Mitos y evidencias en el empleo de la toxina botulínica: neurofarmacología y distonías / Myths and evidence on the use of botulinum toxin: neuropharmacology and dystonia

Introducción. La toxina botulínica de tipo A (TBA) ha supuesto una verdadera revolución terapéutica en neurología, y en la actualidad es el tratamiento rutinario en las distonías focales y la espasticidad. Objetivo. Plantear, revisar y responder cuestiones controvertidas en relación con la neurofarmacología de a TBA y su uso en las distonías en la práctica clínica habitual. Desarrollo. Un grupo de expertos en trastornos del movimiento revisó una lista de temas controvertidos relacionados con la farmacología de la TBA y su uso en las distonías. Revisamos la bibliografía e incluimos artículos relevantes especialmente en inglés, pero también, si su importancia lo merece, en castellano y en francés, hasta junio de 2016. El documento se estructuró como un cuestionario que incluyó las preguntas que podrían generar mayor controversia o duda. El borrador inicial del documento fue revisado por los miembros del panel y se realizaron las modificaciones necesarias hasta alcanzar el mayor grado de consenso. Incluimos preguntas sobre diferentes aspectos de la neurofarmacología, especialmente el mecanismo de acción, la bioequivalencia de los diferentes preparados y la inmunogenicidad. En relación con el subapartado de las distonías, se incluyeron aspectos sobre la evaluación y el tratamiento de las distonías focales. Conclusiones. Esta revisión no pretende ser una guía, sino una herramienta práctica destinada a neurólogos y médicos internos residentes interesados en esta área, dentro de diferentes ámbitos específicos del manejo de la TBA (AU)

Introduction. Botulinum toxin type A (BTA) is a bacterial endotoxin, whose therapeutic use has had a dramatic impact on different neurological disorders, such as dystonia and spasticity. Aim. To analyze and summarize different questions about the use of BTA in our clinical practice. Development. A group of experts in neurology developed a list of topics related with the use of BTA. Two groups were considered: neuropharmacology and dystonia. A literature search at PubMed, mainly for English language articles published up to June 2016 was performed. The manuscript was structured as a questionnaire that includes those questions that, according to the panel opinion, could generate more controversy or doubt. The initial draft was reviewed by the expert panel members to allow modifications, and after subsequent revisions for achieving the highest degree of consensus, the final text was then validated. Different questions about diverse aspects of neuropharmacology, such as mechanism of action, bioequivalence of the different preparations, immunogenicity, etc. were included. Regarding dystonia, the document included questions about methods of evaluation, cervical dystonia, blepharospasm, etc. Conclusion. This review does not pretend to be a guide, but rather a tool for continuous training of residents and specialists in neurology, about different specific areas of the management of BTA (AU)

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to ß1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin.

Chloroquine derivatives block the translocation pores and inhibit cellular entry of Clostridium botulinum C2 toxin and Bacillus anthracis lethal toxin.

The pathogenic bacteria Clostridium botulinum and Bacillus anthracis produce the binary protein toxins C2 and lethal toxin (LT), respectively. These toxins consist of a binding/transport (B7) component that delivers the separate enzyme (A) component into the cytosol of target cells where it modifies its specific substrate and causes cell death. The B7 components of C2 toxin and LT, C2IIa and PA63, respectively, are ring-shaped heptamers that bind to their cellular receptors and form complexes with their A components C2I and lethal factor (LF), respectively. After receptor-mediated endocytosis of the toxin complexes, C2IIa and PA63 insert into the membranes of acidified endosomes and form trans-membrane pores through which C2I and LF translocate across endosomal membranes into the cytosol. C2IIa and PA63 also form channels in planar bilayer membranes, and we used this approach earlier to identify chloroquine as a potent blocker of C2IIa and PA63 pores. Here, a series of chloroquine derivatives was investigated to identify more efficient toxin inhibitors with less toxic side effects. Chloroquine, primaquine, quinacrine, and fluphenazine blocked C2IIa and PA63 pores in planar lipid bilayers and in membranes of living epithelial cells and macrophages, thereby preventing the pH-dependent membrane transport of the A components into the cytosol and protecting cells from intoxication with C2 toxin and LT. These potent inhibitors of toxin entry underline the central role of the translocation pores for cellular uptake of binary bacterial toxins and as relevant drug targets, and might be lead compounds for novel pharmacological strategies against severe enteric diseases and anthrax.

