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Formononetin-inhibited IgE-mediated mast cell activation and attenuated IgE/Ag-induced allergic inflammation by suppressing USP5 or USP13 expression and increasing proteasome-mediated FcεRIγ degradation.

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PDF file - 927K, The effect of Aurora kinases inhibitors on HL-60 and KG1a cell lines.

PDF file - 1715K, Aurora kinases inhibitors and mTOR inhibitors had synergistic effect on the expression of cleaved caspase 3 and p62.

PDF file - 1113K, Aurora kinases inhibitors had no effect with mTOR inhibitors on PBMCs.

PDF file - 1100K, Combination of Aurora kinases inhibitors and 2DG had minimal effect on PBMCs.

PDF file - 1470K, Aurora kinases inhibitors had synergistic effect with mTOR inhibitors on the glycolytic metabolism of U937 cells.

PDF file - 2735K, Aurora kinases inhibitors had synergistic effect with mTOR inhibitors on the proliferation of U937 and NB4 cells.

PDF file - 1081K, Cell viability detection in Aurora kinase A knockdown-U937 cells.

We aimed to validate the Children's Empathy Quotient (EQ-C) and Systemizing Quotient (SQ-C) in Mainland China, which can reflect the profiles of empathizing and systemizing, and describing specific characteristics of autism spectrum disorder (ASD) and gender-typical behaviors in general population. A total of 800 typically developing (TD) children, aged 4–12 years was recruited initially with whose parents/guardians complete the measurements, and 782 TD children who met inclusion criteria were finally included. A 23-item three-factor EQ-C and a 22-item four-factor SQ-C was developed with good internal consistency (Omega total values of 0.87 and 0.86) and test–retest reliability (Pearson correlation coefficients of 0.82 and 0.69). In TD children, girls scored significantly higher on EQ-C (31.4 ± 7.8 vs. 28.2 ± 7.7) but there were no gender differences in SQ-C scores. TD children showed different cognitive styles (empathizing-dominant for girls with 42.6% identified as Type E; systemizing-dominant for boys with 40.7% identified as Type S). A further sample of 222 children with ASD indicated that they scored lower on EQ/SQ-C compared to TD children (13.2 ± 5.1 vs. 29.7 ± 7.9, 12.4 ± 5.8 vs. 23.5 ± 8.3) and were generally systemizing-dominant (Type S: 50.8% for boys and 64.0% for girls). Autistic children scored higher on the SQ-C in those without intellectual disability and with higher paternal education level and family income (14.2 ± 6.1 vs. 10.9 ± 5.0, 13.3 ± 6.2 vs. 11.5 ± 5.1, 13.7 ± 5.6 vs. 11.9 ± 5.8), while there were no differences in the EQ-C. This study indicated good reliability and validity of the Chinese version of EQ/SQ-C, which can be used in Chinese children with and without ASD. Lay Summary: We developed the Chinese version of the Children's Empathy Quotient (EQ-C) and Systemizing Quotient (SQ-C) in 782 typically developing (TD) children aged 4–12 years in Mainland China, yielding a 23-item, 3-factor EQ-C and a 22-item, 4-factor SQ-C with good psychometric properties. In TD children, we found gender difference only in scores of EQ-C. Further analyses of 222 autistic children indicated that differences were found in scores of SQ-C when considering their gender, intelligence and socio-economic status.

The mechanisms and origin of the chemoselectivity of Cu(i)- and Au(i)-catalyzed oxidation of diynes for the divergent syntheses of two different N-heterocycles, substituted pyrroles and dihydroindeno[1,2-c]pyrrol-3(2H)-ones, were elucidated using density functional theory.

The Au(i)-catalyzed intermolecular annulation of 2-(1-alkynyl)-2-alken-1-one 1a with N-allenamide 2a is driven by the higher reactivity of the gold all-carbon 1,3-dipole species A than the gold allylic carbocation B, rather than the easier access to A.

The flavoring agent dihydrocoumarin suppressed IgE-induced mast cell activation via down-regulating MAPK, NF-κB and AP-1 pathways. Dihydrocoumarin may represent a novel MC-suppressing strategy for the treatment of IgE-mediated allergic diseases.

