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The conducting airways are lined by distinct cell types, comprising basal, secretory, ciliated, and rare cells, including ionocytes, solitary cholinergic chemosensory cells, and solitary and clustered (neuroepithelial bodies) neuroendocrine cells. Airway neuroendocrine cells are in clinical focus since they can give rise to small cell lung cancer. They have been implicated in diverse functions including mechanosensation, chemosensation, and regeneration, and were recently identified as regulators of type 2 immune responses via the release of the neuropeptide calcitonin gene-related peptide (CGRP). We here assessed the expression of the chemokine CXCL13 (B cell attracting chemokine) by these cells by RT-PCR, in silico analysis of publicly available sequencing data sets, immunohistochemistry, and immuno-electron microscopy. We identify a phenotype of neuroendocrine cells in the naïve mouse, producing the chemokine CXCL13 predominantly in solitary neuroendocrine cells of the tracheal epithelium (approx. 70% CXCL13+) and, to a lesser extent, in the solitary neuroendocrine cells and neuroepithelial bodies of the intrapulmonary bronchial epithelium (+). In silico analysis of published sequencing data of murine tracheal epithelial cells was consistent with the results obtained by immunohistochemistry as it revealed that neuroendocrine cells are the major source of Cxcl13-mRNA, which was expressed by 68–79% of neuroendocrine cells. An unbiased scRNA-seq data analysis of overall gene expression did not yield subclusters of neuroendocrine cells. Our observation demonstrates phenotypic heterogeneity of airway neuroendocrine cells and points towards a putative immunoregulatory role of these cells in bronchial-associated lymphoid tissue formation and B cell homeostasis.

Governments around the world face policy issues that require strategies and solutions using new technologies, access to data, new analytical tools and techniques and social crowdsourcing analysis for the successful implementation and acceptance of public policies and their later assessment. Data is a fundamental resource for carrying out all government activities and governments everywhere, and at all levels, are looking into the opportunities of data-driven innovation. In this context, the PolicyCLOUD environment will introduce a holistic methodology for policy modelling and management based on data, providing a toolkit that allows both stakeholders and engaged citizens to create policies by exploiting the PolicyCLOUD models and analytical tools on various datasets, contexts and policy models. The document D7.2: Market Analysis and Business Potentials is the first deliverable of the PolicyCLOUD business- related activities, setting the market context where the project’s results will be exploited and giving an initial insight about these results. Following the proven Atos’ “Innovation Hub Methodology”, that divides the project time period in two different phases (analysis and strategy), the Consortium has conducted several activities, notable amongst those is the Value Proposition Canvas Workshop. This was an interactive session during First General Assembly, held remotely on the 7th May 2020, focusing on the PolicyCLOUD core offering and stakeholders that could obtain the greatest value of the solution. Initial versions of deliverables D2.1: State of the art and requirement analysis and D2.2: Conceptual Model and Reference Architecture have given input to this deliverable, especially in the analysis of the technological context and in the identification of the project’s outcomes and their main features. The results of this deliverable D7.2 will in turn come back to the technical Work Packages (WPs) and will influence the architecture design with the aim of solving real problems that users have. Ultimately, this is the goal of the business activities of the project, ensure that the finished technology is usable, engaging end users from the initial phases of the project and facilitating its further use. The feedback expected from the Impact Creation Board in relation to this deliverable will be key to design next activities and to support project’s innovation.

