FcεRI Cluster Size Determines Effective Mast Cell Desensitization without Effector Responses in vitro

2021 ◽  
pp. 1-9
Author(s):  
Yuka Nagata ◽  
Ryo Suzuki
Keyword(s):  
Mast Cell ◽  
Molecular Mechanisms ◽  
Cluster Formation ◽  
Tolerance Induction ◽  
Specific Ige ◽  
Cell Surface Expression ◽  
Optimal Size ◽  
Allergen Specific Immunotherapy ◽  
The Impact

<b><i>Background:</i></b> Allergen-specific desensitization of mast cell (MC) IgE receptors (FcεRI) is an important mechanism of allergen-specific immunotherapy that enables tolerance induction via systemic desensitization. Experimental in vitro IgE-mediated MC desensitization is a potential tool to understand the molecular mechanisms underlying this therapy. Desensitized MCs exhibit internalized IgE and its FcεRI receptors in response to suboptimal doses of allergen without provoking activation. The ovalbumin (OVA) allergen exhibits altered allergenicity upon heat treatment. MC reactions are fundamentally regulated by allergen features (i.e., allergenicity); however, the effects of allergenicity on desensitization remain unclear. <b><i>Objectives:</i></b> This study aimed to examine the impact of allergenicity on the establishment of in vitro MC desensitization using naive OVA (nOVA) and heated OVA (hOVA), which could induce varying MC effector responses. <b><i>Method:</i></b> Bone marrow-derived MCs (BMMCs) were sensitized with OVA-specific IgE, desensitized with sequentially increasing doses of nOVA or hOVA at 10-min intervals, and challenged with nOVA. To evaluate desensitization, the cell surface expression level and subcellular localization of FcεRI-bound IgE were analyzed before and after the final nOVA challenge. MC activation was determined by measuring the release of β-hexosaminidase into supernatants. <b><i>Results:</i></b> Desensitized cells exhibited impaired activation following OVA challenge. Both nOVA and hOVA induced BMMC desensitization under different conditions. Formation of small IgE-FcεRI cluster BMMCs, which adequately represent the desensitized state, was significant. The size of the internalized IgE-FcεRI clusters might be correlated with the desensitized state of MCs. <b><i>Conclusions:</i></b> We demonstrate that the optimal size of IgE-FcεRI clusters for in vitro BMMC desensitization differed significantly depending on allergenicity, and the efficacy of desensitization was reflected by IgE-FcεRI cluster formation. Our study provides information on the characteristics of IgE-FcεRI internalization for successful desensitization in vitro.

scholarly journals Denatonium Benzoate Exposure Promotes IgE-Mediated Mast Cell Degranulation and Allergy By Upregulating FcεRIα Expression

2021 ◽  
Author(s):  
Huaping Xu ◽  
Xiaoyun Shi ◽  
Mengting Xie ◽  
Shiyu Xiao ◽  
Xin Li ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Surface ◽  
Surface Expression ◽  
Specific Ige ◽  
Mast Cell Degranulation ◽  
Cell Surface Expression ◽  
Denatonium Benzoate ◽  
Ige Mediated

Abstract Background: Denatonium benzoate (DB), one of the bitterest compounds known to man, is currently added to a wide range of products and is also used for alcohol denaturation. Some reports demonstrated that asthmatic symptoms are associated with DB exposure but the possible links between DB and IgE-mediated allergy susceptibility have not been examined to date. We investigated the effects of DB on IgE-mediated mast cell degranulation in vitro and in the ovalbumin (OVA)-induced mouse model of allergy.Methods: DB treatments were given to RBL-2H3 IgE-sensitized rat mast cell/basophil cells and KU812 human basophilic cells together with OVA-induced allergic BALB/c mice. Allergic mediator release, Ca2+ influx and OVA-specific IgE anaphylactic shock symptoms were measured along with the cell-surface expression of the α-subunit of high-affinity IgE receptor FcεRI on mast cells.Results: DB increases β-hexosaminidase (β-hex) release and Ca2+ mobilization in IgE-mediated activated RBL-2H3 and KU812 cells, and enhanced the cell-surface expression of FcεRIα. DB also promoted the severity of OVA-induced anaphylactic and diarrheic symptoms which was accompanied by mucus thickness in jejunum and the levels of β-hex, histamine and OVA-specific IgE in allergy mice, as well as the levels of FcεRIα mRNA and the FcεRIα proteinin isolated mucosal mast cells. Conclusions: DB treatments can promote the IgE-mediated mast cell degranulation in vitro and OVA-induced allergic susceptibility in mice by upregulating mast-cell-surface FcεR1α expression, providing evidence for DB exposure in promoting allergy susceptibility.

