Mouse Peritoneal Research Articles

Inhibition of Orai Channel Function Regulates Mas-Related G Protein-Coupled Receptor-Mediated Responses in Mast Cells.

Mast cells (MCs) are tissue resident immune cells that play important roles in the pathogenesis of allergic disorders. These responses are mediated via the cross-linking of cell surface high affinity IgE receptor (FcϵRI) by antigen resulting in calcium (Ca2+) mobilization, followed by degranulation and release of proinflammatory mediators. In addition to FcϵRI, cutaneous MCs express Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MrgprB2). Activation of MRGPRX2/B2 by the neuropeptide substance P (SP) is implicated in neurogenic inflammation, chronic urticaria, mastocytosis and atopic dermatitis. Although Ca2+ entry is required for MRGPRX2/B2-mediated MC responses, the possibility that calcium release-activated calcium (CRAC/Orai) channels participate in these responses has not been tested. Lentiviral shRNA-mediated silencing of Orai1, Orai2 or Orai3 in a human MC line (LAD2 cells) resulted in partial inhibition of SP-induced Ca2+ mobilization, degranulation and cytokine/chemokine generation (TNF-α, IL-8, and CCL-3). Synta66, which blocks homo and hetero-dimerization of Orai channels, caused a more robust inhibition of SP-induced responses than knockdown of individual Orai channels. Synta66 also blocked SP-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation and abrogated cytokine/chemokine production. It also inhibited SP-induced Ca2+ mobilization and degranulation in primary human skin MCs and mouse peritoneal MCs. Furthermore, Synta66 attenuated both SP-induced cutaneous vascular permeability and leukocyte recruitment in mouse peritoneum. These findings demonstrate that Orai channels contribute to MRGPRX2/B2-mediated MC activation and suggest that their inhibition could provide a novel approach for the modulation of SP-induced MC/MRGPRX2-mediated disorders.

Phytochemical investigation on Vitex negundo leaves and their anti-inflammatory and analgesic activities

The phytochemical investigation on Vitex negundo leaves has led to the isolation of one new iridoid glucoside (8α-hydroxy-4-carboxyl-5βH-9βH-iridoid-1α-O-(6′-O-(6,7-dihydrofoliamenthonyl)-β-ᴅ-glucopyranoside, 3), together with three known compounds, namely agnuside (1), 6′-O-E-caffeoylmussaenosidic acid (2), and 3,5-dicaffeoylquinic acid (4). The HPLC analytical study was also performed to quantify the content of agnuside (1) in dried leaves. The results indicated the very high content of 1 (3.04 ± 0.02%). The method was also validated by various parameters, including linearity (R2= 0.9999), precision (intra-day RSD ≤ 2.50%, inter-day RSD= 0.76%), and accuracy (recovery rates 96.58-101.86%). The animal testing data showed that the extract did not reduce pain at the doses of 9.6 and 28.8 g /kg (leaf weight/body weight) in the hot plates and pain measuring models but showed the pain reduction in the acetic acid-induced pain model. The extract at the dose of 5.6 g/kg (leaf weight/body weight) also had effects on the acute inflammation in the carrageenin-induced edema model. The extract at the dose 9.6 and 28.8 g/kg (leaf weight/body weight) also showed significant chronic anti-inflammation, comparable to methylprednisolone at the dose 10 mg/kg on the mouse peritoneal.