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic immunological response to Aspergillus fumigatus (Af) exposure, which induces a strong T helper 2 (Th2) response via mechanisms that have yet to be elucidated. The aim of the present study was to investigate the hypothesis that T2 ribonuclease from Af (Af RNASET2) induces M2‑type macrophage polarization to produce a T helper 2 (Th2) immune response. Recombinant Af RNASET2 (rAf RNASET2) was expressed and purified in a prokaryotic pET system and BALB/c mice were immunized with rAf RNASET2 for in vivo analyses. Expression levels of M2 polarization factors were evaluated in RAW264.7 macrophages treated with rAf RNASET2 in vitro using flow cytometry, reverse transcription‑quantitative PCR, and western blot analysis. The results predicted that the mature Af RNASET2 protein (382 amino acids; GenBank no. MN593022) contained two conserved amino acid sequence (CAS) domains, termed CAS‑1 and CAS‑2, which are also characteristic of the RNASET2 family proteins. The protein expression levels of the Th2‑related cytokines interleukin (IL)‑4, IL‑10, and IL‑13 were upregulated in mice immunized with rAf RNASET2. RAW264.7 macrophages treated with rAf RNASET2 showed increased mRNA expression levels of M2 factors [arginase 1, Il‑10, and Il‑13]; however, there was no difference in cells treated with rAf RNASET2 that had been inactivated with a ribonuclease inhibitor (RNasin). The protein expression levels of IL‑10 in macrophage culture supernatant were also increased following stimulation with rAf RNASET2. In addition, rAf RNASET2 upregulated the expression of phosphorylated mitogen activated protein kinases (MAPKs) in RAW264.7 cells, whereas MAPK inhibitors attenuated rAf RNASET2‑induced IL‑10 expression in RAW264.7 cells. In conclusion, the present study reveals that high rAf RNASET2 activity is required for rAf RNASET2‑induced M2 polarization of macrophages and suggests an important immune regulatory role for Af RNASET2 in ABPA pathogenesis.

This study aims to validate the simplified Chinese version of the Social Communication Questionnaire (SCQ) in children aged 2-12 years from both general and clinical populations. We recruited 819 Chinese children in this study, including 505 typically developing (TD) children, 202 children with autism spectrum disorder (ASD) and 112 children with non-ASD neurodevelopmental disorders. All the children's parents completed the simplified Chinese version of the SCQ and all children with ASD were additionally assessed for intelligence and the Childhood Autism Rating Scale to confirm their diagnosis. We have developed a 40-item, 4-factor structure of SCQ with two domains (social communication and social interaction; and restricted, repetitive, and stereotyped patterns of behavior), which showed adequate goodness of fit (comparative fit index [CFI] = 0.96, Tucker-Lewis index [TLI] = 0.95, standardized root mean squared residual [SRMR] = 0.07, root mean square error of approximation [RMSEA] = 0.05), with good internal consistency (Cronbach's alpha = 0.92). We have provided different cut-offs to distinguish ASD cases from TD children (11 for children under 4 years [sensitivity: 0.96, specificity: 0.95], 12 for children 4 years and above [sensitivity: 0.93, specificity: 0.98]) or children with other neurodevelopmental disorders (14 [sensitivity: 0.85, specificity: 0.88]). Through this large sample validation, we confirmed that the simplified Chinese version of the SCQ could be used for children aged 2-12 years with relatively good psychometric properties. LAY SUMMARY: We aimed to develop the simplified Chinese version of the Social Communication Questionnaire (SCQ) for Chinese children aged 2-12 years as a screening tool to identified potential risk of autism spectrum disorder (ASD). We have developed a 40-item, 4-factor structure of SCQ with two domains, which showed adequate goodness of fit and good psychometric properties. We also provided different cut-offs to identify ASD cases in general or clinical populations.

During an exploration of plant pathogens in vegetables occuring in Zhejiang province, China, a novel fungal species, was found. Three strains ZJUP0033-4, ZJUP0038-3 and ZJUP0132 were isolated from black round lesions in the stems and leaves of Amaranthus sp. Phylogenetic analyses based on sequences from four genes including rDNA internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α), histone (HIS) and β-tubulin (TUB) indicated that D. sinensis clustered in a distinct clade closely related to D. neoarctii, D. angelicae, D. subordinaria, D. arctii, D. cuppatea, D. lusitanicae, D. novem, D. infecunda, D. ganjae and D. manihotia. Morphologically, D. sinensis is distinguished by brown, scattered, globose pycnidia and ellipsoid alpha conidia with bi- to multiguttulate.