scholarly journals Role of Zinc Signaling in the Regulation of Mast Cell-, Basophil-, and T Cell-Mediated Allergic Responses

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Keigo Nishida ◽  
Ryota Uchida
Keyword(s):  
Mast Cell ◽  
T Cell ◽  
Molecular Mechanisms ◽  
Neuronal Degeneration ◽  
Zinc Homeostasis ◽  
Zinc Signaling ◽  
The Impact

Zinc is essential for maintaining normal structure and physiological function of cells. Its deficiency causes growth retardation, immunodeficiency, and neuronal degeneration. Zinc homeostasis is tightly regulated by zinc transporters and metallothioneins that control zinc concentration and its distribution in individual cells and contributes to zinc signaling. The intracellular zinc signaling regulates immune reactions. Although many molecules involved in these processes have zinc-binding motifs, the molecular mechanisms and the role of zinc in immune responses have not been elucidated. We and others have demonstrated that zinc signaling plays diverse and specific rolesin vivoandin vitroin studies using knockout mice lacking zinc transporter function and metallothionein function. In this review, we discuss the impact of zinc signaling focusing particularly on mast cell-, basophil-, and T cell-mediated inflammatory and allergic responses. We also describe zinc signaling dysregulation as a leading health problem in inflammatory disease and allergy.

VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Aikebaier Maimaiti ◽  
Amier Aili ◽  
Hureshitanmu Kuerban ◽  
Xuejun Li
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Gallic Acid ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Dependent Manner ◽  
Lung Carcinoma Cells ◽  
Induced Apoptosis ◽  
The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

scholarly journals The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiroaki Kanzaki ◽  
Tetsuhiro Chiba ◽  
Junjie Ao ◽  
Keisuke Koroki ◽  
Kengo Kanayama ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Progression Free Survival ◽  
Erk Signaling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antitumor Effects ◽  
The Impact

AbstractFGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib.

scholarly journals Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Federico Tinarelli ◽  
Elena Ivanova ◽  
Ilaria Colombi ◽  
Erica Barini ◽  
Edoardo Balzani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Neuronal Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Neuronal Cultures ◽  
Functional Impairments ◽  
After Hours ◽  
The Impact

Abstract Background DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

scholarly journals Curcumin: Could This Compound Be Useful in Pregnancy and Pregnancy-Related Complications?

Nutrients ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 3179 ◽  
Author(s):  
Tiziana Filardi ◽  
Rosaria Varì ◽  
Elisabetta Ferretti ◽  
Alessandra Zicari ◽  
Susanna Morano ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Curcuma Longa ◽  
Growth Disorders ◽  
Beneficial Effects ◽  
Toxic Agents ◽  
Health Promoting ◽  
Short And Long Term ◽  
The Impact ◽  
In Pregnancy

Curcumin, the main polyphenol contained in turmeric root (Curcuma longa), has played a significant role in medicine for centuries. The growing interest in plant-derived substances has led to increased consumption of them also in pregnancy. The pleiotropic and multi-targeting actions of curcumin have made it very attractive as a health-promoting compound. In spite of the beneficial effects observed in various chronic diseases in humans, limited and fragmentary information is currently available about curcumin’s effects on pregnancy and pregnancy-related complications. It is known that immune-metabolic alterations occurring during pregnancy have consequences on both maternal and fetal tissues, leading to short- and long-term complications. The reported anti-inflammatory, antioxidant, antitoxicant, neuroprotective, immunomodulatory, antiapoptotic, antiangiogenic, anti-hypertensive, and antidiabetic properties of curcumin appear to be encouraging, not only for the management of pregnancy-related disorders, including gestational diabetes mellitus (GDM), preeclampsia (PE), depression, preterm birth, and fetal growth disorders but also to contrast damage induced by natural and chemical toxic agents. The current review summarizes the latest data, mostly obtained from animal models and in vitro studies, on the impact of curcumin on the molecular mechanisms involved in pregnancy pathophysiology, with the aim to shed light on the possible beneficial and/or adverse effects of curcumin on pregnancy outcomes.