Delayed Administration of Nintedanib Ameliorates Fibrosis Progression in CG-Induced Peritoneal Fibrosis Mouse Model

Background: A multiple-target tyrosine kinase inhibitor, nintedanib, which is approved for treatment of interstitial pulmonary disease, has been demonstrated to have anti-fibrotic activity outside of the lungs. We explored its therapeutic effect in a murine model of peritoneal fibrosis. Methods: Daily intraperitoneal injections of chlorhexidine gluconate (CG) induced peritoneal fibrosis in mice. The effects of delayed administration of nintedanib (given at day 21 after CG injection and then given daily for 14 days) were determined by immunohistochemical staining, ELISA, and immunoblot analysis. Results: Delayed administration of nintedanib significantly inhibited peritoneal fibrosis progression as indicated by decreasing deposition and expression of extracellular matrix (ECM) proteins (fibronectin and type I collagen). Treatment with nintedanib also upregulated MMP-2 and reciprocally downregulated TIMP-2, along with reducing expression of α-SMA, β-vimentin, and two transcription factors (Snail and Twist), and retaining E-cadherin expression. Nintedanib also inhibited co-expression of β-vimentin with Snail or Twist as shown by immunofluorescent staining. Moreover, nintedanib decreased the number of CD31-positive blood vessels and CD31 expression in the injured peritoneum. Moreover, delayed application of nintedanib inhibited the expression of several cytokines/chemokines, including monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6, and infiltration of CD68⁺ macrophages to the injured peritoneum. Finally, nintedanib blocked phosphorylation of STAT3, NF-κB, and Smad3 during the development of peritoneal fibrosis. Conclusions: Delayed administration of nintedanib inhibits progression of peritoneal fibrosis and partially reverses established peritoneal fibrosis by attenuating epithelial-mesenchymal transition, inflammation, and angiogenesis, as well as promoting ECM degradation. We conclude that nintedanib has a therapeutic potential to treat peritoneal fibrosis.

Loss of JNK-Associated Leucine Zipper Protein Promotes Peritoneal Dialysis-Related Peritoneal Fibrosis

Background: Peritoneal dialysis-related peritoneal fibrosis is the leading cause of peritoneal ultrafiltration failure. Multitude factors and pathological processes have been implicated in peritoneal fibrosis development and progression, whereas the intrinsic anti-fibrotic mechanism has rarely been explored. JNK-associated leucine zipper protein (JLP) has been recently found possessing powerful anti-fibrotic merits of overall antagonizing TGF-β-induced profibrotic effects. Objectives: We wondered whether JLP is expressed in the peritoneum, and if so, whether it exerts the anti-fibrotic effects similar to those in the kidney. Method: Here, we examined and confirmed JLP expression in peritoneum tissue of mice. Then, we established a peritoneal fibrosis model in Jlp wild-type and Jlp global deficient mice and observed the different effects of Jlp on peritoneal fibrosis progression. In vitro studies were performed on peritoneal mesothelial HMrSV5 cells with or without Jlp knockdown to investigate the underlying mechanism by which Jlp exerts anti-fibrotic effects. Results: We found that the expression of JLP decreased in a high-glucose peritoneal dialysis solution (HGPDS)-induced peritoneal fibrosis mouse model and in HGPDS-treated peritoneal mesothelial cell HMrSV5. JLP deletion exacerbated HGPDS-induced peritoneal fibrosis in peritoneal fibrosis mice, and knockdown of JLP resulted in an increased profibrotic response to HGPDS stimulation in HMrSV5 cells, which was associated with epithelial-to-mesenchymal transition, elevated autophagy, and apoptosis, as well as enhanced TGF-β1/Smad signaling activation. Conclusions: Our findings revealed a new anti-fibrotic factor of Jlp involved in peritoneal fibrosis induction and shed light on novel therapeutic targets in peritoneal ultrafiltration failure.

Development of a refined ex vivo model of peritoneal adhesion formation, and a role for connexin 43 in their development.

