Botulinum toxin antibody titres: measurement, interpretation, and practical recommendations.

Botulinum toxin (BT) therapy may be blocked by antibodies (BT-AB) resulting in BT-AB induced therapy failure (ABF). BT-AB may be detected by the mouse lethality assay (MLA), the mouse diaphragm assay (MDA) and the sternocleidomastoid test (SCMT). For the first time, we wanted to compare all three BT-AB tests and correlate them to subjective complaint of complete or partial secondary therapy failure in 37 patients with cervical dystonia (25 females, 12 males, age 51.

Significantly lower antigenicity of incobotulinumtoxin than abo- or onabotulinumtoxin.

Background: For many indications, BoNT/A is repetitively injected with the risk of developing neutralizing antibodies (NABs). Therefore, it is important to analyze whether there is a difference in antigenicity between the different licensed BoNT/A preparations.

Methods: In this cross-sectional study, the prevalence of NABs was tested by means of the sensitive mouse hemidiaphragm assay (MHDA) in 645 patients.

The immunology of botulinum toxin therapy: A brief summary.

Like all proteins foreign to the human body, also botulinum toxin (BT) is antigenic and may stimulate an immune response with formation of antibodies (BT-AB). Affected patients may no longer respond to BT therapy and various degrees of BT-AB related therapy failure (ABF) may result. We want to review the immunological interactions between BT and BT-AB, the prevalence, the time course and the risk factors for BT-AB formation as they are related to the treatment algorithms, the patient's immune system and to exogenic factors.

Long-term adherence and response to botulinum toxin in different indications.

Objective: The objective of the study was the analysis of adherence and self-perceived treatment response to long-term botulinum neurotoxin type A (BoNT-A) treatment in different neurological indications.

Methods: In this retrospective, monocentric, observational study, cross-sectional and longitudinal data of 1351 patients documenting 20705 injection appointments at the BoNT outpatient clinic of Heinrich Heine University Duesseldorf between 1989 and 2014 were retrospectively analyzed. Patients had been treated with BoNT for neurological conditions, including cervical dystonia (CD), blepharospasm (BSP), other dystonia (ODT), hemifacial spasm (HFS), and spasticity (SPAS).

Clinical relevance of neutralizing antibodies in botulinum toxin long-term treated still-responding patients with cervical dystonia.

Background: The aim of the study was to test the clinical relevance of neutralizing antibodies (NABs) in patients with cervical dystonia (CD) still responding to repeat injections with botulinum toxin type A (BoNT/A).

Methods: Enzyme-linked immunosorbent assay (ELISA)-test evidence from a cross-sectional study on 221 CD-patients with treatment durations of between 2 and 21 years and still responding to repeat BoNT/A-injections showed the presence of antibodies against BoNT/A in 39 patients. A mouse hemi-diaphragm (MHDA) confirmation test was performed in these 39 ELISA-positive patients, and demographic (age, sex, age at onset of CD) and treatment-related (duration of treatment, mean dose of the last 10 injections, TSUI-score, patient's subjective scoring of the treatment effect, patient's scoring of quality of life by means of the CDQ24-questionnaire) data from these 39 patients were compared with data from ELISA-negative patients.

Comparing lanbotulinumtoxinA (Hengli) with onabotulinumtoxinA (Botox) and incobotulinumtoxinA (Xeomin) in the mouse hemidiaphragm assay.

LanbotulinumtoxinA (LAN) is manufactured and registered in China since 1994. Despite its widespread use in China and its increasing use in other Asian countries and in South America, it is not yet well known elsewhere. We wanted to compare its potency labelling using the mouse diaphragm assay (MDA), an isolated muscle model for botulinum toxin (BT) potency measurements, which is superior to clinical tests and which was recently refined as an alternative batch release assay for BT manufacturing.

The role of human serum albumin and neurotoxin associated proteins in the formulation of BoNT/A products.

Botulinum neurotoxin (BoNT) is synthesized as a progenitor toxin complex (PTC) by Clostridium botulinum. This PTC comprises, in addition to the neurotoxin itself, neurotoxin associated proteins (NAPs) which are composed of three hemagglutinins and one non-toxic, non-hemagglutinin protein. After oral ingestion, these NAPs protect the neurotoxin from the low pH and proteases in the gastrointestinal tract and play a role in the entry via the intestinal barrier.

Botulinum toxin type D blocks autonomic cholinergic synapses in humans: discussion of a potential therapeutic use.

Based on epidemiological data it was believed that botulinumtoxin type D (BT-D) may not block human cholinergic synapses. We wanted to investigate BT-D's effect on the autonomic cholinergic synapse in humans. For this, we compared in four volunteers intraindividually the hypohidrotic effect of intradermal BT-D and BT-A in Minor's iodine starch sweat test.

Do complexing proteins provide mechanical protection for botulinum neurotoxins?