Uso de toxina botulínica paravertebral para detener la progresión de escoliosis en pollos pinealectomizados: la columna vertebral como sistema de tensegridad / Use of paravertebral injection of botulinum toxin to control scoliosis progression in pinealectomized chicken: the spine as a tensegrity system

Antecedentes y Objetivos. La escoliosis idiopática infantil no tiene un tratamiento satisfactorio que permita reducir la importante morbilidad y mortalidad asociada a los casos más severos y progresivos de la enfermedad. El desarrollo de procedimientos que puedan ralentizar la progresión de la enfermedad durante el desarrollo del niño puede influir favorablemente en el crecimiento y retrasar el tratamiento definitivo de la deformidad al momento de la madurez músculoesquelética. Presentamos un estudio sobre la influencia de la toxina botulínica en el desarrollo de deformidad en un modelo animal de escoliosis progresiva. Material y Método. Utilizamos 52 pollos Broiler hembra, en los que se practicó pinealectomía para producir escoliosis. Comparamos la evolución de la deformidad entre un grupo control y un grupo intervención asignado a recibir toxina botulínica paravertebral en la concavidad de la curva, bajo control electromiográfico. Realizamos estudios radiográficos y anatomopatológicos de los animales para evaluar los resultados. El grado de escoliosis se midió utilizando el método del ángulo de Cobb. Resultados. Cinco animales no sobrevivieron al estudio (1 en el grupo control y 4 en el de intervención). En el grupo control observamos una deformidad media de 32.9º (n= 25) y en el grupo intervención de 18.8º (n=22), encontrando diferencias estadísticamente significativas (p<0.05). Por tanto, la aplicación de toxina botulínica en la concavidad de la deformidad de pollos pinealectomizados frena la progresión de escoliosis. Conclusiones. La consideración de la columna vertebral y sus tejidos blandos asociados como una estructura de tensegridad puede explicar el fenómeno mediante el desequilibrio generado entre los componentes de tensión (músculos y ligamentos) y compresión (vértebras) que conforman el sistema. Estos resultados justifican nuevos estudios en investigación clínica para explorar una nueva alternativa para el tratamiento de la escoliosis idiopática infantil (AU)

Background and Objectives. Severe and progressive adolescent idiopathic scoliosis has no satisfactory treatment since high rates of morbidity and mortality are associated. Development of procedures that might slow down the progression of the deformity in the growing children may postpone definitive surgery to the end of musculoskeletal maturity period. A study about the influence of botulinum toxin in the development of deformity in a progressive scoliosis animal model is reported. Methods. Surgical pinealectomy was performed in 52 Broiler chickens to induce progressive scoliosis. Scoliosis progression among a control group and an intervention group assigned to paravertebral injection of botulinum toxin in curve´s concavity electromyographycally assisted is compared. Conventional x-ray and anatomopathologic studies were conducted to evaluate results. Cobb angle method was used to measure spine deformation. Results. Five animals died (1 in the control group and 4 in the intervention group). Mean scoliosis values observed were 32.9 degrees (n= 25) and 18.8 degrees (n= 22) for control and intervention groups respectively (p<0.05). Therefore, the use of botulinum toxin in the deformity´s concavity restrains scoliosis progression in pinealectomized chickens. Conclusions. The assumption of the spine and its associated soft tissues as a tensegrity structure may explain these results, through the induced imbalance between the tension (muscles and ligaments) and compression (vertebrae) components that shape the system. Further studies are necessary to determine clinical applications of this therapy in adolescent idiopathic scoliosis (AU)

Median nerve motor entry points in the forearm - clinical application

Superficial forearm flexors receive primary and secondary branches from the median nerve (mn). These branches vary in number, size and motor entry points (m) into the muscles. Knowledge of these points is essential for maximal compound muscle action potential (cmap) recording from these muscles. Spasticity of these flexors is treated using botulinum toxin (bt) injection or selective partial neurotomy (spn) of the nerve branches to the spastic muscles. Twenty human cadaveric forearms were dissected. The location of the motor entry points of the median nerve to the superficial forearm flexor muscles was expressed as a distance from the medial (me) and lateral (le) epicondyles of the humerus. Fifty apparently healthy volunteers (25 males and 25 females) underwent cmap recording from the superficial forearm flexors. Thirty patients (15 males and 15 females) with spastic hyperflexion of the wrist and fingers underwent bt injection or spn. Pronator teres (pt) had 2-4 m, flexor carpi radialis (fcr) had 1-3 m and flexor digitorum superficialis (fds) had 3-8 m. Variable shapes of the cmap were recorded from them (monophasic, biphasic or multiphasic). Based on the anatomical results, bt injection was done at 5 points (p1-p5); pt was injected at p1, fcr was injected at p2, and fds is a large muscle and was injected at p3, p4, p5 (proximal, middle, distal), giving good results in 85% of cases; spn was done in severe cases refractory to bt injection with excellent to good results in 80% of cases. The patterns of branching of mn differ from the classically described patterns. Therefore, revising the innervation patterns of the superficial forearm flexors is mandatory, since the variations observed are more diverse than has been described. Identification of the branches and the motor entry points of mn are essential for cmap recording from the superficial forearm flexors, bt injection, spn and tendon transfer

No disponible

Botulinum Toxin as a Pain Killer: Players and Actions in Antinociception.