Despite many advances across the surgical sciences, post-surgical peritoneal adhesions still pose a considerable risk in modern-day procedures and are highly undesirable. We have developed a novel mouse peritoneal strip ex vivo adhesion model which may serve to bridge the gap between single cell culture systems and in vivo animal drug testing for the assessment of potential anti-adhesion agents, and study of causality of the process. We investigated the optimal conditions for adhesion formation with mouse peritoneal tissue strips by modifying an existing ex vivo rat model of peritoneal adhesions. We assessed the impact of the following conditions on the formation of adhesions: contact pressure, abrasions, and the presence of clotted blood. Macroscopic adhesions were detected in all mouse peritoneal strips exposed to specific conditions, namely abrasions and clotted blood, where peritoneal surfaces were kept in contact with pressure using cotton gauze in a tissue cassette. Adhesions were confirmed microscopically. Interestingly, connexin 43, a gap junction protein, was found to be upregulated at sites of adhesions. Key features of this model were the use of padding the abraded tissue with gauze and the use of a standardised volume of clotted blood. Using this model, peritoneal strips cultured with clotted blood between abraded surfaces were found to reproducibly develop adhesion bands at 72h. Our goal is to develop a model that can be used in genetically modified mice in order to dissect out the role of particular genes in adhesion formation and to test drugs to prevent adhesion formation.

Spirostanol Saponins from Flowers of Allium Porrum and Related Compounds Indicating Cytotoxic Activity and Affecting Nitric Oxide Production Inhibitory Effect in Peritoneal Macrophages.

Saponins, a diverse group of natural compounds, offer an interesting pool of derivatives with biomedical application. In this study, three structurally related spirostanol saponins were isolated and identified from the leek flowers of Allium porrum L. (garden leek). Two of them were identical with the already known leek plant constituents: aginoside (1) and 6-deoxyaginoside (2). The third one was identified as new component of A. porrum; however, it was found identical with yayoisaponin A (3) obtained earlier from a mutant of elephant garlic Allium ampeloprasun L. It is a derivative of the aginoside (1) with additional glucose in its glycosidic chain, identified by MS and NMR analysis as (2α, 3β, 6β, 25R)-2,6-dihydroxyspirostan-3-yl β-D-glucopyranosyl-(1 → 3)-β-D-glucopranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosyl]-(1 → 4)-β-D-galactopyranoside, previously reported also under the name alliporin. The leek native saponins were tested together with other known and structurally related saponins (tomatonin and digitonin) and with their related aglycones (agigenin and diosgenin) for in vitro cytotoxicity and for effects on NO production in mouse peritoneal cells. The highest inhibitory effects were exhibited by 6-deoxyaginoside. The obtained toxicity data, however, closely correlated with the suppression of NO production. Therefore, an unambiguous linking of obtained bioactivities of saponins with their expected immunobiological properties remained uncertain.

Increased expression of epimorphin in a peritoneal fibrosis mouse model

Long-term peritoneal dialysis results in functional and histopathological alterations of the peritoneal membrane, leading to peritoneal fibrosis (PF). The mechanism of PF has not been fully elucidated, and at present there is no effective therapy for PF. Epimorphin is a mesenchymal protein that not only regulates morphogenesis in organ development but is implicated in tissue repair. However, the role of epimorphin in PF has not yet been clarified. PF was induced in C57/Bl6 mice by intraperitoneal injection of chlorhexidine gluconate (CG-injected mice) three times a week for 3 weeks. The parietal peritoneum was subsequently dissected and assessed by Masson's trichrome staining, and epimorphin expression was analysed by immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR). Furthermore, epimorphin-positive regions were analysed by multiple immunofluorescence staining using fibrosis-associated markers. In addition, normal rat fibroblast cells (NRK-49F) were treated with transforming growth factor-β (TGF-β) in the presence or absence of epimorphin. The expression of fibrosis-associated markers was assessed by real-time RT-PCR. In CG-injected mice, Masson's trichrome staining showed marked thickening of the submesothelial compact zone. Weak epimorphin expression was observed in the narrow submesothelial compact zone beneath the mesothelial cells in control mice; however, epimorphin expression was stronger in the submesothelial compact zone in CG-injected mice. Epimorphin expression was observed mainly in α-smooth muscle actin (α-SMA)-positive myofibroblasts. Epimorphin suppressed the TGF-β-induced upregulation of α-SMA and platelet-derived growth factor receptor-β in cultured cells. Our results suggest that epimorphin may be a therapeutic target for fibrotic diseases of the peritoneum.