Governments around the world face policy issues that require strategies and solutions using new technologies, access to data, new analytical tools and techniques and social crowdsourcing analysis for the successful implementation and acceptance of public policies and their later assessment. Data is a fundamental resource for carrying out all government activities and governments everywhere, and at all levels, are looking into the opportunities of data-driven innovation. In this context, the PolicyCLOUD environment will introduce a holistic methodology for policy modelling and management based on data, providing a toolkit that allows both stakeholders and engaged citizens to create policies by exploiting the PolicyCLOUD models and analytical tools on various datasets, contexts and policy models. The document D7.2: Market Analysis and Business Potentials is the first deliverable of the PolicyCLOUD business- related activities, setting the market context where the project’s results will be exploited and giving an initial insight about these results. Following the proven Atos’ “Innovation Hub Methodology”, that divides the project time period in two different phases (analysis and strategy), the Consortium has conducted several activities, notable amongst those is the Value Proposition Canvas Workshop. This was an interactive session during First General Assembly, held remotely on the 7th May 2020, focusing on the PolicyCLOUD core offering and stakeholders that could obtain the greatest value of the solution. Initial versions of deliverables D2.1: State of the art and requirement analysis and D2.2: Conceptual Model and Reference Architecture have given input to this deliverable, especially in the analysis of the technological context and in the identification of the project’s outcomes and their main features. The results of this deliverable D7.2 will in turn come back to the technical Work Packages (WPs) and will influence the architecture design with the aim of solving real problems that users have. Ultimately, this is the goal of the business activities of the project, ensure that the finished technology is usable, engaging end users from the initial phases of the project and facilitating its further use. The feedback expected from the Impact Creation Board in relation to this deliverable will be key to design next activities and to support project’s innovation., This version is submitted to the EC, not yet aproved, {"references":["IG POLICY CANVAS. 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Cholinergic chemosensory cells (CCC) are infrequent epithelial cells with immunosensor function, positioned in mucosal epithelia preferentially near body entry sites in mammals including man. Given their adaptive capacity in response to infection and their role in combatting pathogens, we here addressed the time points of their initial emergence as well as their postnatal development from first exposure to environmental microbiota (i.e., birth) to adulthood in urethra and trachea, utilizing choline acetyltransferase (ChAT)-eGFP reporter mice, mice with genetic deletion of MyD88, toll-like receptor-2 (TLR2), TLR4, TLR2/TLR4, and germ-free mice. Appearance of CCC differs between the investigated organs. CCC of the trachea emerge during embryonic development at E18 and expand further after birth. Urethral CCC show gender diversity and appear first at P6-P10 in male and at P11-P20 in female mice. Urethrae and tracheae of MyD88- and TLR-deficient mice showed significantly fewer CCC in all four investigated deficient strains, with the effect being most prominent in the urethra. In germ-free mice, however, CCC numbers were not reduced, indicating that TLR2/4-MyD88 signaling, but not vita-PAMPs, governs CCC development. Collectively, our data show a marked postnatal expansion of CCC populations with distinct organ-specific features, including the relative impact of TLR2/4-MyD88 signaling. Strong dependency on this pathway (urethra) correlates with absence of CCC at birth and gender-specific initial development and expansion dynamics, whereas moderate dependency (trachea) coincides with presence of first CCC at E18 and sex-independent further development.

The thymus is the site of T cell maturation which includes positive selection in the cortex and negative selection in the medulla. Acetylcholine is locally produced in the thymus and cholinergic signaling influences the T cell development. We recently described a distinct subset of medullary epithelial cells in the murine thymus which express the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and components of the canonical taste transduction cascade, i.e. transient receptor potential melastatin-like subtype 5 channel (TRPM5), phospholipase Cβ(2), and Gα-gustducin. Such a chemical phenotype is characteristic for chemosensory cells of mucosal surfaces which utilize bitter receptors for detection of potentially hazardous compounds and cholinergic signaling to initiate avoidance reflexes. We here demonstrate mRNA expression of bitter receptors Tas2r105, Tas2r108, and Tas2r131 in the murine thymus. Using a Tas2r131-tauGFP reporter mouse we localized the expression of this receptor to cholinergic cells expressing the downstream elements of the taste transduction pathway. These cells are distinct from the medullary thymic epithelial cells which promiscuously express tissue-restricted self-antigens during the process of negative selection, since double-labeling immunofluorescence showed no colocalization of autoimmune regulator (AIRE), the key mediator of negative selection, and TRPM5. These data demonstrate the presence of bitter taste-sensing signaling in cholinergic epithelial cells in the thymic medulla and opens a discussion as to what is the physiological role of this pathway.

Specialized epithelial cells in the respiratory tract such as solitary chemosensory cells and brush cells sense the luminal content and initiate protective reflexes in response to the detection of potentially harmful substances. The majority of these cells are cholinergic and utilize the canonical taste signal transduction cascade to detect “bitter” substances such as bacterial quorum sensing molecules. Utilizing two different mouse strains reporting expression of choline acetyltransferase (ChAT), the synthesizing enzyme of acetylcholine (ACh), we detected cholinergic cells in the submucosal glands of the murine larynx and trachea. These cells were localized in the ciliated glandular ducts and were neither found in the collecting ducts nor in alveolar or tubular segments of the glands. ChAT expression in tracheal gland ducts was confirmed by in situ hybridization. The cholinergic duct cells expressed the brush cell marker proteins, villin and cytokeratin-18, and were immunoreactive for components of the taste signal transduction cascade (Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel = TRPM5, phospholipase C β2 ), but not for carbonic anhydrase IV. Furthermore, these cells expressed the bitter taste receptor Tas2r131, as demonstrated utilizing an appropriate reporter mouse strain. Our study identified a previously unrecognized presumptive chemosensory cell type in the duct of the airway submucosal glands that likely utilizes ACh for paracrine signaling. We propose that these cells participate in infection-sensing mechanisms and initiate responses assisting bacterial clearance from the lower airways.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Members of the calcitonin peptide family-calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin2/intermedin (IMD)-exert modulatory effects upon monocytes and macrophages of various extrapulmonary origins. Utilizing the rat alveolar macrophage (AMφ) cell line NR8383, we here set out to determine to which extent these three peptides and their receptors are differentially regulated in AMφ and what specific effects they have on AMφ key functions. LPS treatment differentially up-regulated expression of the peptides and receptors. Among the three peptides, IMD mRNA content was lowest both in primary rat AMφ and NR8383 cells, whereas IMD peptide dominated in basal and LPS-stimulated secretion from NR8383 cells. Fcγ receptor-mediated phagocytosis and TNF-α production were inhibited by AM, IMD, and CGRP, whereas pro-IL-1β mRNA was slightly down-regulated exclusively by CGRP. Neither of these peptides affected IL-6 or IL-10 production. None increased intracellular calcium concentration, but AM significantly inhibited store-operated calcium entry. In conclusion, the rat AMφ cell line NR8383 is both a source and a target of the calcitonin peptide family members AM, IMD, and CGRP. Despite sharing proteins of the receptor complexes, AM, IMD, and CGRP each showed a characteristic pattern of effects and regulation, suggesting that these closely related peptides are not just redundant members of one common signaling pathway but act in concert by addressing parallel signaling cascades. Since peptide and receptor expression are up-regulated by LPS, these signaling pathways might act as inhibitory feedback mechanisms in pulmonary bacterial infection.