Botulinum neurotoxins (BoNTs) have been widely used to treat a variety of clinical ailments associated with pain. The inhibitory action of BoNTs on synaptic vesicle fusion blocks the releases of various pain-modulating neurotransmitters, including glutamate, substance P (SP), and calcitonin gene-related peptide (CGRP), as well as the addition of pain-sensing transmembrane receptors such as transient receptor potential (TRP) to neuronal plasma membrane. In addition, growing evidence suggests that the analgesic and anti-inflammatory effects of BoNTs are mediated through various molecular pathways. Recent studies have revealed that the detailed structural bases of BoNTs interact with their cellular receptors and SNAREs. In this review, we discuss the molecular and cellular mechanisms related to the efficacy of BoNTs in alleviating human pain and insights on engineering the toxins to extend therapeutic interventions related to nociception.

Explanation of timing of botulinum neurotoxin effects, onset and duration, and clinical ways of influencing them.

While the steps in the action of botulinum neurotoxin (BoNT) are well known, the factors underlying the timing of these steps are not fully understood. After toxin is injected into a muscle, it resides in the extracellular space and must be taken up into the nerve terminals. More toxin will be taken up if near the endplate. Toxin is distributed mainly by convection and there is likely little diffusion. Toxin that is not taken up will go into the general circulation where it may have a slight systemic effect. The uptake is activity and temperature dependent. Encouraging the unwanted muscle contractions after injection should be helpful. Cooling will decrease the uptake. The times for washout from the extracellular space and uptake of the toxin are not well established, but are likely measured in minutes. Toxin in the general circulation has a long half time. The time from injection to weakness is determined by how long it takes to get sufficient damage of the SNARE proteins to interfere with synaptic release. Toxins are zinc dependent proteases, and supplemental zinc may produce a greater effect. There will be weakness as long as there is residual toxin in the nerve ending.

Factores que influyen en los resultados urodinámicos de la toxina botulínica en el tratamiento de la hiperactividad neurógena / Factors that influence the urodynamic results of botulinum toxin in the treatment of neurogenic hyperactivity

Objetivos: Determinar la eficacia urodinámica y los factores que influyen en los resultados urodinámicos del tratamiento de la hiperactividad neurógena del detrusor con inyección intradetrusoriana de toxina botulínica tipo A (TXB-A) en pacientes con lesión medular (LM). Material y métodos: Se realizó un estudio retrospectivo en una cohorte de 70 pacientes formada por 40 varones y 30 mujeres con LM estable de 39 ± 13,3 años de edad (media ± desviación típica), sometidos a inyección intradetrusoriana de 300 UI de TXB-A. Se realizó un estudio urodinámico previo y otro a los 6 ± 4,3 meses del tratamiento. Posteriormente se realizaron nuevos estudios urodinámicos hasta un intervalo de 16 ± 12,2 meses. Resultados: La TXB-A aumentó significativamene (p < 0,05) la capacidad vesical cistomanométrica, el volumen vesical de la primera contracción involuntaria del detrusor y el residuo posmiccional. Se observó una disminución con tendencia hacia la significación estadística (p < 0,1) de la presión máxima miccional del detrusor y el flujo miccional máximo. No varió significativamente la acomodación vesical ni el índice de resistencia uretral (BOOI). El aumento de la capacidad vesical se mantuvo en el 50% de la muestra más de 32 meses. La edad, el sexo, el tratamiento anticolinérgico y la antigüedad de la lesión no mostraron influencia respecto del aumento de la capacidad vesical. La sonda a permanencia (SVP) fue el único factor negativo estadísticamente significativo. Conclusiones: El efecto urodinámico de la TXB-A se mantiene durante un considerable intervalo de tiempo. La SVP influye negativamente en el resultado del tratamiento