Mitochonic acid-5 ameliorates chlorhexidine gluconate-induced peritoneal fibrosis in mice.

Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis, attributable to inflammation and mitochondrial dysfunction. Mitochonic acid-5 (MA-5), an indole-3-acetic acid derivative, improves mitochondrial dysfunction and has therapeutic potential against various diseases including kidney diseases. However, whether MA-5 is effective against peritoneal fibrosis remains unclear. Therefore, we investigated the effect of MA-5 using a peritoneal fibrosis mouse model. Peritoneal fibrosis was induced in C57BL/6 mice via intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 3weeks. MA-5 was administered daily by oral gavage. The mice were divided into control, MA-5, CG, and CG + MA-5 groups. Following treatment, immunohistochemical analyses were performed. Fibrotic thickening of the parietal peritoneum induced by CG was substantially attenuated by MA-5. The number of α-smooth muscle actin-positive myofibroblasts, transforming growth factor β-positive cells, F4/80-positive macrophages, monocyte chemotactic protein 1-positive cells, and 4-hydroxy-2-nonenal-positive cells was considerably decreased. In addition, reduced ATP5a1-positive and uncoupling protein 2-positive cells in the CG group were notably increased by MA-5. MA-5 may ameliorate peritoneal fibrosis by suppressing macrophage infiltration and oxidative stress, thus restoring mitochondrial function. Overall, MA-5 has therapeutic potential against peritoneal fibrosis.

The effect of folate-appended methyl-β-cyclodextrin increases on survival rates in a peritoneal dissemination mouse models of human ovarian cancer

The effect of folate-appended methyl-β-cyclodextrin increases on survival rates in a peritoneal dissemination mouse models of human ovarian cancer

IgE cross-linking induces activation of human and mouse mast cell progenitors

The concept of innate and adaptive effector cells that are repleted by maturing inert progenitor cell populations is changing. Mast cells develop from rare mast cell progenitors populating peripheral tissues at homeostatic conditions, or as a result of induced recruitment during inflammatory conditions. Because FcεRI-expressing mast cell progenitors are the dominating mast cell type during acute allergic lung inflammation invivo, we hypothesized that they are activated by IgE cross-linking. Mouse peritoneal and human peripheral blood cells were sensitized and stimulated with antigen, or stimulated with anti-IgE, and the mast cell progenitor population analyzed for signs of activation by flow cytometry. Isolated peritoneal mast cell progenitors were studied before and after anti-IgE stimulation at single-cell level by time-lapse fluorescence microscopy. Lung mast cell progenitors were analyzed for their ability to produce IL-13 by intracellular flow cytometry in a mouse model of ovalbumin-induced allergic airway inflammation. Sensitized mouse peritoneal mast cell progenitors demonstrate increased levels of phosphorylation of tyrosines on intracellular proteins (total tyrosine phosphorylation), and spleen tyrosine kinase (Syk) phosphorylation after antigen exposure. Anti-IgE induced cell surface-associated lysomal-associated membrane protein-1 (LAMP-1) in naive mast cell progenitors, and prompted loss of fluorescence signal and altered morphology of isolated cells loaded with lysotracker. In human mast cell progenitors, anti-IgE increased total tyrosine phosphorylation, cell surface-associated LAMP-1, and CD63. Lung mast cell progenitors from mice with ovalbumin-induced allergic airway inflammation produce IL-13. Mast cell progenitors become activated by IgE cross-linking and may contribute to the pathology associated with acute allergic airway inflammation.