Specialized epithelial cells with a tuft of apical microvilli (“brush cells”) sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect “bitter” substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cβ2. Thymic “cholinergic chemosensory cells” were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall’s corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

In the thymus, T cell maturation is influenced by cholinergic signaling, and the predominantly expressed receptor is theα3-subunit of nicotinic acetylcholine receptors, encoded by thechrna3gene. We here determined its cellular distribution utilizing an appropriate eGFP-expressing reporter mouse strain. Neither T cells (CD4, CD8) nor mesenchymal cells (desmin-positive) expressed eGFP. In the thymic medulla, eGFP-positive cells either were scattered or, more frequently, formed small clusters resembling Hassall’s corpuscles. Immunolabeling revealed that these cells were indeed terminally differentiated epithelial cells expressing keratin 10 (K10) but neither typical cortical (K8, K18) nor medullary keratins (K5, K14). These labeling patterns reflected those in the epidermis of the skin, where overlap of K10 and eGFP expression was seen in the stratum granulosum, whereas underlying basal cells displayed K5-immunoreactivity. A substantial portion of thymic eGFP-positive cells was also immunoreactive to chromogranin A, a peptide previously reported in epidermal keratinocytes in the stratum granulosum. Its fragment catestatin has multiple biological activities, including suppression of proinflammatory cytokine release from macrophages and inhibition ofα3β4 nAChR. The present findings suggest that its thymic production and/or release are under cholinergic control involving nAChR containing theα3-subunit.

To describe a patient who developed pancytopenia and lactic acidosis after receiving linezolid therapy.A 32-year-old man with past medical history of heart failure, hypertension received a 14-day course of linezolid for treatment of empyema with broncho-pleural fistula. During his follow up visit, he was found to be septic and further evaluation showed pancytopenia, acidosis and elevated liver enzymes. With the exclusion of other possible etiologies, the time course to development of these laboratory abnormalities correlated with the use of linezolid. Antibiotics were switched subsequently and the labs improved after reaching a nadir on the fourth day after stopping linezolid.Linezolid was noted to cause time and dose-dependent reversible myelosuppression in preclinical studies. There are several case reports of throinbocytopenia occurring with linezolid use, but only few cases of pancytopenia and lactic acidosis are reported. Exact mechanism of toxicity is unknown, but many hypothesize it to be similar to chloramphenicol marrow toxicity because of the cross-reactivity to mitochondrial ribosomes.Clinicians should be aware of linezolid as a drug capable of causing pancytopenia, lactic acidosis and elevated liver enzymes. While recovery is usually complete following withdrawal of the drug, we need to still monitor for these rare but severe complications; and consider treating only for a shorter duration, if possible.

Gamma radiation is known to induce cell death in several organs. This damage is associated with endonuclease-mediated DNA fragmentation; however, the enzyme that produces the latter and is likely to cause cell death is unknown. To determine whether the most abundant cytotoxic endonuclease DNase I mediates gamma-radiation-induced tissue injury, we used DNase I knockout mice and zinc chelate of 3,5-diisopropylsalicylic acid (Zn-DIPS), which, as we show, has DNase I inhibiting activity in vitro. The study demonstrated for the first time that inactivation or inhibition of DNase I ameliorates radiation injury to the white pulp of spleen, intestine villi and bone marrow as measured using a quantitative TUNEL assay. The spleen and intestine of DNase I knockout mice were additionally protected from radiation by Zn-DIPS, perhaps due to the broad radioprotective effect of the zinc ions. Surprisingly, the main DNase I-producing tissues such as the salivary glands, pancreas and kidney showed no effect of DNase I inactivation. Another unexpected observation was that even without irradiation, DNA fragmentation and cell death were significantly lower in the intestine of DNase I knockout mice than in wild-type mice. This points to the physiological role of DNase I in normal cell death in the intestinal epithelium. In conclusion, our results suggested that DNase I-mediated mechanism of DNA damage and subsequent tissue injury are essential in gamma-radiation-induced cell death in radiosensitive organs.

