Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4.

Background: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle tocause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective: This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods: We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP14 were assayed on the activated mast cells. Betahexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results: Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogenactivated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by betahexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase,and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions: Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition

Myo1f, an Unconventional Long-Tailed Myosin, Is a New Partner for the Adaptor 3BP2 Involved in Mast Cell Migration.

Mast cell chemotaxis is essential for cell recruitment to target tissues, where these cells play an important role in adaptive and innate immunity. Stem cell factor (SCF) is a major chemoattractant for mast cells. SCF binds to the KIT receptor, thereby triggering tyrosine phosphorylation in the cytoplasmic domain and resulting in docking sites for SH2 domain-containing molecules, such as Lyn and Fyn, and the subsequent activation of the small GTPases Rac that are responsible for cytoskeletal reorganization and mast cell migration. In previous works we have reported the role of 3BP2, an adaptor molecule, in mast cells. 3BP2 silencing reduces FcεRI-dependent degranulation, by targeting Lyn and Syk phosphorylation, as well as SCF-dependent cell survival. This study examines its role in SCF-dependent migration and reveals that 3BP2 silencing in human mast cell line (LAD2) impairs cell migration due to SCF and IgE. In that context we found that 3BP2 silencing decreases Rac-2 and Cdc42 GTPase activity. Furthermore, we identified Myo1f, an unconventional type-I myosin, as a new partner for 3BP2. This protein, whose functions have been described as critical for neutrophil migration, remained elusive in mast cells. Myo1f is expressed in mast cells and colocalizes with cortical actin ring. Interestingly, Myo1f-3BP2 interaction is modulated by KIT signaling. Moreover, SCF dependent adhesion and migration through fibronectin is decreased after Myo1f silencing. Furthermore, Myo1f silencing leads to downregulation of β1 and β7 integrins on the mast cell membrane. Overall, Myo1f is a new 3BP2 ligand that connects the adaptor to actin cytoskeleton and both molecules are involved in SCF dependent mast cell migration

High-dose intravenous immunoglobulins might modulate inflammation in COVID-19 patients

High-dose of IVIG show immunomodulatory properties that may be useful for controlling the hyperinflammatory status observed in COVID-19 patients. The use of high-dose of intravenous immunoglobulins (IVIGs) as immunomodulators for the treatment of COVID-19-affected individuals has shown promising results. IVIG reduced inflammation in these patients, who progressively restored respiratory function. However, little is known about how they may modulate immune responses in COVID-19 individuals. Here, we have analyzed the levels of 41 inflammatory biomarkers in plasma samples obtained at day 0 (pretreatment initiation), 3, 7, and 14 from five hospitalized COVID-19 patients treated with a 5-d course of 400 mg/kg/d of IVIG. The plasmatic levels of several cytokines (Tumor Necrosis Factor, IL-10, IL-5, and IL-7), chemokines (macrophage inflammatory protein-1α), growth/tissue repairing factors (hepatic growth factor), complement activation (C5a), and intestinal damage such as Fatty acid-binding protein 2 and LPS-binding protein showed a progressive decreasing trend during the next 2 wk after treatment initiation. This trend was not observed in IVIG-untreated COVID-19 patients. Thus, the administration of high-dose IVIG to hospitalized COVID-19 patients may improve their clinical evolution by modulating their hyperinflammatory and immunosuppressive status

Mitigation of coupled wind-wave-earthquake responses of a 10 MW fixed-bottom offshore wind turbine

