Oromandibular dystonia as a cause of temporomandibular disorder (Case report) / Distonia oromandibular como causa de disfunção temporomandibular (Relato de caso)

Temporomandibular disorders (TMD) can have multiple etiologies, including oromandibular dystonia (OMD). However, in a few cases, the OMD can evolve from cervical dystonia (CD), leading to severe bone degeneration. The purpose of this case report of a 64-year-old woman presenting to the Outpatient Neurology Clinic of the Federal University of Bahia is to illustrate the development of oromandibular dystonia with temporomandibular joint (TMJ) dysfunction after 10 years of cervical dystonia. Clinical examination showed bone degeneration of the mandibular ramus and right TMJ click, a prevalent sound in patients with temporomandibular disorders when they open their mouths or chew. After onabotulinum toxin type A injections in the right lateral pterygoid muscle, the patient improved in swallowing and pain. This case highlights the importance of close follow-up of cervical dystonia patients to identify new dystonic muscles. In our patient, lateral pterygoid muscle involvement was followed by several comorbidities, such as dysphagia and jawbone abnormalities.

Os distúrbios temporomandibulares (DTM) podem ter múltiplas etiologias, incluindo a distonia oromandibular (DO). No entanto, em raros casos, a DO pode evoluir a partir da distonia cervical (DC) e raramente pode levar a degeneração óssea. O objetivo deste relato de caso de uma mulher de 64 anos atendida no Ambulatório de Neurologia da universidade Federal da Bahia é ilustrar o desenvolvimento de distonia oromandibular com disfunção da articulação temporomandibular (ATM) após 10 anos de distonia cervical. O exame clínico mostrou degeneração óssea do ramo mandibular e clique na ATM direita, um som prevalente em pacientes com distúrbios temporomandibulares quando abrem a boca ou mastigam. Após injeções de toxina botulínica tipo A no músculo pterigoideo lateral direito, a paciente apresentou melhora na deglutição e na dor. Este caso destaca a importância do acompanhamento próximo de pacientes com distonia cervical para identificar novos músculos distônicos. Em nossa paciente, o envolvimento do músculo pterigoide lateral foi seguido por várias comorbidades, como disfagia e anormalidades ósseas da mandíbula.

Botulinum neurotoxin serotype A heavy chain intervenes in the H3 acetylation: A preliminary study / 中国比较医学杂志

Objective To investigate the effect and molecular mechanism of botulinum neurotoxin serotype A (BoNT/A) heavy chain on neuron regeneration. Methods Cell culture, rats, immunofluorescence, SDS-PAGE and western blot, etc. were adopted in this study to explore the alterations of histone-3 acetylation (acetyl-H3 ) by local treatment of BoNT/A heavy chain to spinal cord injury (SCI) in rats (in vivo) or by adding it into cell culture (in vitro). Meanwhile, the relevance of acetyl-H3 to neurite out-growth based on SCI and cell culture with BoNT/A heavy chain application was approached as well. Results The application of BoNT/A heavy chain to cultured Neuro-2a cells increased the level of H3 acetylation. The increase of H3 acetylation was paralleled with the growth of neuritogenesis. Also, the neuronal treatment of BoNT/A heavy chain to SCI promoted the re-growth of neuronal processes surrounding the lesions. The growth of neuronal processes was positively correlated to the level of H3 acetylation. During the periods of BoNT/A heavy chain treatment in vivo or in vitro, the increase of H3 acetylation showed two peaks. Conclusions BoNT/A heavy chain increased the H3 acetylation, which might be one of its neuritogenic mechanisms.

Type C waterborne botulism outbreaks in buffaloes (Bubalus bubalis) in the Amazon region / Surtos de botulismo hídrico tipo C em búfalos (Bubalus bubalis) na região amazônica

Botulism is a poisoning caused by botulinum neurotoxins (BoNTs). BoNTs serotypes C and D are involved in botulism outbreaks in cattle in several countries. Despite the high number of buffaloes worldwide, the real impact of botulism in buffaloes is not known, because it is not a notifiable disease in Brazil and only few studies have evaluated the occurrence of the disease in buffaloes. Those studies did not conduct diagnostic tests to confirm the presence of BoNTs. The objective of the present study was to describe three outbreaks of botulism in buffaloes in the Brazilian Amazon region considering epidemiological and clinical data as well as laboratory diagnosis to confirm the presence of BoNTs. The results of the bioassay were negative in the tissues and in feed samples, but positive for BoNT C in water samples. Confirmation of the occurrence of botulism in buffaloes allows the implementation of preventive strategies in susceptible herds. Waterborne botulism in buffaloes is prevented by ensuring the constant circulation of water collections and restricting the presence of dead animals and bones in order to prevent the accumulation of organic matter and the development of anaerobic conditions, which might favor the replication of Clostridium botulinum. Another measure that can be adopted is the shading of the pasture, in order to maintain the thermal comfort for the buffaloes and to avoid the excess of permanence of them in the water pools.(AU)

Botulismo é uma intoxicação causada por neurotoxinas botulínicas (BoNTs). Os sorotipos C e D de BoNTs estão envolvidos em surtos de botulismo em bovídeos em vários países. Apesar do elevado número de búfalos em todo o mundo, o real impacto do botulismo em búfalos não é conhecido; pois não é uma doença de notificação obrigatória no Brasil e poucos estudos avaliaram a incidência desta doença em búfalos. Além disso, estes estudos não realizaram testes diagnósticos para confirmar a presença de BoNTs. O objetivo do presente estudo foi descrever três surtos de botulismo em búfalos na região amazônica brasileira, considerando dados epidemiológicos e clínicos, bem como o diagnóstico laboratorial para confirmar a presença de BoNTs. Os resultados do bioensaio em camundongos foram negativos em todos os tecidos e nas amostras de alimentos testados; no entanto foram positivos para BoNT C nas amostras de água. A confirmação da ocorrência de botulismo em búfalos permite a implementação de estratégias preventivas nos rebanhos. O botulismo hídrico nos búfalos pode ser prevenido assegurando-se que coleções de água fossem mantidas limpas, sem a presença de animais mortos e ossadas no seu interior e não permitindo o acúmulo de matéria orgânica e condições de anaerobiose favoráveis à multiplicação de Clostridium. botulinum. Outra medida que pode ser adotada é o sombreamento das pastagens, a fim de manter o conforto térmico dos búfalos e assim evitar o excesso de sua permanência dentro das fontes de água.(AU)

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.

