Endothelin-1 mediates Aspergillus fumigatus induced airway inflammation and remodelling

BACKGROUND: Asthma is a chronic inflammatory condition of the airways and patients sensitised to airborne fungi such as Aspergillus fumigatus have more severe asthma. Thickening of the bronchial subepithelial layer is a contributing factor to asthma severity for which no current treatment exists. Airway epithelium acts as an initial defence barrier to inhaled spores, orchestrating an inflammatory response and contributing to subepithelial fibrosis. OBJECTIVE: We aimed to analyse the production of profibrogenic factors by airway epithelium in response to A. fumigatus, in order to propose novel anti-fibrotic strategies for fungal-induced asthma. METHODS: We assessed the induction of key profibrogenic factors, TGFβ1, TGFβ2, periostin and endothelin-1, by human airway epithelial cells and in mice exposed to A. fumigatus spores or secreted fungal factors. RESULTS: A. fumigatus specifically caused production of endothelin-1 by epithelial cells in vitro but not any of the other profibrogenic factors assessed. A. fumigatus also induced endothelin-1 in murine lungs, associated with extensive inflammation and airway wall remodelling. Using a selective endothelin-1 receptor antagonist, we demonstrated for the first time, that endothelin-1 drives many features of airway wall remodelling and inflammation elicited by A. fumigatus. CONCLUSION: Our findings are consistent with the hypothesis that elevated endothelin-1 levels contribute to subepithelial thickening and highlight this factor as a possible therapeutic target for difficult-to-treat fungal-induced asthma

Differential proinflammatory responses to Aspergillus fumigatus by airway epithelial cells in vitro are protease dependent

Aspergillus fumigatus is an important human respiratory mould pathogen. In addition to a barrier function, airway epithelium elicits a robust defence against inhaled A. fumigatus by initiating an immune response. The manner by which A. fumigatus initiates this response and the reasons for the immunological heterogeneity with different isolates are unclear. Both direct fungal cell wall–epithelial cell interaction and secretion of soluble proteases have been proposed as possible mechanisms. Our aim was to determine the contribution of fungal proteases to the induction of epithelial IL-6 and IL-8 in response to different A. fumigatus isolates. Airway epithelial cells were exposed to conidia from a low or high protease-producing strain of A. fumigatus, and IL-6 and IL-8 gene expression and protein production were quantified. The role of proteases in cytokine production was further determined using specific protease inhibitors. The proinflammatory cytokine response correlated with conidia germination and hyphal extension. IL-8 induction was significantly reduced in the presence of matrix metalloprotease or cysteine protease inhibitors. With a high protease-producing strain of A. fumigatus, IL-6 release was metalloprotease dependent. Dectin-1 antagonism also inhibited the production of both cytokines. In conclusion, A. fumigatus-secreted proteases mediate a proinflammatory response by airway epithelial cells in a strain-dependent manner

Detection of human neutrophil elastase (HNE) on wound dressings as marker of inflammation

Chronic wound fluids have elevated concentration of human neutrophil elastase (HNE) which can be used as inflammation/infection marker. Our goal is to develop functional materials for fast diagnosis of wound inflammation/infection by using HNE as a specific marker. For that, fluorogenic peptides with a HNE-specific cleavage sequence were incorporated into traditional textile dressings, to allow real-time detection of the wound status. Two different fluorogenic approaches were studied in terms of intensity of the signal generated upon HNE addition: a fluorophore 7-amino-4-trifluormethylcoumarin (AFC) conjugated to a HNE-specific peptide and two fluorophore/quencher pairs (FAM/Dabcyl and EDANS/Dabcyl) coupled to a similar peptide as a Förster resonance energy transfer (FRET) strategy. Also, two immobilization methods were tested: sonochemistry immobilization onto a cotton bandage and glutaraldehyde (GTA)-assisted chemical crosslinking onto a polyamide dressing. The immobilized fluorogenic AFC peptide showed an intense fluorescence emission in the presence of HNE. HNE also induced an enhanced fluorescent signal with the EDANS/Dabcyl FRET peptide which showed to be a more sensitive and effective strategy than the AFC peptide. However, its chemical immobilization onto the polyamide dressing greatly decreased its detection, mainly due to the more difficult access of the enzyme to the cleavage sequence of the immobilized peptide. After optimization of the in situ immobilization, it will be possible to use these fluorescence-functionalized dressings for an effective and specific monitoring of chronic wounds by simply using a portable ultraviolet (UV) light source. We envision that the development of this point-of-care medical device for wound control will have a great impact on patients life quality and reduction of costs on health care system.This study was funded by the European project InFact-Functional materials for fast diagnosis of wound infection (FP7-NMP-2013-SME-7-grant agreement no. 604278). The work done at Centre of Biological Engineering (CEB) was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte 2020-Programa Operacional Regional do Norte

Allergens induce enhanced bronchoconstriction and leukotriene production in C5 deficient mice