scholarly journals Impact of Hypothermia on Differentiation and Maturation of Neutrophils

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2393-2393
Author(s):  
Yusuke Torikoshi ◽  
Asumi Yokota ◽  
Naoka Kamio ◽  
Atsushi Sato ◽  
Tsukimi Shouji ◽  
...  
Keyword(s):  
Body Temperature ◽  
Host Defense ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Environmental Temperature ◽  
Cecal Ligation ◽  
The Body ◽  
Neutrophil Differentiation ◽  
The Impact

Abstract Accumulating evidence has suggested that low body temperature is associated with the risk of infection. Unintentional drops in the body temperature known as "accidental hypothermia" are occasionally accompanied with infections. Patients under therapeutic hypothermia for post-cardiac arrest care are also susceptible to infections. In addition, secondary hypothermia caused by severe sepsis is significantly associated with higher mortality. These observations suggest the negative impact of hypothermia on host defense. Neutrophils are continuously produced in the bone marrow (BM) and supplied to the peripheral blood (PB) or tissues, where they fight against microorganisms. In addition to the neutrophil functions, sufficient supply of neutrophils is a critical determinant of host defense. However, little is known about the impact of hypothermia on granulopoiesis, the process of neutrophil production in the BM. In this study, we investigated the changes in granulopoiesis under hypothermic conditions. We first analyzed the neutrophils in the PB of mice exposed to low environmental temperature (4 °C). Under this condition, rectal temperature of the mice significantly declined from 36.7±0.4 °C to 35.5±0.4 °C. After 72-hour exposure to the low environmental temperature, PB neutrophil counts were significantly decreased. In order to understand the reason for the decrease, we analyzed their BMs by flow cytometry. Previously we developed a unique strategy to divide cells undergoing granulopoiesis into 5 subpopulations based on the expression of c-kit and Ly6G, which reflect successive differentiation/maturation from #1 (c-kithi Ly6G-) to #5 (c-kit- Ly6Ghi) (Satake S and Hirai H et al. J Immunol, 2012). In BM cells of the mice exposed to the low environmental temperature, a significant decrease in mature neutrophils (#5) and a significant increase in cellular intermediates (#3 and #4) were observed, while total BM cell numbers were unchanged. In order to clarify whether these changes were cell-intrinsic or -extrinsic, total BM cells were cultured in vitro at either 35 °C or 37 °C in the presence of G-CSF. Flow cytometric analysis of these cultured BM cells at 72 hours revealed the increase in the intermediates (#2 to #4) and a decrease in the mature subpopulation (#5), suggesting that these alterations were cell-intrinsic phenomena. When neutrophil precursors (#1 or #2) were purified by cell sorter and subjected to in vitro culture at 35 °C for 48 hours, the number of resultant mature neutrophils (#5) were significantly less than those induced at 37 °C. These results clearly indicate that hypothermia delayed neutrophil differentiation/maturation. Interestingly, mice with sepsis induced by cecal ligation and puncture (CLP) accompanied with lower body temperature revealed significantly fewer PB granulocytes and shorter survival when compared to those mice which maintained normal body temperature after CLP. In order to understand the molecular mechanisms underlying the differentiation/maturation delay induced by hypothermia, we performed RNA sequencing of purified neutrophil precursors (#2) after 24-hour culture either at 35 °C or 37 °C. Interestingly, we found alterations in amino acid metabolic pathways and target genes of C/EBP, which is the transcription factor family required for granulopoiesis and cellular metabolism. Collectively, these results indicate hypothermia causes neutropenia through delayed neutrophil differentiation/maturation. We are currently analyzing metabolic changes to understand more precise molecular mechanisms by which hypothermia regulates granulopoiesis. This study will facilitate the understanding of host defense at low body temperature, and shed novel insight into the management of hypothermia in patients. Disclosures Kashiwagi: Takara Bio Inc.: Employment. Hirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding.