Objectives: To determine the urodynamic efficacy and factors that influence the urodynamic results of treatment of neurogenic detrusor hyperactivity with intradetrusor injection of botulinum toxin type A (BTX-A) in patients with spinal cord injury (SCI). Material and methods: A retrospective study was conducted with a cohort of 70 patients composed of 40 men and 30 women with stable SCI (mean age, 39 ± 13.3 years) who underwent an intradetrusor injection of 300 IUs of BTX-A. A urodynamic study was conducted prior to the injection and 6 ± 4.3 months after the treatment. New urodynamic studies were subsequently performed up to an interval of 16 ± 12.2 months. Results: The BTX-A significantly increased (p < .05) the cystomanometric bladder capacity, the bladder volume of the first involuntary contraction of the detrusor and the postvoid residue. We observed a decrease that tended towards statistical significance (p < .1) of the maximum detrusor pressure and the maximum urine flow. Neither the bladder accommodation nor the urethral resistance index (bladder outlet obstruction index) varied significantly. The increase in vesical capacity was maintained in 50% of the sample for more than 32 months. Age, sex, anticholinergic treatment and lesion age showed no influence in terms of the increase in bladder capacity. The indwelling urinary catheter (IUC) was the only statistically significant negative factor. Conclusions: The urodynamic effect of BTX-A is maintained for a considerable time interval. The IUC negatively influences the result of the treatment

Guía de práctica clínica para el tratamiento de la espasticidad espinal con toxina botulínica / Clinical practice guideline for the treatment of spinal spasticity with botulinum toxin

Introducción. Hasta en un 70% de los pacientes con lesión medular aparece espasticidad; la necesidad de tratarla va aumentando progresivamente con el tiempo de evolución. La espasticidad espinal suele ser generalizada y la toxina botulínica puede ser útil como tratamiento adyuvante al farmacológico para resolver problemas concretos. Las guías de práctica clínica son instrumentos que facilitan la toma de decisiones y mejoran los resultados clínicos. Material y método. Presentamos el protocolo de valoración y tratamiento con toxina botulínica definido tomando como base las peculiaridades clínicas propias de la espasticidad asociada a la lesión medular. Resultados y conclusiones. El uso de esta guía en nuestro centro nos ha permitido estandarizar la selección del paciente con espasticidad espinal y establecer unos objetivos claros de tratamiento y un plan apropiado de seguimiento, facilitando la valoración objetiva de los resultados (AU)

Introduction. Spasticity occurs in approximately 70% of patients with a spinal cord injury. The need for treatment progressively increases from the time of injury. In spinal spasticity, which is usually generalized, botulinum toxin is useful as an adjuvant therapy in specific situations. Clinical practice guidelines are tools that enhance decision-making and improve clinical outcomes. Material and methods. We present a protocol for spinal spasticity assessment and treatment with botulinum toxin designed according to the specific features of spasticity associated with spinal cord injury. Results and conclusions. The use of this protocol at our center has allowed us to standardize the selection of patients with spinal spasticity, to define clear treatment aims and to develop an accurate follow up schedule, thus aiding objective assessment of results (AU)

Botulinum toxin complex increases paracellular permeability in intestinal epithelial cells via activation of p38 mitogen-activated protein kinase.

Clostridium botulinum produces a large toxin complex (L-TC) that increases paracellular permeability in intestinal epithelial cells by a mechanism that remains unclear. Here, we show that mitogen-activated protein kinases (MAPKs) are involved in this permeability increase. Paracellular permeability was measured by FITC-dextran flux through a monolayer of rat intestinal epithelial IEC-6 cells, and MAPK activation was estimated from western blots. L-TC of C. botulinum serotype D strain 4947 increased paracellular dextran flux and activated extracellular signal-regulated kinase (ERK), p38, but not c-Jun N-terminal kinase (JNK) in IEC-6 cells. The permeability increase induced by L-TC was abrogated by the p38 inhibitor SB203580. These results indicate that L-TC increases paracellular permeability by activating p38, but not JNK and ERK.

Therapeutic effectiveness of botulinum neurotoxin A: potent blockade of autonomic transmission by targeted cleavage of only the pertinent SNAP-25.