Abstract P111: Effects Of Magnetic Field On Abca1-mediated Cellular Lipid Release And Adult Brain Cell Generation In Mice

Aim: This project aims to explore a novel magnetic field (MF) health care based on the cholesterol metabolism. Methods: Mouse peritoneal macrophages foam cells were incubated in CO 2 -independent culture medium containing apoA-I and were exposed in a MF (0.4 T) using the electromagnet of EPR spectrometer. C57BL/6N mice (8 week-old) were peritoneally injected F-ara-EdU (133μg/g mouse body weight) then exposed to MF under the identical condition. Mouse plasma, cerebrospinal fluid, liver and brain were harvested on the 28 th day. Lipoprotein profiles were examined enzymatic detection followed by a gel-permeable-HPLC (Skylight Biotech Inc.). Amyloid β40 and β42 levels were determined by WAKO ELISA kit. Fixed mouse brain sections (40um) were prepared by Leica CM1900 cryostat. Alexa fluor® 488 conjugates were link to F-ara-EdU by a click chemistry method and fluor positive nucleus images were captured by a confocal super resolution SpinSR10 (Olympus, Inc.). Results: ABCA1-apoA-I mediated cellular cholesterol release was not affected or reduced by the exposure in the MF of 0.4 T. The Pcsk9 expression showed significant reduction in the hepatocytes. Reduction of Abca1 expression and increase of Scarb1 were obtained non significantly. The newly generated hippocampal dentate gyrus cells in the control mice group and the MF exposed group were 1.6±1.17, and 0.47±0.62 cells per dentate gyrus section, respectively (P=0.003) despite bulk of new cells in olfactory bulbs in the both groups. Discussions: No substantial increase in cellular cholesterol export was observed by MF irradiation of mouse peritoneal macrophage cells. In addition, the lipoprotein profile showed a decrease in HDL cholesterol even after 28 days. As a result of gene expression in the liver, a decrease in ABCA1 expression and an increase in SR-BI expression inferred reduction of HDL. The number of new cells detected in the granular zone and the subgranular zone in the dentate gyrus of the hippocampus was significantly reduced in the mice subjected to the MF treatment. In the future, it is necessary to investigate in detail whether these newborn cells are neuron or glial cells.

Lingonberry Fruit Ethanol Extract Ameliorates DSS-Induced Ulcerative Colitis In Vivo and In Vitro

Ulcerative colitis (UC) is an inflammatory chronic intestinal disease with pathological characteristics, including imbalanced immune function and the overexpression of inflammatory cytokines and mediators. Inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6) were oversecreted in UC condition. Cyclooxygenase (COX)-2 and prostaglandin (PG)E2 were also overexpressed in colon tissue. Lingonberry (LB) (Vaccinium vitis-idaea L.) possesses pharmacological activities, including anti-oxidant, anti-cancer, and anti-obesity effects. To explore LB’s effects on UC, BALB/c mice were administered with 3% (w/v) dextran sulphate sodium (DSS) and LB extract (70% ethanol) orally for nine days. The severity of UC was measured by the change in body weight and colon length. To evaluate LB’s regulatory effect on inflammatory cytokines, the enzyme-linked immunosorbent assay (ELISA) kit was used to measure the inflammatory cytokines in mouse serum. Mouse peritoneal microphages were used to detect LB’s anti-inflammatory effect. The results showed that LB treatment ameliorated less weight loss and longer colon length compared to the DSS-treated group. LB treatment also ameliorated the secretion of inflammatory cytokines. These results indicated that LB has potential as an herbal medicine to treat UC.

