A sensitive cell-based assay for testing potency of botulinum neurotoxin type A.

Botulinum neurotoxin type A (BoNT/A) is a biopharmaceutic widely used for the treatment of neu­rological diseases and in aesthetic medicine to achieve months-long paralysis of target muscles and glands. Large numbers of mice are used in the mouse bioassay (MBA) for various botulinum-related applications including batch release potency testing, antitoxin testing, countermeasure development, and basic research. BoNT/A intoxication causes severe suffering to the mice used for testing, and application-specific, non-animal alternatives are urgently needed.

Membrane Capacitance Measurements of Stimulus-Evoked Exocytosis in Adrenal Chromaffin Cells.

Research using membrane capacitance (C) measurements in adrenal chromaffin cells has transformed our understanding of the molecular mechanisms controlling regulated exocytosis. This is in part due to the exquisite temporal resolution of the technique, and the possibility of combining quantification of exo-/endocytosis at the whole-cell level, with the ability to simultaneously monitor and control the calcium signals triggering vesicle fusion. In this regard, experiments performed with C measurements complement amperometry experiments that give a measure of secreted transmitter and the behavior of the fusion pore, and fluorescent microscopy studies used to monitor vesicle and protein dynamics in imaged regions of the cell.

Glucagon-like peptide-1 receptor controls exocytosis in chromaffin cells by increasing full-fusion events.

Agonists for glucagon-like-peptide-1 receptor (GLP-1R) are currently used for the treatment of type 2 diabetes and obesity. Their benefits have been centered on pancreas and hypothalamus, but their roles in other organ systems are not well understood. We studied the action of GLP-1R on secretions of adrenal medulla.

Double-Binding Botulinum Molecule with Reduced Muscle Paralysis: Evaluation in In Vitro and In Vivo Models of Migraine.

With a prevalence of 15%, migraine is the most common neurological disorder and among the most disabling diseases, taking into account years lived with disability. Current oral medications for migraine show variable effects and are frequently associated with intolerable side effects, leading to the dissatisfaction of both patients and doctors. Injectable therapeutics, which include calcitonin gene-related peptide-targeting monoclonal antibodies and botulinum neurotoxin A (BoNT/A), provide a new paradigm for treatment of chronic migraine but are effective only in approximately 50% of subjects.

Mast Cell Purification Protocols.

Studying a tissue-specific mast cell can be of particular benefit given the heterogeneity that is known to exist among mast cells isolated or developed from different sources. Methods for isolating mast cells from a variety of tissues have been in existence for a number of years although, over time, these methodologies have been refined. We have had considerable experience studying mast cells isolated from human lung tissue.

Secretion of Mast Cell Inflammatory Mediators Is Enhanced by CADM1-Dependent Adhesion to Sensory Neurons.

Neuroimmune interactions are important in the pathophysiology of many chronic inflammatory diseases, particularly those associated with alterations in sensory processing and pain. Mast cells and sensory neuron nerve endings are found in areas of the body exposed to the external environment, both are specialized to sense potential damage by injury or pathogens and signal to the immune system and nervous system, respectively, to elicit protective responses. Cell adhesion molecule 1 (CADM1), also known as SynCAM1, has previously been identified as an adhesion molecule which may couple mast cells to sensory neurons however, whether this molecule exerts a functional as well as structural role in neuroimmune cross-talk is unknown.

Orai and TRPC channel characterization in FcRI-mediated calcium signaling and mediator secretion in human mast cells.

Inappropriate activation of mast cells via the FcRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcRI-initiated mediator release.

P2X7 receptors induce degranulation in human mast cells.

Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation.

Mast cell purification protocols.

Studying a tissue-specific mast cell can be of particular benefit given the heterogeneity that is known to exist among mast cells isolated or developed from different sources. Methods for isolating mast cells from a variety of tissues have been in existence for a number of years although, over time, these methodologies have been refined. We have had considerable experience studying mast cells isolated from human lung tissue.

Synaptotagmin IV determines the linear Ca2+ dependence of vesicle fusion at auditory ribbon synapses.

Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca(2+) dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca(2+) sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca(2+) dependence of immature spiking cells.

Functional transient receptor potential melastatin 7 channels are critical for human mast cell survival.

Mast cells play a significant role in the pathophysiology of many diverse diseases such as asthma and pulmonary fibrosis. Ca2+ influx is essential for mast cell degranulation and release of proinflammatory mediators, while Mg2+ plays an important role in cellular homeostasis. The channels supporting divalent cation influx in human mast cells have not been identified, but candidate channels include the transient receptor potential melastatin (TRPM) family.

Differential regulation of endogenous N- and P/Q-type Ca2+ channel inactivation by Ca2+/calmodulin impacts on their ability to support exocytosis in chromaffin cells.

P/Q-type (Ca(V)2.1) and N-type (Ca(V)2.2) Ca2+ channels are critical to stimulus-secretion coupling in the nervous system; feedback regulation of these channels by Ca2+ is therefore predicted to profoundly influence neurotransmission.

Potentiation of exocytosis by phospholipase C-coupled G-protein-coupled receptors requires the priming protein Munc13-1.

The vesicle priming protein Munc13-1 is regulated by diacylglycerol (DAG) and is therefore hypothesized to play a role in the control of neurotransmitter release by phospholipase C (PLC)-coupled receptors. We combined voltage-clamp recordings of voltage-gated Ca2+ channels (VGCCs) and high-resolution capacitance measurements to investigate the mechanism of receptor-mediated modulation of exocytosis in bovine chromaffin cells. Activation of endogenous H1 G(q)-protein-coupled receptors (G(q)PCRs) by histamine potentiated stimulus-coupled secretion despite concurrently inhibiting Ca2+ influx through VGCCs.

Identification of the P2Y(12) receptor in nucleotide inhibition of exocytosis from bovine chromaffin cells.

Nucleotides are released from bovine chromaffin cells and take part in a feedback loop to inhibit further exocytosis. To identify the nucleotide receptors involved, we measured the effects of a range of exogenous nucleotides and related antagonists on voltage-operated calcium currents (I(Ca)), intracellular calcium concentration ([Ca(2+)](i)), and membrane capacitance changes. In comparative parallel studies, we also cloned the bovine P2Y(12) receptor from chromaffin cells and determined its properties by coexpression in Xenopus laevis oocytes with inward-rectifier potassium channels made up of Kir3.