Project description:Correct communication between immune cells and peripheral neurons is crucial for the protection of our bodies. Its breakdown is observed in many common, often painful conditions, including arthritis, neuropathies and inflammatory bowel or bladder disease. Here, we have characterised the immune response in a mouse model of neuropathic pain using flow cytometry and cell-type specific RNA sequencing (RNA-seq). We found few striking sex differences, but a very persistent inflammatory response, with increased numbers of monocytes and macrophages up to 3½ months after the initial injury. This raises the question of whether the commonly used categorisation of pain into “inflammatory” and “neuropathic” is one that is mechanistically appropriate. Finally, we collated our data with other published RNA-seq datasets on neurons, satellite glial cells, macrophages and Schwann cells in naïve and nerve injury states. The result is a practical web-based tool for the transcriptional data-mining of peripheral neuroimmune interactions.
Project description:Constitution of a biobank of tissues, whole blood and plasma samples and stools to identify markers associated with treatment response, postoperative morbidity including neuro-cognitive and mood complications and prognosis of Inflammatory Bowel disease or colorectal cancer.
Project description:To study differentially expressed genes in neuro-ectodermal cell lines MYCN amplification (NMA) is the most important prognostic factor in neuroblastoma (NBL) patients, however 70% of advanced stage NBL are non-NMA and lack known driving oncogenic events. Gene expression profiles (HU133plus2.0 arrays, Affymetrix) of 17 NBL and 5 peripheral neuro-ectodermal cell lines were used to identify potential subgroups of NBL cell lines with a distinct gene signature. One group of non-NMA NBL cell lines was identified with a distinct gene expression profile and characterized by high expression of AXL. AXL is a tyrosine kinase receptor which plays a role in the metastatic process of cancer. We hypothesized that AXL contributes to the metastasizing potential of non-NMA NBL and tested if AXL silencing diminishes malignant properties of high AXL expressing cell lines. AXL was silenced in two non-NMA NBL cell lines by using a lentiviral shRNA construct that was able to transduce these cell lines with >90% infection efficiency. AXL mRNA expression level was efficiently knocked-down resulting in a severe decrease of migration of AXL positive cell lines GI-M-EN and SH-EP-2, and decreased invasion of GI-M-EN. Morphologically, AXL knockdown induced more rounded cells with a loss of contact. Intracellularly, we observed induction of stress fibers (immunofluorescence F-actin) in GI-M-EN. These changes in cytoskelet were associated with decreased migration. No effects were observed for cell proliferation, apoptosis or downstream pathways. In conclusion, AXL is identified as a possible mediator of NBL metastasis. Arrays were performed with 5 different PNET cell lines, which were used as controls for 17 NBL cell lines (GSE22771)
Project description:To study differentially expressed genes in neuro-ectodermal cell lines MYCN amplification (NMA) is the most important prognostic factor in neuroblastoma (NBL) patients, however 70% of advanced stage NBL are non-NMA and lack known driving oncogenic events. Gene expression profiles (HU133plus2.0 arrays, Affymetrix) of 17 NBL and 5 peripheral neuro-ectodermal cell lines were used to identify potential subgroups of NBL cell lines with a distinct gene signature. One group of non-NMA NBL cell lines was identified with a distinct gene expression profile and characterized by high expression of AXL. AXL is a tyrosine kinase receptor which plays a role in the metastatic process of cancer. We hypothesized that AXL contributes to the metastasizing potential of non-NMA NBL and tested if AXL silencing diminishes malignant properties of high AXL expressing cell lines. AXL was silenced in two non-NMA NBL cell lines by using a lentiviral shRNA construct that was able to transduce these cell lines with >90% infection efficiency. AXL mRNA expression level was efficiently knocked-down resulting in a severe decrease of migration of AXL positive cell lines GI-M-EN and SH-EP-2, and decreased invasion of GI-M-EN. Morphologically, AXL knockdown induced more rounded cells with a loss of contact. Intracellularly, we observed induction of stress fibers (immunofluorescence F-actin) in GI-M-EN. These changes in cytoskelet were associated with decreased migration. No effects were observed for cell proliferation, apoptosis or downstream pathways. In conclusion, AXL is identified as a possible mediator of NBL metastasis.
Project description:How a tissue returns to homeostasis following injury remains enigmatic. Here, we show that elevated Ca2+ dynamics among enterocytes (ECs) mediated by nAchRs (nicotinic Acetylcholine (Ach) Receptor) in the adult Drosophila midgut, are essential for the epithelium to return to homeostasis after injury. This bioelectric signal is controlled by 1) changes in EC-responsiveness to Ach; 2) enteric neuro-EC interactions; and 3) epithelial gap junctions. Our findings demonstrate how a bioelectric signal during regeneration, initiated by local neuro-epithelial communication and propagated by gap junctions, is coupling the epithelium into a unified response that promotes return to homeostasis.