In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinum neurotoxin type A (BoNT/A), in treating numerous diseases due to hyper-active autonomic nerves, truncation of its target and inhibition of neurotransmission were studied in rat sympathetic neurons. Tetrodotoxin-sensitive spontaneous cholinergic neurotransmission was blocked >80% by 1 pM BoNT/A despite cleaving <20% of the SNAP-25. A maximum cleavage of ∼60% SNAP-25 could be achieved with >1 nM BoNT/A, despite an absence of non-cleavable SNAP-25 in the detergent-solubilised neurons. In contrast, BoNT/E (100 nM) truncated nearly all the SNAP-25 in the intact cells, but was unable to block neurotransmission at low concentrations like BoNT/A. Chimeras created by inserting the acceptor-binding HC domain of BoNT/A into BoNT/E still cleaved all the SNAP-25, indicating ubiquitous expression of BoNT/A acceptors. Accordingly, SV2 and SNAP-25 were found to be co-expressed and broadly co-localised in neurons, but absent from non-neuronal cells. On the other hand, partial cleavage by the BoNT/A protease persisted upon replacing its HC with counterparts from BoNT/E or BoNT/B. Moreover, limited cleavage of SNAP-25 was conferred onto the protease from BoNT/E when fused to the N-terminus of BoNT/A. Thus, the BoNT/A protease is uniquely well-adapted for selectively inactivating the SNAP-25 directly involved in neurotransmission; this together with the toxin's acceptor and its target being localised on the peri-somatic boutons likely contribute to its exceptional therapeutic utility in the clinic.

The life history of a botulinum toxin molecule.

There is an emerging literature describing the absorption, distribution, metabolism and elimination of botulinum toxin. This work reveals that the toxin can be absorbed by both the oral and inhalation routes. The primary mechanism for absorption is binding and transport across epithelial cells. Toxin that enters the body undergoes a distribution phase, which is quite short, and an elimination phase, which is comparatively long. During the distribution phase, botulinum toxin migrates to the peri-neuronal microcompartment in the vicinity of vulnerable cells, such as cholinergic nerve endings. Only these cells have the ability to selectively accumulate the molecule. When the toxin moves from the cell membrane to the cell interior, it undergoes programmed death. This is coincident with release of the catalytically active light chain that paralyzes transmission. Intraneuronal metabolism of light chain is via the ubiquitination-proteasome pathway. Systemic metabolism and elimination is assumed to be via the liver. The analysis of absorption, distribution, metabolism and elimination of the toxin helps to create a life history of the molecule in the body. This has many benefits, including: a) clarifying the mechanisms that underlie the disease botulism, b) providing insights for development of medical countermeasures against the toxin, and c) helping to explain the meaning of a lethal dose of toxin. It is likely that work intended to enhance understanding of the fate of botulinum toxin in the body will intensify. These efforts will include new and powerful analytic tools, such as single molecule-single cell analyses in vitro and real time, 3-dimensional pharmacokinetic studies in vivo.

Valoración de la marcha mediante plantillas instrumentadas en pacientes con espasticidad de miembros inferiores tras infiltración con toxina botulínica / Walking assessment with instrumented insoles in patients with lower limb spasticity after botulinum toxin infiltration

Introducción: El objetivo del presente trabajo es la valoración funcional y cinemática en pacientes con espasticidad de miembros inferiores tras lesión encefálica o medular con indicación de infiltración con toxina botulínica (TBA). Material y métodos: Diez pacientes (11 extremidades inferiores) atendidos en unidad de neurorrehabilitación. Examen clínico, funcional (functional ambulation categories, valoración marcha del Hospital de Sagunto) y biomecánico (plantillas instrumentadas Biofoot/IBV versión 5,0) antes y 3 semanas después de la infiltración con TBA. Estadísticos: prueba de la t para muestras relacionadas de las variables clínicas, funcionales y biomecánicas antes y después dela infiltración. Nivel de significación inferior a 0,05. Modo cualitativo para valorar si los cambios en las variables biomecánicas orientan a una aproximación a los parámetros de normalidad. Resultados: La infiltración con TBA mejora el tono muscular, el arco articular y la frecuencia de espasmos (p < 0,01). La población infiltrada muestra un nivel alto de satisfacción con la mejora de los síntomas. No hay cambios funcionales en habilidad para la marcha tras la infiltración. En los parámetros biomecánicos mejora de la cadencia de marcha y la presión máxima en retropié se aproxima a la significación estadística cercana a 0,1. Conclusiones: Con la disminución del tono muscular tras infiltración con TBA, mejoran los síntomas clínicos derivados de esta sin cambios funcionales en escalas de marcha. Los cambios en los parámetros biomecánicos indican la necesidad de ampliar los estudios con plantillas instrumentadas en población con espasticidad tras lesión del sistema nervioso central tratada con infiltración de TBA (AU)