Rehabilitation for Upper Limb Hemiparesis after Stroke: / The Japanese Journal of Rehabilitation Medicine

A multi-institutional study using our protocol of low-frequency repetitive transcranial magnetic stimulation (rTMS) and intensive occupational therapy (OT) showed significant improvement of motor function of the affected upper limb in poststroke patients. The response to the treatment was not influenced by age or time after stroke onset. Our protocol is a safe, feasible, and potentially useful neurorehabilitative intervention for upper limb hemiparesis after stroke. The extent of the improvement seems to be influenced by the baseline severity of upper limb hemiparesis. The results suggest that patients with Brunnstrom stage 4 or 5 upper limb hemiparesis are best suited for this protocol. Botulinum toxin type A (BoNT-A) has been reported to be an effective treatment for limb spasticity after stroke. However, the spasticity reduction after BoNT-A injection alone does not ensure an improvement in the active motor function of the affected limb. Our proposed protocol of a BoNT-A injection, followed by home-based functional training seems to have the potential to improve the active motor function of the affected upper limb after stroke.

Development of immunodetection system for botulinum neurotoxin type B using synthetic gene based recombinant protein.

Background & objectives: Botulinum neurotoxins (A-G) are among most poisonous substances in the world, produced by obligate anaerobic bacteria Clostridum botulinum. Among the seven serotypes A, B, E and F are of human importance. In India, the prevalence of C. botulinum as well as botulism outbreaks have been reported. Due to its extreme toxicity it has been classified in the Category A of biological warfare agent. So far, there is no commercial detection system available in India to detect botulism. The present study aims to develop an immuno detection system for botulinum neurotoxin serotype B using synthetic gene approach. Methods: The truncated fragment of the botulinum neurotoxin type B from amino acid 1-450 was synthesized using PCR overlap primers; the constructed gene was cloned in the pQE30UA vector and transformed to Escherichia coli SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of C. botulinum as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated. Results: The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1:2048000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes. Interpretation & conclusions: There is no commercial immunodetection system available in India to detect botulism. The developed detection system is highly specific. It will be useful for growing food industry to detect botulinum neurotoxin in food samples as well as in clinical samples.

Development of Capture ELISA Using a Biotinylated Monoclonal Antibody for Detection of Botulinum Neurotoxin Type A

A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC19397. For the capture ELISA, the optimized amount (2 microgram/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 microgram/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r(2)) ranged from 0.9951~0.9999 for all assays. The inter-variations were typically 0.50~6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was 0.488 ng/ml, which was close to mouse LD(50). In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens.

Studies on The Receptors of Botulinum Neurotoxins / 生物化学与生物物理进展

Botulinum neurotoxin(BoNT) is the most lethal biotoxin known to mankind. It inhibits acetylcholinerelease from the cholinergic nerve ending by cleavage of SNARE proteins, followed by neuromuscular blockadeand paralysis. Gangliosides are considered to act as a first receptor of BoNT with low affinity.Then the membranebound gangliosides-BoNT complex moves laterally to reach and bind the toxin specific protein receptor,synaptotagmin, with a high affinity constant. At last the gangliosides-BoNT-synaptotagmin complex undergoesreceptor-mediated endocytosis. This double-receptors theory is widely accepted. The research data are summarizedand reviewed.

Soluble expression of reconstructed heavy chain fragment of botulinum neurotoxin serotype A in Escherichia coli / 解放军医学杂志

Objective To clone the reconstructed Hc gene of botulinum neurotoxin type A(BoNT/AHc)and to explore the soluble expression of the reconstructed gene in E.coli.Methods The gene of Hc fragment was synthesized by replacing rare codons with frequent ones in E.coli,while the components of amino acids didn't change,and the contents of AT decreased from 76.4 % to 57.3%.The reconstructed gene was then cloned into the prokaryotic expression vector pQE-60.The recombinant plasmid pQE-60Hc was introduced into E.coli Origami(DE3)that was induced to express the protein.The soluble expression products were then detected by Western Blot.Finally the expressed product of recombination plasmid pQE-60Hc was analyzed with SDS-PAGE after purification through Ni-NTA column.Results The reconstructed Hc gene of BoNT/AHc was amplified by PCR.The expression vector pQE-60Hc was constructed successfully with BamH Ⅰ and Nco Ⅰto ingest both BoNT/AHc and vector pQE-60.Reconstructed gene could be expressed effectively in E.coli in soluble form.The molecular weight of expressed product of recombination plasmid pQE-60Hc analyzed by SDS-PAGE was 52 000,which was the same as anticipated.And the soluble expression product accounted for to 11.5 % of the total bacterial protein.Western blot assay showed that the expression product could bind to specific antibody agent BoNT/A.Conclusion The expression vector has been constructed and the reconstructed gene was expressed successfully and effectively in E.coli,which may provide a foundation for further study on antitoxin and vaccine.