Objective— The present study investigates the role of Src and Syk tyrosine kinases in signaling by G-protein coupled and platelet adhesion receptors. Methods and Results— Using Syk −/− platelets or the Src kinase inhibitor PP2, we demonstrate a critical role for Src and Syk kinases in mediating lamellipodia formation on VWF, collagen, CRP, fibrinogen, and fibronectin. In all cases, the spreading defect was overcome by addition of thrombin. Conversely, platelet aggregation and α IIb β 3 activation induced by thrombin was similar to controls, arguing against a functional role for Src and Syk in α IIb β 3 activation. Unexpectedly, CRP potentiated integrin α IIb β 3 activation and platelet aggregation induced by subthreshold concentrations of thrombin in Syk −/− platelets or in the presence of the Src kinase inhibitor PP2. Potentiation in the presence of PP2 was lost in the absence of FcRγ-chain or GPVI confirming that it was mediated through the immunoglobulin receptor. Further delineation of this PP2-resistant synergy revealed that PAR4 could trigger the enhanced response in combination with CRP. Conclusions— We show that Syk is critical for lamellipodia formation on a range of immobilized proteins but that this can be overcome by addition of thrombin. Further, we reveal a novel role for GPVI in supporting thrombin-induced activation, independent of Syk and Src kinases.

Platelet glycoprotein (GP) Ib-IX is a key receptor in arterial platelet adhesion and thrombus formation. A growing body of evidence suggests the inherent adhesive nature of platelets contribute to pathological events such as inflammation and atherosclerosis. However, the molecular mechanisms by which platelets might participate in these processes remain unknown. In the current study, we used a mouse model of platelet receptor GPIb-IX deficiency (mBSS), an equivalent to the human Bernard-Soulier syndrome, to determine whether the development of atherosclerotic lesions is influenced by normal platelet adhesion. Our hypothesis is that mBSS platelets might have a protective role in the development of atherosclerosis given their abnormal function and lack of a critical platelet adhesion receptor. To test this hypothesis we first generated congenic mBSS mice by backcrossing for 10 generations with wild-type C57BL/6J mice. C57BL/6J-GPIb−/− mice were then crossed to the well-characterized murine model of hypercholesterolemia, LDL-receptor deficient animals (C57BL/6J-LDLrtm1Her). Following genotypic screening and second generation breeding we established a colony of double knockout animals (C57BL/6J-LDLrtm1 Her-GPIb−/−) lacking both the gene for the LDL receptor and platelet GPIbα. Experimental animals were divided into 3 groups:double knockouts (C57BL/6J-LDLrtm1Her-GpIb−/−);animals only missing the LDL receptor gene (C57BL/6J-LDLrtm1Her); andwild-type controls (C57BL/6J). At 5 weeks of age mice were weaned and placed on a Western style atherogenic diet composed of 0.2% cholesterol. We have examined aortas following exposure to this high fat diet for 18 and 24 weeks. Six mice from each group were euthanized and the aortas were dissected. The hearts were perfused with 10 ml of phosphate-buffered saline and aortas were opened longitudinally from the heart to the iliac bifurcation. The specimens were stained with Sudan IV to identify the gross presence of fatty streaks in the intimal layer of aortas. Digital images of the stained aortas were captured. All double knockout animals and LDL receptor-deficient mice developed extensive atherosclerotic lesions throughout the aorta with especially pronounced lesions in the area of the aortic arch and arterial branching points. No lesions were observed in control C57BL/6J aortas. Sudan IV-positive lesions were counted visually and they ranged from 21–23 in double knockout aortas and 16–21 in LDL receptor deficient mice. The lesions were more pronounced at earlier time points in male animals. We also observed a correlation between the development of advanced aortic lesions and clinically sound manifestations, such as skin trophic changes and limb self-amputation in some animals. Overall, these findings do not support our initial hypothesis. In fact, we observed that double knockout mice appear to have exaggerated development of atherosclerotic lesions. This finding has led us to a biochemical analysis of mBSS platelets where we have observed phosphorylated resting platelet proteins not present in resting normal mouse platelets. Thus, we conclude in this model of atherosclerosis the BSS platelet may be functioning as a “pseudo-activated” form of a platelet and might be the molecular basis for the apparent support rather than inhibition of arterial atherogenesis.