Introduction: Botulinum toxin A (BTA) improves the kinematic parameters of gait in patients with spasticity of lower limbs, but there are no studies in which kinetic parameters are measured with instrumented insoles. We therefore used instrumented insoles to perform a functional assessment of therapeutic results in patients with lower limb spasticity after brain injury or spinal cord infiltration indicating BTA. Material and methods: Ten patients (11 lower limbs) seen in a Neurorehabilitation Unit. The tests carried out included clinical examination, gait assessment (Functional Ambulation Categories (FAC); Hospital de Sagunto Gait Scale), and biomechanical assessment (Biofoot / IBV version 5.0), before and three weeks after infiltration with BTA. Statistics: t-test for related samples of clinical variables, functional variables and biomechanical variables before and after infiltration. Level of significance P< .05. Qualitative method to assess whether changes in the biomechanical variables tended toward normal values. Results: BTA improves muscle tone, joint arch and frequency of spasms (P<.01). The patient sample showed a high level of satisfaction with the improvement in symptoms. There were no changes in walking ability after injection. There were no statistically significant changes inbiomechanical parameters, but there was improved gait cadence. The relatively small statistical significance close to P=.1 of the peak pressure in the heel after injection indicates the need for further studies with instrumented insoles in people with spasticity due to central nervous system injury. Conclusions: With the decrease in muscle tone after infiltration with BTA the clinical symptoms associated with muscle tone improved without any functional changes in gait scales. The changes in the biomechanical parameters show that larger studies using instrumented insoles should be performed in the population with spasticity after a central nervous system injury treated with BTA infiltration (AU)

Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis.

Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.

Analysis of the mechanisms that underlie absorption of botulinum toxin by the inhalation route.

Botulinum toxin is a highly potent oral and inhalation poison, which means that the toxin must have an efficient mechanism for penetration of epithelial barriers. To date, three models for toxin passage across epithelial barriers have been proposed: (i) the toxin itself undergoes binding and transcytosis; (ii) an auxiliary protein, HA35, transports toxin from the apical to the basal side of epithelial cells; and (iii) an auxiliary protein, HA35, acts on the basal side of epithelial cells to disrupt tight junctions, and this permits paracellular flux of toxin. These models were evaluated by studying toxin absorption following inhalation exposure in mice. Three types of experiments were conducted. In the first, the potency of pure neurotoxin was compared with that of progenitor toxin complex, which contains HA35. The results showed that the rate and extent of toxin absorption, as well as the potency of absorbed toxin, did not depend upon, nor were they enhanced by, the presence of HA35. In the second type of experiment, the potencies of pure neurotoxin and progenitor toxin complex were compared in the absence or presence of antibodies on the apical side of epithelial cells. Antibodies directed against the neurotoxin protected against challenge, but antibodies against HA35 did not. In the final type of experiment, the potency of pure neurotoxin and toxin complex was compared in animals pretreated to deliver antibodies to the basal side of epithelial cells. Once again, antibodies directed against the neurotoxin provided resistance to challenge, but antibodies directed against HA35 did not. Taken collectively, the data indicate that the toxin by itself is capable of crossing epithelial barriers. The data do not support any hypothesis in which HA35 is essential for toxin penetration of epithelial barriers.

Efectos de la toxina botulínica A (Botox®) intraarticular en la artrosis de rodilla avanzada / No disponible

Objetivos: la osteoartritis es la enfermedad articular más común y una de los problemas de salud más frecuentes y sintomáticos en la madurez y senectud. Este trabajo describe nuestra experiencia clínica con inyecciones intraarticulares de toxina botulínica tipo A (TBA) en dolor de rodilla artrósico y refractario. Materiales y métodos: doce mujeres con gonartrosis avanzada y refractaria, acudieron a consulta para el manejo del dolor de rodilla de moderado a severo. Las pacientes estuvieron en seguimiento 16 semanas, durante las que la mejoría de su estado fue valorado comparando el dolor basal respecto al dolor en el momento de la visita mediante diferentes escalas estandarizadas. Simultáneamente, se evaluaron la funcionalidad, la cantidad de medicación analgésica diaria y los efectos secundarios. Resultados: el consumo de medicación decreció de 3,6 a 2 tomas/día. No se observaron efectos secundarios. Conclusión: se evidenció un alivio del dolor que se inició en la semana 2 y se mantuvo hasta 12 semanas tras la inyección de TBA. La mediana del cambio de la escala total de WOMAC y de la EVA fue de –7,5 y –1,9 respectivamente, con una disminución significativa del dolor a las 16 semanas. No se registraron cambios en la escala WOMAC en 5 pacientes, mientras que 3 casos mostraron un descenso en dicha escala mayor del 20% y los en los 4 restantes fue superior al 40%. Las que respondieron a la infiltración notaron una mejora en las actividades de la vida diaria gracias a este alivio. El consumo de medicación decreció de 3,6 a 2 tomas/ día. No se observaron efectos secundarios (AU)