BACKGROUND: Previous genetic analysis has shown that a deletion in the complement component 5 gene-coding region renders mice more susceptible to allergen-induced airway hyperresponsiveness (AHR) due to reduced IL-12 production. We investigated the role of complement in a murine model of asthma-like pulmonary inflammation. METHODS: In order to evaluate the role of complement B10 mice either sufficient or deficient in C5 were studied. Both groups of mice immunized and challenged with a house dust extract (HDE) containing high levels of cockroach allergens. Airways hyper-reactivity was determined with whole-body plesthysmography. Bronchoalveolar lavage (BAL) was performed to determine pulmonary cellular recruitment and measure inflammatory mediators. Lung homogenates were assayed for mediators and plasma levels of IgE determined. Pulmonary histology was also evaluated. RESULTS: C5-deficient mice showed enhanced AHR to methylcholine challenge, 474% and 91% increase above baseline Penh in C5-deficient and C5-sufficient mice respectively, p < 0.001. IL-12 levels in the lung homogenate (LH) were only slightly reduced and BAL IL-12 was comparable in C5-sufficient and C5-deficient mice. However, C5-deficient mice had significantly higher cysteinyl-leukotriene levels in the BAL fluid, 1913 +/- 246 pg/ml in C5d and 756 +/- 232 pg/ml in C5-sufficient, p = 0.003. CONCLUSION: These data demonstrate that C5-deficient mice show enhanced AHR due to increased production of cysteinyl-leukotrienes

In vitro and in vivo characterization of highly purified Human Mesothelioma derived cells

<p>Abstract</p> <p>Background</p> <p>Malignant pleural mesothelioma is a rare disease known to be resistant to conventional therapies. A better understanding of mesothelioma biology may provide the rationale for new therapeutic strategies. In this regard, tumor cell lines development has been an important tool to study the biological properties of many tumors. However all the cell lines established so far were grown in medium containing at least 10% serum, and it has been shown that primary cell lines cultured under these conditions lose their ability to differentiate, acquire gene expression profiles that differ from that of tissue specific stem cells or the primary tumor they derive from, and in some cases are neither clonogenic nor tumorigenic. Our work was aimed to establish from fresh human pleural mesothelioma samples cell cultures maintaining tumorigenic properties.</p> <p>Methods</p> <p>The primary cell cultures, obtained from four human pleural mesotheliomas, were expanded in vitro in a low serum proliferation-permissive medium and the expression of different markers as well as the tumorigenicity in immunodeficient mice was evaluated.</p> <p>Results</p> <p>The established mesothelioma cell cultures are able to engraft, after pseudo orthotopic intraperitoneal transplantation, in immunodeficient mouse and maintain this ability to after serial transplantation. Our cell cultures were strongly positive for CD46, CD47, CD56 and CD63 and were also strongly positive for some markers never described before in mesothelioma cell lines, including CD55, CD90 and CD99. By real time PCR we found that our cell lines expressed high mRNA levels of typical mesothelioma markers as mesothelin (MSLN) and calretinin (CALB2), and of BMI-1, a stemness marker, and DKK1, a potent Wingless [WNT] inhibitor.</p> <p>Conclusions</p> <p>These cell cultures may provide a valuable in vitro and in vivo model to investigate mesothelioma biology. The identification of new mesothelioma markers may be useful for diagnosis and/or prognosis of this neoplasia as well as for isolation of mesothelioma tumor initiating cells.</p

Membrane Docking Geometry of GRP1 PH Domain Bound to a Target Lipid Bilayer: An EPR Site-Directed Spin-Labeling and Relaxation Study

The second messenger lipid PIP3 (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP3-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP3-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP3 lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP3. The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP3 target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP3 on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP3 headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP3 headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP3 headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP3-bound GRP1 PH domain on supported lipid bilayers

A short history of the 5-HT2C receptor: from the choroid plexus to depression, obesity and addiction treatment

This paper is a personal account on the discovery and characterization of the 5-HT2C receptor (first known as the 5- HT1C receptor) over 30 years ago and how it translated into a number of unsuspected features for a G protein-coupled receptor (GPCR) and a diversity of clinical applications. The 5-HT2C receptor is one of the most intriguing members of the GPCR superfamily. Initially referred to as 5-HT1CR, the 5-HT2CR was discovered while studying the pharmacological features and the distribution of [3H]mesulergine-labelled sites, primarily in the brain using radioligand binding and slice autoradiography. Mesulergine (SDZ CU-085), was, at the time, best defined as a ligand with serotonergic and dopaminergic properties. Autoradiographic studies showed remarkably strong [3H]mesulergine-labelling to the rat choroid plexus. [3H]mesulergine-labelled sites had pharmacological properties different from, at the time, known or purported 5-HT receptors. In spite of similarities with 5-HT2 binding, the new binding site was called 5-HT1C because of its very high affinity for 5-HT itself. Within the following 10 years, the 5-HT1CR (later named 5- HT2C) was extensively characterised pharmacologically, anatomically and functionally: it was one of the first 5-HT receptors to be sequenced and cloned. The 5-HT2CR is a GPCR, with a very complex gene structure. It constitutes a rarity in theGPCR family: many 5-HT2CR variants exist, especially in humans, due to RNA editing, in addition to a few 5-HT2CR splice variants. Intense research led to therapeutically active 5-HT2C receptor ligands, both antagonists (or inverse agonists) and agonists: keeping in mind that a number of antidepressants and antipsychotics are 5- HT2CR antagonists/inverse agonists. Agomelatine, a 5-HT2CR antagonist is registered for the treatment of major depression. The agonist Lorcaserin is registered for the treatment of aspects of obesity and has further potential in addiction, especially nicotine/ smoking. There is good evidence that the 5-HT2CR is involved in spinal cord injury-induced spasms of the lower limbs, which can be treated with 5-HT2CR antagonists/inverse agonists such as cyproheptadine or SB206553. The 5-HT2CR may play a role in schizophrenia and epilepsy. Vabicaserin, a 5-HT2CR agonist has been in development for the treatment of schizophrenia and obesity, but was stopped. As is common, there is potential for further indications for 5-HT2CR ligands, as suggested by a number of preclinical and/or genome-wide association studies (GWAS) on depression, suicide, sexual dysfunction, addictions and obesity. The 5-HT2CR is clearly affected by a number of established antidepressants/antipsychotics and may be one of the culprits in antipsychotic-induced weight gain

NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization

<p>Abstract</p> <p>Background</p> <p>Nitrogen dioxide (NO₂) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO₂is also produced endogenously in the lung during acute inflammatory responses. NO₂can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c⁺antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c⁺cells in NO₂-promoted allergic sensitization.</p> <p>Methods</p> <p>We systemically depleted CD11c⁺cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO₂followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c⁺cells from wildtype mice were studied after exposure to NO₂and ovalbumin for cellular phenotype by flow cytometry and <it>in vitro </it>cytokine production.</p> <p>Results</p> <p>Transient depletion of CD11c⁺cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c⁺cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO₂exposure. By 48 hours, CD11c⁺MHCII⁺DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c⁺CD11b^-and CD11c⁺CD11b⁺pulmonary cells exposed to NO₂<it>in vivo </it>increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647⁺CD11c⁺MHCII⁺DCs present in MLN from NO₂-exposed mice by 48 hours. Co-cultures of ova-specific CD4⁺T cells from naïve mice and CD11c⁺pulmonary cells from NO₂-exposed mice produced IL-1, IL-12p70, and IL-6 <it>in vitro </it>and augmented antigen-induced IL-5 production.</p> <p>Conclusions</p> <p>CD11c⁺cells are critical for NO₂-promoted allergic sensitization. NO₂exposure causes pulmonary CD11c⁺cells to acquire a phenotype capable of increased antigen uptake, migration to the draining lymph node, expression of MHCII and co-stimulatory molecules required to activate naïve T cells, and secretion of polarizing cytokines to shape a Th2/Th17 response.</p

Soy Isoflavones Genistein and Daidzein Exert Anti-Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat1–86 Exposure

HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity.We demonstrate in primary cortical cell cultures that 17β-estradiol or isoflavones (genistein or daidzein) attenuate Tat(1-86)-induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERα or ERβ specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERβ selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling.Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons

Revised Lithostratigraphy of the Sonsela Member (Chinle Formation, Upper Triassic) in the Southern Part of Petrified Forest National Park, Arizona

BACKGROUND: Recent revisions to the Sonsela Member of the Chinle Formation in Petrified Forest National Park have presented a three-part lithostratigraphic model based on unconventional correlations of sandstone beds. As a vertebrate faunal transition is recorded within this stratigraphic interval, these correlations, and the purported existence of a depositional hiatus (the Tr-4 unconformity) at about the same level, must be carefully re-examined. METHODOLOGY/PRINCIPAL FINDINGS: Our investigations demonstrate the neglected necessity of walking out contacts and mapping when constructing lithostratigraphic models, and providing UTM coordinates and labeled photographs for all measured sections. We correct correlation errors within the Sonsela Member, demonstrate that there are multiple Flattops One sandstones, all of which are higher than the traditional Sonsela sandstone bed, that the Sonsela sandstone bed and Rainbow Forest Bed are equivalent, that the Rainbow Forest Bed is higher than the sandstones at the base of Blue Mesa and Agate Mesa, that strata formerly assigned to the Jim Camp Wash beds occur at two stratigraphic levels, and that there are multiple persistent silcrete horizons within the Sonsela Member. CONCLUSIONS/SIGNIFICANCE: We present a revised five-part model for the Sonsela Member. The units from lowest to highest are: the Camp Butte beds, Lot's Wife beds, Jasper Forest bed (the Sonsela sandstone)/Rainbow Forest Bed, Jim Camp Wash beds, and Martha's Butte beds (including the Flattops One sandstones). Although there are numerous degradational/aggradational cycles within the Chinle Formation, a single unconformable horizon within or at the base of the Sonsela Member that can be traced across the entire western United States (the "Tr-4 unconformity") probably does not exist. The shift from relatively humid and poorly-drained to arid and well-drained climatic conditions began during deposition of the Sonsela Member (low in the Jim Camp Wash beds), well after the Carnian-Norian transition