Lactic acid suppresses MRGPRX2 mediated mast cell responses

MAS related G-protein coupled receptor X2 (MRGPRX2) is a G-protein coupled receptor (GPCR) expressed in human mast cells that has been implicated to play an important role in causing pseudo-allergic reactions as well as exacerbating inflammation during asthma and other allergic diseases. Lactic acid, a byproduct of glucose metabolism, is abundantly present in inflamed tissues and has been shown to regulate functions of several immune cells. Because the endogenous ligands for MRGPRX2 (substance P and LL-37) are elevated during pathologic conditions, such as cancer and asthma, and given that lactic acid levels are also enhanced in these patients, we explored the role of lactic acid in regulating mast cells response via MRGPRX2 and MrgprB2, the mouse orthologue of the human receptor. We found that lactic acid suppressed both the early (Ca2+ mobilization and degranulation) and late (chemokine/cytokine release) phases of mast cell activation; this data was confirmed in LAD2, human skin and mouse peritoneal mast cells. In LAD2 cells, the reduction in degranulation and chemokine/cytokine production mediated by lactic acid was dependent on pH. In agreement with our in vitro studies, lactic acid also reduced passive systemic anaphylaxis to compound 48/80 (a known MRGPRX2/MrgprB2 ligand) and skin inflammation in a mouse model of rosacea that is dependent on MrgprB2 expression on skin mast cells. Our data thus suggest that lactic acid may serve to inhibit mast cell-mediated inflammation during asthma and reduce immune response during cancer by affecting mast cell activation through MRGPRX2.

A novel receptor for platelet-activating factor and lysophosphatidylcholine in Trypanosoma cruzi.

The lipid mediators, platelet-activating factor (PAF) and lysophosphatidylcholine (LPC), play relevant pathophysiological roles in Trypanosoma cruzi infection. Several species of LPC, including C18:1 LPC, which mimics the effects of PAF, are synthesized by T. cruzi. The present study identified a receptor in T. cruzi, which was predicted to bind to PAF, and found it to be homologous to members of the progestin and adiponectin family of receptors (PAQRs). We constructed a three-dimensional model of the T. cruzi PAQR (TcPAQR) and performed molecular docking to predict the interactions of the TcPAQR model with C16:0 PAF and C18:1 LPC. We knocked out T. cruzi PAQR (TcPAQR) gene and confirmed the identity of the expressed protein through immunoblotting and immunofluorescence assays using an anti-human PAQR antibody. Wild-type and knockout (KO) parasites were also used to investigate the in vitro cell differentiation and interactions with peritoneal mouse macrophages; TcPAQR KO parasites were unable to react to C16:0 PAF or C18:1 LPC. Our data are highly suggestive that PAF and LPC act through TcPAQR in T. cruzi, triggering its cellular differentiation and ability to infect macrophages.

TGF-β1 Receptor Inhibitor SB525334 Attenuates the Epithelial to Mesenchymal Transition of Peritoneal Mesothelial Cells via the TGF-β1 Signaling Pathway.

We investigated the effect of SB525334 (TGF-β receptor type 1 (TβRI) inhibitor) on the epithelial to mesenchymal transition (EMT) signaling pathway in human peritoneal mesothelial cells (HPMCs) and a peritoneal fibrosis mouse model. In vitro experiments were performed using HPMCs. HPMCs were treated with TGF-β1 and/or SB525334. In vivo experiments were conducted with male C57/BL6 mice. The 0.1% chlorhexidine gluconate (CG) was intraperitoneally injected with or without SB52534 administration by oral gavage. Mice were euthanized after 28 days. EMT using TGF-β1-treated HPMCs included morphological changes, cell migration and invasion, EMT markers and collagen synthesis. These pathological changes were reversed by co-treatment with SB525334. CG injection was associated with an increase in peritoneal fibrosis and thickness, which functionally resulted in an increase in the glucose absorption via peritoneum. Co-treatment with SB525334 attenuated these changes. The levels of EMT protein markers and immunohistochemical staining for fibrosis showed similar trends. Immunofluorescence staining for EMT markers showed induction of transformed cells with both epithelial and mesenchymal cell markers, which decreased upon co-treatment with SB525334. SB525334 effectively attenuated the TGF-β1-induced EMT in HPMCs. Cotreatment with SB525334 improved peritoneal thickness and fibrosis and recovered peritoneal membrane function in a peritoneal fibrosis mouse model.