Objective: osteoarthritis is the most common joint disease and it is among the most disabling health problems for middle aged and older people. Purpose: to test the effects of a single intraarticular injection of Botulinum Toxin A (BTA) on refractory knee osteoarthritis. Material and method: twelve women (mean age: 72) with refractory knee osteoarthritis who had failed other treatments and without possibilities of arthroplasty were injected with 100 units of BTA (Botox®-Allergan). Patients were followed-up for 16 weeks. They were assessed by means of VAS, WOMAC questionnaire, daily analgesic consumption, and a verbal relief scale. Side effects were also evaluated. Results: a peak of pain relief was evident 2 weeks after BTA injection and lasted typically for 16 weeks. The median change in WOMAC total score and VAS were –7.5 and –1.9 respectively, with a statistically significant pain decrease over 16 weeks. Five out of 12 do not found any decrease in total Womac score; 3 patients described at least 20% of relief and the remaining 4 found more than 40% of reduction in that scale. Those cases who responded to the BTA injection noted improved function in activities of daily living. Daily analgesic necessity droped from 3.6 to 2 doses. No side effects were noted. Conclusion: beneficial effects lasted for at least 12 weeks and gave pain relief. Based on the positive findings of this study, we believe that further, randomized, controlled trials for the use of BTA in knee osteoarthritic pain would be warranted (AU)

Evidence for anterograde transport and transcytosis of botulinum neurotoxin A (BoNT/A).

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that blocks synaptic transmission via the cleavage of SNAP-25 (synaptosomal-associated protein of 25 kDa). BoNT/A is successfully used in clinical neurology for the treatment of several neuromuscular pathologies and pain syndromes. Despite its widespread use, relatively little is known on BoNT/A intracellular trafficking in neurons. Using the visual pathway as a model system, here we show that catalytically active BoNT/A is capable of undergoing anterograde axonal transport and transcytosis. Following BoNT/A injection into the rat eye, significant levels of BoNT/A-cleaved SNAP-25 appeared in the retinorecipient layers of the superior colliculus (SC). Anterograde propagation of BoNT/A effects required axonal transport, ruling out a systemic spread of the toxin. Cleaved SNAP-25 was present in presynaptic structures of the tectum, but retinal terminals were devoid of the immunoreactivity, indicative of transcytosis. Experiments based on sequential administration of BoNT/A and BoNT/E showed a persistent catalytic activity of BoNT/A in tectal cells following its injection into the retina. Our findings demonstrate that catalytically active BoNT/A is anterogradely transported from the eye to the SC and transcytosed to tectal synapses. These data are important for a more complete understanding of the mechanisms of action of BoNT/A.

Efecto protector de la toxina botulínica en colgajos cutáneos en colgajos cutáneos / The protective effect of botulinum toxin on skin flaps

Los colgajos cutáneos de patrón vascular randomizado son de gran utilidad en Cirugía Reconstructiva; un aporte vascular adecuado es el factor más importante para la supervivencia de los mismos. Realizamos un estudio experimental levantando un total de 36 colgajos en el dorso de 9conejos (4 en cada conejo): a) 9 controles, b) 9 con aplicación de toxina botulínica tipo A transoperatoria a lo largo del colgajo, c) 9 con toxina botulínica tipo A aplicada 7 días antes del procedimiento quirúrgico, y, d) 9 con aplicación de toxina botulínica tipo A transoperatoria más epinefrina. El análisis de los colgajos se realizó a través de imágenes digitales que se examinaron con el programa ImageJ. Efectuamos el análisis estadístico con la prueba T de Student. La evaluación final de los colgajos se realizó al séptimo día, tiempo en el cual se obtuvo: a) una media de área necrótica en los controles del 72.17±3.9%; b) en los que se aplicó toxina botulínica transoperatoria el porcentaje de aérea necrótica fue del 5.09±1.2% p=0.032; c) en aquellos en los que la toxina botulínica se aplicó 7 días antes del procedimientos reportó necrosis del 24.97±2.7% p=0.041 y d)en los que se aplicó toxina botulínica transoperatoria más epinefrina el porcentaje de necrosis fue del 23.90±3.9%p=0.045. Concluimos que la toxina botulínica tipo A actúa como protector contra el proceso de sufrimiento - isquemia de los tejidos, debido a su acción vasodilatadora, recomendando su aplicación al inicio del procedimiento quirúrgico (AU)