Project description:Airway cholinergic nerves play a key role in airway physiology and disease. In asthma and other diseases of the respiratory tract, airway cholinergic neurons undergo plasticity and contribute to airway hyperresponsiveness and mucus secretion. We currently lack mechanistic understanding of airway cholinergic neuroplasticity due to the absence of human in vitro models. Here, we developed a human in vitro model for peripheral cholinergic neurons using human pluripotent stem cell (hPSC) technology. hPSCs were differentiated towards vagal neural crest precursors and subsequently directed towards functional airway cholinergic neurons using the neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergic neurons were characterized by ChAT and VAChT expression, and responded to chemical stimulation with changes in Ca2+ mobilization. To culture these cells, allowing axonal separation from the neuronal cell bodies, a two-compartment PDMS microfluidic chip was subsequently fabricated . The two compartments were connected via microchannels to enable axonal outgrowth. On-chip cell culture did not compromise phenotypical characteristics of the cells compared to standard culture plates. When the hPSC-derived peripheral cholinergic neurons were cultured in the chip, axonal outgrowth was visible, while the somal bodies of the neurons were confined to their compartment. The microfluidic chip developed in this study represents a human in vitro platform to model neuro-effector interactions in the airways that may be used for mechanistic studies into neuroplasticity in asthma and other lung diseases.
Project description:Neuroimmune crosstalk is critical for intestinal and tissue homeostasis. Yet the role of molecularly distinct subsets of gut-innervating neurons in regulating the activity of gut immunocytes, and the mechanisms of this neuroimmune signaling remain unclear. Here, we performed a chemogenetic and flow cytometry-based analysis of mice targeting eight different peripheral neuron subsets to assess how gut immunocytes are altered following neural activation. We found that distinct neurons modulated discreet anatomical populations of immunocytes in the gut in ileum, cecum, and colon. Nos1+ neuron activation decreased the percentage of RORg+ ileal conventional CD4+ T cells, whereas ChAT+ neuron activation decreased ileal neutrophils. Trpv1+ neuron activation displayed the most robust immunomodulatory phenotype, causing downregulation of RORg+ T regulatory cells in the colon and cecum. The immune cells exhibited decreased proliferation, enhanced cell stress, and altered cell activation markers. Further genetic and pharmacological approaches showed that spinal afferent Trpv1+ neurons specifically decreased Treg cells by signaling via the neuropeptide CGRP. Our study provided a comprehensive understanding of neuro-immune interactions, revealing a role for mechanisms by which Trpv1+ neurons regulate gut Treg cells.
Project description:Background : Candida albicans is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common disease-causing microbial agent. Results: We have developed a toolbox allowing in vivo protein tagging by PCR-mediated homologous recombination with TAP, HA and MYC tags. The transformation cassettes were designed to accommodate a common set of integration primers. The tagged proteins can be used to perform tandem affinity purification (TAP) or chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP). Tandem affinity purification of C. albicans Nop1 revealed the high conservation of the small processome composition in yeasts. Data obtained with in vivo TAP-tagged Tbf1, Cbf1 and Mcm1 recapitulates previously published genome-wide location profiling by ChIP-CHIP. We also designed a new reporter system for in vivo analysis of transcriptional activity of gene loci in C. albicans. Conclusion: This toolbox provides a basic setup to perform purification of protein complexes and increase the number of annotated transcriptional regulators and genetic circuits in C. albicans. Two independent biological replicates of ChIP-CHIP of Mcm1-TAP in yeast and hyphal states. ChIP-CHIP of Cbf1-TAP and Tbf1-TAP.
Project description:We present LASSIM, which is a toolbox built to build and infer parameters within mechanistic models on a genomic scale. This is made possible due to a property shared across biological systems, namely the existence of a subset of master regulators, here denoted the core system. The introduction of a core system of genes simplifies the inference into small solvable sub-problems, and implies that all main regulatory actions on peripheral genes come from a small set of regulator genes. This separation allows substantial parts of computations to be solved in parallel, i.e. permitting the use of a computer cluster, which substantially reduces the time for the computation to finish.
Project description:The American Cancer Society and The Centers for Disease Control and Prevention in collaboration with The National Colorectal Cancer Roundtable published "How to Increase Colorectal Cancer Screening rates in Practice: A Primary Care Clinician’s Evidence-Based Toolbox and Guide" in 2005. This toolbox outlines evidence-based interventions aimed at increasing colorectal cancer screening by primary care providers and their office staff. The Toolbox contains the tools to design a multifaceted intervention to increase primary care physician rates of colorectal cancer screening (CRCS). This is a pilot study to look at implementing the toolbox and its affects.