Botulinum toxin (BT) consists of botulinum neurotoxin and complexing proteins (CPs). CPs might provide mechanical protection for botulinum neurotoxin. As incobotulinumtoxinA (INCO, Xeomin®) does not contain CPs, we wanted to compare its mechanical stability to that of onabotulinumtoxinA (ONA, Botox®) containing CPs.

High prevalence of neutralizing antibodies after long-term botulinum neurotoxin therapy.

Objective: To investigate the prevalence of neutralizing antibodies (NAbs) against botulinum neurotoxin type A (BoNT/A) during long-term BoNT/A treatment in different neurologic indications.

Methods: In this monocentric, observational cross-sectional study, 596 outpatients treated with BoNT/A for different indications were tested for BoNT/A binding antibodies by ELISA. Positive samples were investigated for NAbs with the mouse hemidiaphragm test.

Comparing incobotulinumtoxinA (Xeomin®) and onabotulinumtoxinA (Botox®): identical potency labelling in the hemidiaphragm assay.

Botulinum toxin (BT) is provided by several manufacturers producing a number of different drugs. Their potency is given in internationally standardised mouse units (MU). Clinical practise, however, reveals that the potency labelling of different BT drugs may not be identical.

Long-term stability of reconstituted incobotulinumtoxinA: how can we reduce costs of botulinum toxin therapy?

Botulinum neurotoxin (BNT), the biologically active component of botulinum toxin (BT), is a large double-stranded protein susceptible to various physical and chemical influences. All BT type A (BT-A) drugs are stored as powders allowing shelf lives from 24 to 36 months. After reconstitution, the specified shelf life is reduced to 8-24 h.

Immunological aspects of botulinum toxin therapy.

Botulinum toxin (BT) is used in many medical specialties to treat muscle hyperactivity, exocrine gland hyperactivity and pain disorders. BT drugs consist of botulinum neurotoxin (BNT), complexing proteins (CP) and excipients. Antibodies can be formed against BNT and CP.

Reconstituting botulinum toxin drugs: shaking, stirring or what?

Most botulinum toxin (BT) drugs are stored as powders which need to be reconstituted with normal saline before clinical use. As botulinum neurotoxin (BNT), the therapeutically active ingredient, is a large double-stranded protein the process of reconstitution should be performed with special attention to mechanical stress applied. We wanted to test the mechanical stability of BNT during the reconstitution process.

Botulinum toxin therapy: reduction of injection site pain by pH normalisation.

Botulinum toxin (BT) is injected intramuscularily and may produce injection site pain (ISP). We wanted to explore whether the pH value of the reconstituted BT drug contributes to ISP and, if so, what strategies can be applied to reduce it. In part 1 of the study, pH values of different reconstitution solutions and of major BT drugs reconstituted with different reconstitution solutions were assessed.

Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN).

The historical method for the detection of botulinum neurotoxin (BoNT) is represented by the mouse bioassay (MBA) measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN) assay, employs the isolated N. phrenicus-hemidiaphragm tissue.

Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca²⁺ and GTPγS.

Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency.

An enzyme-linked immunosorbent assay for detection of botulinum toxin-antibodies.

Introduction: Antibodies against botulinum neurotoxin (BNT-AB) can be detected by the mouse protection assay (MPA), the hemidiaphragm assay (HDA), and by enzyme-linked immunosorbent assays (ELISA). Both MPA and HDA require sacrifice of experimental animals, and they are technically delicate and labor intensive. We introduce a specially developed ELISA for detection of BNT-A-AB and evaluate it against the HDA.

IncobotulinumtoxinA (Xeomin(®)) can produce antibody-induced therapy failure in a patient pretreated with abobotulinumtoxinA (Dysport(®)).

IncobotulinumtoxinA has not produced a single case of antibody-induced therapy failure after 8 years of worldwide usage. We are reporting a patient with progressive hereditary juvenile onset generalised dystonia who was pretreated with abobotulinumtoxinA for 15 years, before she received incobotulinumtoxinA. To the fifth and sixth applications, she responded with complete therapy failure.

Human mast cell line-1 (HMC-1) cells exhibit a membrane capacitance increase when dialysed with high free-Ca(2+) and GTPγS containing intracellular solution.

An increase in cytosolic free calcium concentration [Ca(2+)]i initiates the exocytotic activity in various types of secretory cells. The guanosine 5'-O-[3-thio]triphosphate (GTPγS), a non-hydrolysable analogue of GTP (guanosine 5'-triphosphate), is an effective secretagogue for different cell types of different species, like mast cells, neutrophils or eosinophils. Consequently, the internal administration of GTPγS causes degranulation of mouse and rat mast cells.

Exchanging the minimal cell binding fragments of tetanus neurotoxin in botulinum neurotoxin A and B impacts their toxicity at the neuromuscular junction and central neurons.

The modular four domain structure of clostridial neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial neurotoxins circumventing the passage of the blood-brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain HC.

Botulinum toxin: application, safety, and limitations.

Botulinum neurotoxin type A (BoNT/A), despite its high toxicity, is approved for therapy of many neurological (e.g., dystonia, spasticity) and non-neurological (e.