Random-pattern cutaneous flaps are very useful in Reconstructive Surgery; an adequate vascular contribution is an important factor for the survival of the flaps. We designed an experimental study elevating a total of 36 dorsal flaps, divided in 4 groups: a) 9 were controls, to which 0.9%physiological solution was applied, b) 9 with a transoperatory application of type A botulinum toxin throughout the length of the flap, c) 9 with an application of type A botulinumtox in 7 days before surgery and d), 9 with type A botulinumtoxin with epinephrine applied transsurgery in all the flap. The skin flaps were analyzed 7 days after surgery with Image J program and the T Student test. We take a medium of the necrotic area: a) for the controls of 72.17±3.9%, b)botulinum toxin transsurgery 5.09±1.2% p=0.032, c) botulinumtoxin applied 7 days before 24.97±2.7% p=0.041 and) botulinum toxin with epinephrine 23.90±3.9% p=0.045.We conclude that botulinum toxin acts as a protector against the suffering process – ischemia of the tissues, thanks to its vasodilator action and that it should be applied in the beginning of the surgical procedure (AU)

Comparison of oral toxicological properties of botulinum neurotoxin serotypes A and B.

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. Of the seven known serotypes (A-G) of BoNT, serotypes A, B and E cause most of the foodborne intoxications in humans. BoNTs in nature are associated with non-toxic accessory proteins known as neurotoxin-associated proteins (NAPs), forming large complexes that have been shown to play important roles in oral toxicity. Using mouse intraperitoneal and oral models of botulism, we determined the dose response to both BoNT/B holotoxin and complex toxins, and compared the toxicities of BoNT/B and BoNT/A complexes. Although serotype A and B complexes have similar NAP composition, BoNT/B formed larger-sized complexes, and was approximately 90 times more lethal in mouse oral intoxications than BoNT/A complexes. When normalized by mean lethal dose, mice orally treated with high doses of BoNT/B complex showed a delayed time-to-death when compared with mice treated with BoNT/A complex. Furthermore, we determined the effect of various food matrices on oral toxicity of BoNT/A and BoNT/B complexes. BoNT/B complexes showed lower oral bioavailability in liquid egg matrices when compared to BoNT/A complexes. In summary, our studies revealed several factors that can either enhance or reduce the toxicity and oral bioavailability of BoNTs. Dissecting the complexities of the different BoNT serotypes and their roles in foodborne botulism will lead to a better understanding of toxin biology and aid future food risk assessments.

A phase I/IIa clinical trial of a recombinant Rho protein antagonist in acute spinal cord injury.

Multiple lines of evidence have validated the Rho pathway as important in controlling the neuronal response to growth inhibitory proteins after central nervous system (CNS) injury. A drug called BA-210 (trademarked as Cethrin(®)) blocks activation of Rho and has shown promise in pre-clinical animal studies in being used to treat spinal cord injury (SCI). This is a report of a Phase I/IIa clinical study designed to test the safety and tolerability of the drug, and the neurological status of patients following the administration of a single dose of BA-210 applied during surgery following acute SCI. Patients with thoracic (T2-T12) or cervical (C4-T1) SCI were sequentially recruited for this dose-ranging (0.3 mg to 9 mg Cethrin), multi-center study of 48 patients with complete American Spinal Injury Association assessment (ASIA) A. Vital signs; clinical laboratory tests; computed tomography (CT) scans of the spine, head, and abdomen; magnetic resonance imaging (MRI) of the spine, and ASIA assessment were performed in the pre-study period and in follow-up periods out to 1 year after treatment. The treatment-emergent adverse events that were reported were typical for a population of acute SCI patients, and no serious adverse events were attributed to the drug. The pharmacokinetic analysis showed low levels of systemic exposure to the drug, and there was high inter-patient variability. Changes in ASIA motor scores from baseline were low across all dose groups in thoracic patients (1.8±5.1) and larger in cervical patients (18.6±19.3). The largest change in motor score was observed in the cervical patients treated with 3 mg of Cethrin in whom a 27.3±13.3 point improvement in ASIA motor score at 12 months was observed. Approximately 6% of thoracic patients converted from ASIA A to ASIA C or D compared to 31% of cervical patients and 66% for the 3-mg cervical cohort. Although the patient numbers are small, the observed motor recovery in this open-label trial suggests that BA-210 may increase neurological recovery after complete SCI. Further clinical trials with Cethrin in SCI patients are planned, to establish evidence of efficacy.