© 2020 Elsevier Ltd In this paper we present a study on the mitigation of dynamic responses of a 10 MW monopile offshore wind turbine under coupled wind-wave-earthquake excitations. We have developed and validated the generic seismic coupled analysis and structural control architecture tool to overcome the limitation of numerical tools when examining the wind-wave-earthquake coupling effects. We investigated the dynamic responses of a 10 MW monopile offshore wind turbine under different loading combinations and found that the earthquake loading increases the tower-top displacement and pile-cap moment by 47.6% and 95.1%, respectively, compared to the wind-wave-only condition. It is found that the earthquake-induced vibration in the fore-aft direction is mitigated by the wind and wave loadings due to the energy dissipated by the aerodynamic and hydrodynamic damping. In addition, the tower responses are dominated by the earthquake excitation. In order to alleviate the tower vibration induced by the earthquake, we implemented the structural control capability within the tool using tuned mass dampers. The tuned mass dampers with appropriately selected design parameters achieve a larger mitigation on the tower-top displacement for the earthquake-only condition compared to the coupled-loading scenario. The reason is that the tuned mass damper is only effective in mitigating tower vibration, and it is not capable of reducing the tower elastic deformation which is the major contribution of the tower displacement for the coupled-loading condition. In addition, we have found that a heavier tuned mass damper requires a lower tuned frequency to achieve a larger mitigation. A configuration for the mitigation control of the 10 MW offshore wind turbine is suggested by using a 5% mass ratio of the tuned mass damper

Stable neutralizing antibody levels 6 months after mild and severe COVID-19 episodes

Background. Understanding mid-term kinetics of immunity to SARS-CoV-2 is the cornerstone for public health control of the pandemic and vaccine development. However, current evidence is rather based on limited measurements, losing sight of the temporal pattern of these changes. Methods. We conducted a longitudinal analysis on a prospective cohort of COVID-19 patients followed up for >6 months. Neutralizing activity was evaluated using HIV reporter pseudoviruses expressing SARS-CoV-2 S protein. IgG antibody titer was evaluated by ELISA against the S2 subunit, the receptor binding domain (RBD), and the nucleoprotein (NP). Statistical analyses were carried out using mixed-effects models. Findings. We found that individuals with mild or asymptomatic infection experienced an insignificant decay in neutralizing activity, which persisted 6 months after symptom onset or diagnosis. Hospitalized individuals showed higher neutralizing titers, which decreased following a 2-phase pattern, with an initial rapid decline that significantly slowed after day 80. Despite this initial decay, neutralizing activity at 6 months remained higher among hospitalized individuals compared to mild symptomatic. The slow decline in neutralizing activity at mid-term contrasted with the steep slope of anti-RBD, S2, or NP antibody titers, all of them showing a constant decline over the follow-up period. Conclusions. Our results reinforce the hypothesis that the quality of the neutralizing immune response against SARS-CoV-2 evolves over the post-convalescent stage.info:eu-repo/semantics/acceptedVersio

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective: This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods: We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP1-4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results: Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions:Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition

Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals

Mass vaccination campaigns reduced COVID-19 incidence and severity. Here, we evaluated the immune responses developed in SARS-CoV-2-uninfected patients with predominantly antibody-deficiencies (PAD) after three mRNA-1273 vaccine doses. PAD patients were classified based on their immunodeficiency: unclassified primary antibody-deficiency (unPAD, n = 9), common variable immunodeficiency (CVID, n = 12), combined immunodeficiency (CID, n = 1), and thymoma with immunodeficiency (TID, n = 1). unPAD patients and healthy controls (HCs, n = 10) developed similar vaccine-induced humoral responses after two doses. However, CVID patients showed reduced binding and neutralizing titers compared to HCs. Of interest, these PAD groups showed lower levels of Spike-specific IFN-γ-producing cells. CVID individuals also presented diminished CD8+T cells. CID and TID patients developed cellular but not humoral responses. Although the third vaccine dose boosted humoral responses in most PAD patients, it had limited effect on expanding cellular immunity. Vaccine-induced immune responses in PAD individuals are heterogeneous, and should be immunomonitored to define a personalized therapeutic strategies.info:eu-repo/semantics/publishedVersio

Novel Spike-stabilized trimers with improved production protect K18-hACE2 mice and golden Syrian hamsters from the highly pathogenic SARS-CoV-2 Beta variant