Innate-like self-reactive B cells infiltrate human renal allografts during transplant rejection

Intrarenal B cells in human renal allografts indicate transplant recipients with a poor prognosis, but how these cells contribute to rejection is unclear. Here we show using single-cell RNA sequencing that intrarenal class-switched B cells have an innate cell transcriptional state resembling mouse peritoneal B1 or B-innate (Bin) cells. Antibodies generated by Bin cells do not bind donor-specific antigens nor are they enriched for reactivity to ubiquitously expressed self-antigens. Rather, Bin cells frequently express antibodies reactive with either renal-specific or inflammation-associated antigens. Furthermore, local antigens can drive Bin cell proliferation and differentiation into plasma cells expressing self-reactive antibodies. These data show a mechanism of human inflammation in which a breach in organ-restricted tolerance by infiltrating innate-like B cells drives local tissue destruction.

Study of Antibody-Dependent Reactions of Mast Cells In Vitro and in a Model of Severe Influenza Infection in Mice.

We investigated the reaction of mouse peritoneal mast cells (MCs) in vitro after IgG-containing immune complex introduction using A/H5N1 and A/H1N1pdm09 influenza viruses as antigens. The sera of immune mice served as a source of IgG antibodies. The concentration of histamine in the supernatants was determined at 4 hours after incubation with antisera and virus. We compared the contribution of MCs to the pathogenesis of post-immunization influenza infection with A/H5N1 and A/H1N1 influenza viruses in mice. The mice were immunized parenterally with inactivated viruses and challenged with lethal doses of drift A/H5N1 and A/H1N1 influenza viruses on the 14th day after immunization. Simultaneously, half of the mice were injected intraperitoneally with a mixture of histamine receptor blockers (chloropyramine and quamatel). In in vitro experiments, the immune complex formed by A/H5N1 virus and antiserum caused a significant increase in the histamine release compared to immune serum or the virus alone. With regard to the A/H1N1 virus, such an increase was not significant. A/H1N1 immunization caused detectable HI response in mice at 12th day after immunization, in contrast to the A/H5N1 virus. After challenge of A/H5N1-immunized mice, administration of antihistamines increased the survival rate by up to 90%. When infecting the A/H1N1-immunized mice, 90% of the animals were already protected from lethal infection by day 14; the administration of histamine receptor blockers did not increase survival. Histological examination of the lungs has shown that toluidine blue staining allows to estimate the degree of MC degranulation. The possibility of in vitro activation of murine MCs by IgG-containing immune complexes has been shown. In a model of influenza infection, it was shown that the administration of histamine receptor blockers increased survival. When the protection was formed faster due to the earlier production of HI antibodies, the administration of histamine receptor blockers did not significantly affect the course of the infection. These data allow to propose that even if there are antibody-dependent MC reactions, they can be easily stopped by the administration of histamine receptor blockers.

P2X7R in Mast Cells is a Potential Target for Salicylic Acid and Aspirin in Treatment of Inflammatory Pain.

BackgroundMast cells are well known for their role in inflammatory pain. P2X7 receptor (P2X7R) has attracted much attention due to its prominent role in inflammatory diseases. Salicylates are commonly used anti-inflammatory and analgesic drugs. Until now, little has been known about whether P2X7R in mast cells is involved in inflammatory pain and whether it is a potential target for salicylates.MethodsFirst, the expression of P2X receptors in mouse peritoneal mast cells was detected by using RT-PCR, immunofluorescence, calcium imaging and electrophysiological technique. In addition, the functions of P2X receptors, especially P2X7R, in mast cells were studied by using QPCR, ELISA and behavioral tests. Furthermore, P2X7R was used as a target to screen for some anti-inflammatory monomers that could inhibit its activity. At last, the effect of salicylic acid (SA) and aspirin (ASA) on the activity of P2X7R was studied by using calcium imaging, electrophysiological technique, ELISA, real-time PCR, behavioral tests, immunofluorescence and molecular docking.ResultsWe found that P2X1, P2X3, P2X4 and P2X7 receptors were expressed in mouse peritoneal mast cells. The functions of different P2X receptors were various. Activation of P2X7R in mouse mast cells induced the release of inflammatory mediators, such as histamine, IL-1β, and CCL3. In addition, inflammation pain induced by high concentrations of ATP could be alleviated by P2X7R blockers or mast cell defects. Interestingly, SA or ASA could reduce high concentrations of ATP-induced inward current, P2X7R upregulation, mediators release, and inflammatory pain. SA or ASA also inhibited the inward current evoked by P2X7R agonist, BZATP. Molecular docking showed that SA or ASA had affinity for the cytoplasmic GDP-binding region of P2X7R.ConclusionP2X7R in mast cells was involved in inflammation pain by releasing inflammatory mediators, and P2X7R might be a potential target for SA and ASA analgesia.