scholarly journals NEDD4 Regulates Ubiquitination and Stability of the Cell adhesion Molecule IGPR-1 via Lysosomal Pathway

2021 ◽  
Author(s):  
Linzi Sun ◽  
Razie Amraei ◽  
Nader Rahimi
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Molecular Mechanisms ◽  
E3 Ligase ◽  
Cell Surface Expression ◽  
Significant Implication ◽  
Ubiquitin E3 Ligase

ABSTRACTThe cell adhesion molecule immunoglobulin and proline-rich receptor-1 (IGPR-1) regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy. However, the molecular mechanisms governing IGPR-1 cell surface expression levels remains unknown. In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Similarly, knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Furthermore, deletion of 57 amino acids encompassing polyproline rich (PPR) motif on the C-terminus of IGPR-1 nullified the binding of NEDD4 with IGPR-1. Moreover, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulated lysosomal degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine and ammonium chloride increased IGPR-1 levels in the HEK-293 cells ectopically expressing IGPR-1 and in multiple human skin melanoma cell lines. Hence, these findings suggest that ubiquitin E3 ligase NEDD4 is a key regulator of IGPR-1 with a significant implication in the therapeutic targeting of IGPR-1.

Ligation of E-cadherin on in vitro–generated immature Langerhans-type dendritic cells inhibits their maturation

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4276-4284 ◽  
Author(s):  
Elisabeth Riedl ◽  
Johannes Stöckl ◽  
Otto Majdic ◽  
Clemens Scheinecker ◽  
Walter Knapp ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Transforming Growth Factor ◽  
Cluster Formation ◽  
Surface Expression ◽  
Transforming Growth Factor Β1 ◽  
Immature Stage ◽  
Cell Surface Expression ◽  
Regional Lymph Nodes ◽  
E Cadherin

Abstract Epithelial tissues of various organs contain immature Langerhans cell (LC)-type dendritic cells, which play key roles in immunity. LCs reside for long time periods at an immature stage in epithelia before migrating to T-cell–rich areas of regional lymph nodes to become mature interdigitating dendritic cells (DCs). LCs express the epithelial adhesion molecule E-cadherin and undergo homophilic E-cadherin adhesion with surrounding epithelial cells. Using a defined serum-free differentiation model of human CD34+hematopoietic progenitor cells, it was demonstrated that LCs generated in vitro in the presence of transforming growth factor β1 (TGF-β1) express high levels of E-cadherin and form large homotypic cell clusters. Homotypic LC clustering can be inhibited by the addition of anti–E- cadherin monoclonal antibodies (mAbs). Loss of E-cadherin adhesion of LCs by mechanical cluster disaggregation correlates with the rapid up-regulation of CD86, neo-expression of CD83, and diminished CD1a cell surface expression by LCs—specific phenotypic features of mature DCs. Antibody ligation of E-cadherin on the surfaces of immature LCs after mechanical cluster disruption strongly reduces the percentages of mature DCs. The addition of mAbs to the adhesion molecules LFA-1 or CD31 to parallel cultures similarly inhibits homotypic LC cluster formation, but, in contrast to anti–E-cadherin, these mAbs fail to inhibit DC maturation. Thus, E-cadherin engagement on immature LCs specifically inhibits the acquisition of mature DC features. E-cadherin–mediated LC maturation suppression may represent a constitutive active epithelial mechanism that prevents the uncontrolled maturation of immature LCs.

scholarly journals Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis

2017 ◽  
Vol 114 (34) ◽  
pp. E7082-E7091 ◽  
Author(s):  
Liang Xu ◽  
Wei Wang ◽  
Jiabin Wu ◽  
Ji Hyun Shin ◽  
Pengcheng Wang ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Minor Groove ◽  
Dna Lesions ◽  
Dna Alkylation ◽  
Lesion Bypass ◽  
Pol Ii ◽  
The Impact

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O2-, N3-, and O4-ethylthymidine (O2-, N3-, and O4-EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O2-EtdT and N3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O2-EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O4-EtdT lesion. For the N3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove–sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.

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