Most COVID-19 vaccines are based on the SARS-CoV-2 Spike glycoprotein (S) or their subunits. However, S shows some structural instability that limits its immunogenicity and production, hampering the development of recombinant S-based vaccines. The introduction of the K986P and V987P (S-2P) mutations increases the production and immunogenicity of the recombinant S trimer, suggesting that these two parameters are related. Nevertheless, S-2P still shows some molecular instability and it is produced with low yield. Here we described a novel set of mutations identified by molecular modeling and located in the S2 region of the S-2P that increase its production up to five-fold. Besides their immunogenicity, the efficacy of two representative S-2P-based mutants, S-29 and S-21, protecting from a heterologous SARS-CoV-2 Beta variant challenge was assayed in K18-hACE2 mice (an animal model of severe SARS-CoV-2 disease) and golden Syrian hamsters (GSH) (a moderate disease model). S-21 induced higher level of WH1 and Delta variants neutralizing antibodies than S-2P in K18-hACE2 mice three days after challenge. Viral load in nasal turbinate and oropharyngeal samples were reduced in S-21 and S-29 vaccinated mice. Despite that, only the S-29 protein protected 100% of K18-hACE2 mice from severe disease. When GSH were analyzed, all immunized animals were protected from disease development irrespectively of the immunogen they received. Therefore, the higher yield of S-29, as well as its improved immunogenicity and efficacy protecting from the highly pathogenic SARS-CoV-2 Beta variant, pinpoint the S-29 mutant as an alternative to the S-2P protein for future SARS-CoV-2 vaccine development

La proteïna adaptadora 3BP2 com a regulador de l’activitat cel·lular a través dels receptors de membrana FcεRI i KIT

