Fleck D. Sesardic Division of Bacteriology, National Institute for Biological Standards and Control, Health Protection Agency, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK. ' MRC Centre for Developmental Neurobiology, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK. ' King's College London, The Graduate School, Waterloo Bridge, 150 Stamford Street, London, SE1 9NN, UK. ' Cell Biology Imaging, National Institute for Biological Standards and Control, Health Protection Agency, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK. ' Division of Bacteriology, National Institute for Biological Standards and Control, Health Protection Agency, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK BoNT botulinum neurotoxins neurotransmitter release SH-SY5Y cell line human neuroblastoma cells differentiation cell based assay synaptic vesicles recycling neuronal cells. 2011-08-06T23:20:50-05:00 2 1 30 48 2011-08-06T23:20:50-05:00 http://www.inderscience.com/link.php?id=41815 Botulinum toxin type A is a complex composed of the biologically active neurotoxin, several hemagglutinins and other nontoxic proteins. After intramuscular injection these complexing proteins do not have any therapeutic effect. However, they protect the neurotoxin from harsh environmental conditions, e.g., low intragastral pH after oral ingestion. NT201, a BoNT/A drug product devoid of complexing proteins was tested in real-time and accelerated stability studies. NT201 was found to be stable without refrigeration for 48 months and even not affected by short-term temperature stress up to 60°C, demonstrating that complexing proteins are not required for the stability of BoNT/A preparations. Stability of botulinum neurotoxin type A, devoid of complexing proteins
Swen Grein, Gerd J. Mander, Klaus Fink
The Botulinum J., Vol. 2, No. 1 (2011) pp. 49 - 58
Botulinum toxin type A is a complex composed of the biologically active neurotoxin, several hemagglutinins and other nontoxic proteins. After intramuscular injection these complexing proteins do not have any therapeutic effect. However, they protect the neurotoxin from harsh environmental conditions, e.g., low intragastral pH after oral ingestion. NT201, a BoNT/A drug product devoid of complexing proteins was tested in real-time and accelerated stability studies. NT201 was found to be stable without refrigeration for 48 months and even not affected by short-term temperature stress up to 60°C, demonstrating that complexing proteins are not required for the stability of BoNT/A preparations.

]]> 10.1504/TBJ.2011.041815 The Botulinum J., Vol. 2, No. 1 (2011) pp. 49 - 58 Swen Grein Gerd J. Mander Klaus Fink Merz Pharmaceuticals GmbH, Department Biotechnology, Eckenheimer Landstr 100, 60318 Frankfurt, Germany. ' Merz Pharmaceuticals GmbH, Department Biotechnology, Eckenheimer Landstr 100, 60318 Frankfurt, Germany. ' Merz Pharmaceuticals GmbH, Department Biotechnology, Eckenheimer Landstr 100, 60318 Frankfurt, Germany botulinum neurotoxin type A BoNT&#47 A stability complexing proteins temperature stress ICH guideline botulinum neurotoxins. 2011-08-06T23:20:50-05:00 2 1 49 58 2011-08-06T23:20:50-05:00 http://www.inderscience.com/link.php?id=41816 Botulinum Neurotoxin A (BoNT/A) is produced by Clostridium botulinum as a complex with Neurotoxin-Associated Proteins (NAPs) which protect the toxin from proteases and promote adsorption in the gut. Hemagglutinin 33 (Hn-33) makes up the largest fraction of NAPs in the BoNT/A complex. We characterised single domain antibodies (sdAb) which bind Hn-33; the dissociation constant (Kd) varied from 3.8 × 10<SUP align=right>−9</SUP> M to 1.3 × 10<SUP align=right>−10</SUP> M. Sandwich assays showed that the six sdAb bind two distinct epitopes on Hn-33. Circular dichroism was used to monitor sdAb's secondary structure, thermal denaturing upon heating and rapid refolding upon cooling. Evaluation of anti-hemagglutinin Hn-33 single domain antibodies: kinetics, binding epitopes, and thermal stability
George P. Anderson, Dan Zabetakis, Rachael D. Bernstein, Shuowei Cai, Bal Ram Singh, Ellen R. Goldman
The Botulinum J., Vol. 2, No. 1 (2011) pp. 59 - 71
Botulinum Neurotoxin A (BoNT/A) is produced by Clostridium botulinum as a complex with Neurotoxin-Associated Proteins (NAPs) which protect the toxin from proteases and promote adsorption in the gut. Hemagglutinin 33 (Hn-33) makes up the largest fraction of NAPs in the BoNT/A complex. We characterised single domain antibodies (sdAb) which bind Hn-33; the dissociation constant (Kd) varied from 3.8 × 10<SUP align=right>−9</SUP> M to 1.3 × 10<SUP align=right>−10</SUP> M. Sandwich assays showed that the six sdAb bind two distinct epitopes on Hn-33. Circular dichroism was used to monitor sdAb's secondary structure, thermal denaturing upon heating and rapid refolding upon cooling.