Synergistic Activity of Colistin Combined With Auranofin Against Colistin-Resistant Gram-Negative Bacteria.

Colistin-resistant (Col-R) bacteria are steadily increasing, and are extremely difficult to treat. New drugs or therapies are urgently needed to treat infections caused by these pathogens. Combination therapy with colistin and other old drugs, is an important way to restore the activity of colistin. This study aimed to investigate the activity of colistin in combination with the anti-rheumatic drug auranofin against Col-R Gram-negative bacteria. The results of checkerboard analysis demonstrated that auranofin synergized with colistin against Col-R Gram-negative bacteria. Time-kill assays showed significant synergistic antimicrobial activity of colistin combined with auranofin. Electron microscopy revealed that the combination resulted in more cellular structural alterations compared to each drug alone. Auranofin enhanced the therapeutic effectiveness of colistin in mouse peritoneal infection models. These results suggested that the combination of colistin and auranofin might be a potential alternative for the treatment of Col-R Gram-negative bacterial infections.

Murepavadin, a Small Molecule Host Defense Peptide Mimetic, Activates Mast Cells via MRGPRX2 and MrgprB2.

Pseudomonas aeruginosa is a frequent cause of hospital-acquired wound infection and is difficult to treat because it forms biofilms and displays antibiotic resistance. Previous studies in mice demonstrated that mast cells (MCs) not only contribute to P. aeruginosa eradication but also promote wound healing via an unknown mechanism. We recently reported that host defense peptides (HDPs) induce human MC degranulation via Mas-related G protein-coupled receptor-X2 (MRGPRX2). Small molecule HDP mimetics have distinct advantages over HDPs because they are inexpensive to synthesize and display high stability, bioavailability, and low toxicity. Murepavadin is a lipidated HDP mimetic, (also known as POL7080), which displays antibacterial activity against a broad panel of multi-drug-resistant P. aeruginosa. We found that murepavadin induces Ca2+ mobilization, degranulation, chemokine IL-8 and CCL3 production in a human MC line (LAD2 cells) endogenously expressing MRGPRX2. Murepavadin also caused degranulation in RBL-2H3 cells expressing MRGPRX2 but this response was significantly reduced in cells expressing missense variants within the receptor’s ligand binding (G165E) or G protein coupling (V282M) domains. Compound 48/80 induced β-arrestin recruitment and promoted receptor internalization, which resulted in substantial decrease in the subsequent responsiveness to the MRGPRX2 agonist. By contrast, murepavadin did not cause β-arrestin-mediated MRGPRX2 regulation. Murepavadin induced degranulation in mouse peritoneal MCs via MrgprB2 (ortholog of human MRGPRX2) and caused increased vascular permeability in wild-type mice but not in MrgprB2-/- mice. The data presented herein demonstrate that murepavadin activates human MCs via MRGPRX2 and murine MCs via MrgprB2 and that MRGPRX2 is resistant to β-arrestin-mediated receptor regulation. Thus, besides its direct activity against P. aeruginosa, murepavadin may contribute to bacterial clearance and promote wound healing by harnessing MC’s immunomodulatory property via the activation of MRGPRX2.