[cat] Les molècules adaptadores són essencials en l'organització de les molècules de senyalització i en la coordinació i compartimentació de la seva activitat . 3BP2 és una proteïna adaptadora citoplasmàtica expressada ubiqüament però principalment en cèl·lules hematopoètiques. S'ha demostrat que actua com un regulador positiu transduïnt la senyal cel·lular en cèl·lules T , B i NK. 3BP2 és un important regulador en l’alliberació de grànuls citotòxics en les cèl·lules NK. Els mastòcits es desgranulen de manera similar després de l'agregació depenent d'antigen de l'alta afinitat del receptor per IgE (FceRI) a la superfície cel·lular. L'activació d'aquestes cèl·lules indueix l'alliberament de mediadors inflamatoris preformats i la síntesi de novo i també la secreció de citocines i quimiocines. El receptor KIT forma part dels receptors factors de creixement tirosin cinasa. El lligand de KIT és el SCF (stem cell factor), considerat com el factor de creixement més rellevant en mastòcits. L’activació de KIT a través del SCF és crucial per al desenvolupament, la diferenciació i la supervivència dels mastòcits, així com per a una òptima desgranulació depenent d'IgE i producció de citocines. En aquesta tesis s’estudia el paper del 3BP2 en la regulació de l’activitat cel·lular a través dels receptors de membrana, FcepcilonRI i KIT. Amb els resultats podem concloure que el 3BP2 és necessari per obtenir una òptima resposta del mastòcit, tan a nivell de les respostes immediates, com la desgranulació, com en les respostes tardanes com la secreció de IL-8 i de GM-CSF. En la transducció de senyals a traves del FceRI, el 3BP2 és un important regulador de l’activació de mastòcits humans. Per tant, 3BP2 podria ser una diana terapèutica potencial per les malalties inflamatòries mediades pels mastòcits dependents d'IgE. Per altre banda, el silenciament del 3BP2 redueix l’expressió del receptor KIT, tant en la superfície com a nivell transcripcional, això implica un paper del 3BP2 en aquest procés. De fet, el silenciament del 3BP2 condueix a una reducció de MITF, factor de transcripció implicat en l’expressió de KIT. Les mutacions d’activació en KIT s'han associat amb diverses malalties com la mastocitosi, per tant analitzem la capacitat de 3BP2 per regular l'expressió de KIT en la línia cel·lular HMC-1 on KIT es troba mutat. Trobem que el silenciament de 3BP2 en aquestes cèl·lules també redueix l’expressió de KIT. Curiosament el silenciament de KIT i la inhibició de l’activitat cinasa del receptor redueix l’expressió de 3BP2, tot això suggereix una estreta relació entre ambdues molècules. Per tant, 3BP2 podria ser una possible diana terapèutica, en malalties inflamatòries mediades per mastòcits i en trastorns on el KIT esta desregulat. Una d’aquestes malalties associades a la desregulació del receptor KIT son els tumors de cèl·lules de l'estroma gastrointestinal (GIST). La majoria dels GIST presenten mutacions en KIT (85%) o en el receptor PDGFRa (5%) que comporten un increment de l’activitat cinasa d’ambdós receptors. En el present treball es mostra que 3BP2 s'expressa en línies cel·lulars procedents de pacients amb GIST i que la reducció de l’expressió de 3BP2 indueix un decrement de l’expressió del KIT. Curiosament, el silenciament de 3BP2 també redueix l’expressió de PDGFRa. Per altre part, 3BP2 coprecipita amb els receptors KIT i PDGFRa indicant que la molècula pot ser mediador de la senyalització d’ambdós receptors. Aquests nous resultats destaquen la importància del 3BP2 en la regulació de l’expressió i senyalització d’aquests dos receptors oncogènics, mostrant un paper com a possible diana terapèutica en GIST, especialment rellevant en els casos de pacients que han adquirit resistència als inhibidors de l’activitat cinasa d’aquests receptors.[eng] 3BP2 is a cytoplasmic adaptor protein mainly expressed by haematopoietic cells that has been shown to act as a positive regulator in T, B and NK cell signal transduction. 3BP2 is an important regulator of cytotoxic granule release in NK cells. Mast cells similarly degranulate following antigen-dependent aggregation of the high affinity receptor for IgE (FceRI) on the cell surface. Activation of these cells induces the release of preformed inflammatory mediators and the de novo synthesis and secretion of cytokines and chemokines. The KIT receptor whose ligand is the stem cell factor (SCF) is necessary for mast cell development, proliferation and survival as well as for optimal IgE dependent signal. This thesis studied 3BP2 role in regulating cellular activity through membrane receptors, FcepcilonRI and KIT. Our findings point to a requirement for 3BP2 in optimal immediate and late mast cells responses such as degranulation and IL-8 or GM-CSF secretion. In signal transduction through FceRI, 3BP2 is an important regulator of the activation of human mast cells. Moreover 3BP2 silencing reduces expression of KIT receptor both on the surface and at the transcriptional level, this implies a role in this process 3BP2. In fact, the silencing of 3BP2 leads to a reduction in MITF, transcription factor involved in expression of KIT. Our results suggest a potential of 3BP2 as a therapeutic target in mast cell-mediated inflammatory diseases and deregulated KIT disorders. One of this disorders are gastrointestinal stromal tumors (GIST). Most GISTS contain oncogenic gain-of­function of KIT (85%) or PDGFRa receptor (5%) which is accompanied by strong expression of the protein products of these oncogenes. We show that 3BP2 is also expressed in cell lines form GIST patients and there are an interaction with3BP2 and both receptors. Interestingly, silencing of 3BP2 reduce oncogenic KIT and PDGFRa expression. These novel findings highlight the importance of 3BP2 in the regulation of the two oncogenic receptors expression and signalling indicating a role as a potential target in GIST, including those cases containing mutations that confer resistance to the KIT/ PDGFRa kinase inhibitors

Phosphatidyl inositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways are modulated by prostaglandin E₂ in mannitol-stimulated mast cells.

<p>Cells were treated in Tyrode’s buffer for 15 minutes with either 10% mannitol stimulation and/or PGE₂ at 10 µM. EP receptor antagonist pretreatment was done 10 minutes before any cell stimulation as follows: AH6809 (10 µM) blocking receptors EP₁ and EP₂, L826266 (30 µM) blocking receptor EP₃, and AH23848 (10 µM) blocking receptor EP₄. Western Blot analysis was performed in LAD2 cells (A) and HMC-1 (B) to explore (1) AKT phosphorylation as a surrogate marker for PI3K activation, and (2) MAPK activation via JNK, ERK1/2, and p38 phosphorylation. Blot against β-actin was performed (as a loading control). The data is representative of three independent experiments.</p