Ashish Shelar

BackgroundMUPPITS-2 was a randomized, placebo-controlled clinical trial that demonstrated mepolizumab (anti-interleukin-5) reduced exacerbations and blood and airway eosinophils in urban children with severe eosinophilic asthma. Despite this reduction in eosinophilia, exacerbation risk persisted in certain patients treated with mepolizumab. This raises the possibility that subpopulations of airway eosinophils exist that contribute to breakthrough exacerbations.ObjectiveDetermine the effect of mepolizumab on airway eosinophils in childhood asthma.MethodsSputum samples were obtained from 53 MUPPITS-2 participants. Airway eosinophils were characterized using mass cytometry and grouped into subpopulations using unsupervised clustering analyses of 38 surface and intracellular markers. Differences in frequency and immunophenotype of sputum eosinophil subpopulations were assessed based on …

Individual neurons or muscle cells express many G-protein-coupled receptors (GPCRs) for neurotransmitters and neuropeptides, yet it remains unclear how cells integrate multiple GPCR signals that all must activate the same few G-proteins. We analyzed this issue in the Caenorhabditis elegans egg-laying system, where multiple GPCRs on muscle cells promote contraction and egg laying. We genetically manipulated individual GPCRs and G-proteins specifically in these muscle cells within intact animals and then measured egg laying and muscle calcium activity. Two serotonin GPCRs on the muscle cells, Gαq-coupled SER-1 and Gαs-coupled SER-7, together promote egg laying in response to serotonin. We found that signals produced by either SER-1/Gαq or SER-7/Gαs alone have little effect, but these two subthreshold signals combine to activate egg laying. We then transgenically expressed natural or …

RATIONALE Although there is interest that eosinophils, particularly at sites of inflammation are heterogeneous in function and contribution to airway inflammation, evidence to support this concept in asthma is limited. Here we present a detailed functional analysis of airway eosinophils subpopulations using multidimensional CyTOF. METHODS Mass cytometry (CyTOF) was performed on sputum collected from 31 children with moderate-to-severe asthma. Expression of 40 surface and intracellular markers was measured, and a manual gating strategy was utilized to define sputum cell types. Unsupervised clustering analysis was applied to eosinophils to identify eosinophil subpopulations among the samples. Functional marker expression was then compared between eosinophil subpopulations to further elucidate the behavior of these distinct eosinophil subpopulations. RESULTS Three eosinophil subpopulations …

NeuroPAL (Neuronal Polychromatic Atlas of Landmarks) is a recently developed transgene that labels each of the 118 classes of neurons in C. elegans with various combinations of four fluorescent proteins. This neuron‐type‐specific labeling helps identify neurons that could otherwise be confused with neighboring neurons. Neuron identification enables researchers to combine new data that they generate on a C. elegans neuron with existing datasets on that same neuron, such as its synaptic connections, neurotransmitters, and transcriptome. An impediment to using NeuroPAL, however, is overcoming the steep learning curve for interpreting three‐dimensional (3D) fluorescence images of crowded neural ganglia within which different neurons may be similarly colored, some neurons are only very faintly labeled, and the positions of some neurons are variable. Here, we provide protocols that allow researchers to …