]]> 10.1504/TBJ.2011.041816 The Botulinum J., Vol. 2, No. 1 (2011) pp. 59 - 71 George P. Anderson Dan Zabetakis Rachael D. Bernstein Shuowei Cai Bal Ram Singh Ellen R. Goldman Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington DC 20375, USA. ' Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington DC 20375, USA. ' Nova Research Inc., 1900 Elkin St # 230, Alexandria, VA 22308, USA. ' Botulinum Research Center, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA. ' Botulinum Research Center, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA. ' Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington DC 20375, USA botulinum neurotoxins hemagglutinin single domain antibody thermal stability luminex surface plasmon resonance circular dichroism kinetics binding epitopes. 2011-08-06T23:20:50-05:00 2 1 59 71 2011-08-06T23:20:50-05:00 http://www.inderscience.com/link.php?id=41817 The Botulinum Toxin ELISA effectively provided presumptive identification of toxin in 1381 investigation samples including clinical specimens, suspect foods, and cultures. Additionally, the ELISA detected all toxins produced by a panel of stock strains representing known subtypes and was negative for non-botulinum toxin producing Clostridium and enteric pathogens. ELISA results were reproducible both within the same kit (CV < 9%) and among different production lots (CV < 23%). Fifty-five of 57 laboratories correctly identified unknown samples in a multi-laboratory study. The ELISA provides a rapid, robust in vitro screening method which will reduce animal dependence during laboratory investigations of botulism. Utility of Botulinum Toxin ELISA A, B, E, F kits for clinical laboratory investigations of human botulism
Susan E. Maslanka, Carolina Luquez, Brian H. Raphael, Janet K. Dykes, Lavin A. Joseph
The Botulinum J., Vol. 2, No. 1 (2011) pp. 72 - 92
The Botulinum Toxin ELISA effectively provided presumptive identification of toxin in 1381 investigation samples including clinical specimens, suspect foods, and cultures. Additionally, the ELISA detected all toxins produced by a panel of stock strains representing known subtypes and was negative for non-botulinum toxin producing Clostridium and enteric pathogens. ELISA results were reproducible both within the same kit (CV < 9%) and among different production lots (CV < 23%). Fifty-five of 57 laboratories correctly identified unknown samples in a multi-laboratory study. The ELISA provides a rapid, robust in vitro screening method which will reduce animal dependence during laboratory investigations of botulism.

]]> 10.1504/TBJ.2011.041817 The Botulinum J., Vol. 2, No. 1 (2011) pp. 72 - 92 Susan E. Maslanka Carolina Luquez Brian H. Raphael Janet K. Dykes Lavin A. Joseph National Botulism Laboratory Preparedness Team, Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA. ' National Botulism Laboratory Preparedness Team, Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA. ' National Botulism Laboratory Preparedness Team, Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA. ' National Botulism Laboratory Preparedness Team, Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA. ' National Botulism Laboratory Preparedness Team, Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA botulinum toxin ELISA foods clinical specimens cultures human botulism in vitro botulinum detection ELISA performance optimisation botulinum toxin subtypes diagnostic antibody production mouse bioassay. 2011-08-06T23:20:50-05:00 2 1 72 92 2011-08-06T23:20:50-05:00