Accurate house dust mite (HDM) genome and transcriptome data would promote our understanding of HDM allergens. We sought to assemble chromosome-level genome and precise transcriptome profiling of Dermatophagoides farinae and identify novel allergens. In this study, genetic material extracted from HDM bodies and eggs were sequenced. Short-reads from next generation sequencing (NGS) and long-reads from PacBio/Nanopore sequencing were used to construct the D. farinae nuclear genome, transcriptome, and mitochondrial genome. The candidate homologs were screened through aligning our assembled transcriptome data with amino acid sequences in the WHO/IUIS database. Our results showed that compared with the D. farinae draft genome, bacterial DNA content in the presently developed sequencing reads was dramatically reduced (from 22.9888% to 1.5585%), genome size was corrected (from 53.55 Mb to 58.77 Mb), and the contig N50 was increased (from 8.54 kb to 9365.49 kb). The assembled genome has 10 contigs with minimal microbial contamination, 33 canonical allergens and 2 novel allergens. Eight homologs (≥50% homology) were cloned; 2 bound HDM allergic-sera and were identified as allergens (Der f 37 and Der f 39). In conclusion, a chromosome-level genome, transcriptome and mitochondrial genome of D. farinae was generated to support allergen identification and development of diagnostics and immunotherapeutic vaccines.

Mast cells (MCs) initiate and maintain allergic inflammation. Upon being stimulated with immunoglobulin (Ig)E and antigen (Ag), MCs exhibit FceRI (high-affinity IgE) receptor-mediated degranulation, cytokine secretion, and increased focal adhesion kinase (FAK) activity. The aims of this study were to examine mechanisms of FAK regulation in IgE-mediated MC activation and the effects of FAK inhibition on MC-mediated allergic responses. FAK activity was manipulated with short hairpin RNA (shRNA) knockdown, FAK overexpression, and the FAK inhibitor PF-431396 (PF). Gene expression and kinase activation were analyzed with quantitative molecular biology assays. PF effects were tested in the passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and allergic conjunctivitis (AC) mouse models. Our results showed that FAK overexpression increased IgE-mediated degranulation and reduced the dexamethasone inhibitory effect on MCs activation. The FAK inhibitor PF diminished MC release of β-hexosaminidase (β-hex), histamine, and inflammatory cytokines, via a mechanism that involves MAPK and NF-κB signaling pathways. CaMKII was identified as a robust FAK-associating protein. Inhibition of CaMKII activation by KN-93 suppressed FAK activity and its downstream pathway. PF attenuated inflammatory responses in our PCA and ASA models, and relieved signs of allergic disease in AC model mice. In conclusions, MC degranulation and production of inflammatory mediators in allergic disease may be consequent to FceRI crosslinking inducing CaMKII-mediated activation of FAK activity. FAK inhibition may represent a new MC-suppressing treatment strategy for the treatment of allergic diseases.

Previously, a ubiquinol-cytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HDM) species Dermatophagoides farinae (Der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein Der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in Der f and human UQCRB was performed. Four different recombinant Der f 24 and hybrid proteins formed by integrating Der f and human UQCRB sequences were expressed in Escherichia coli, purified using Ni-NTA resins, and IgE-binding activity was determined using IgE-western blotting and enzyme-linked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgE-dot blotting and IgE-ELISA with synthetic polypeptides and HDM-allergic sera. Three-dimensional (3D) structural modeling was used to analyze the position of the immuno-dominant IgE epitope. The amino acid sequence homology between Der f 24 and the human UQCRB protein was determined to be 39.34%. IgE-ELISA and western blot analysis showed that all of the Der f-human UQCRB hybrid proteins generated, except for the one lacking 59 residues of the N-terminal region of Der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the N-terminal reacted with IgEs from HDM-allergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32-aa N-terminal region of Der f 24 was localized to a structural protrusion, which may facilitate specific IgE-binding. These results indicate that the immunodominant IgE epitope of Der f 24 is located mainly in a 32-residue region of the N-terminus. These findings may inform the mechanisms of HDM allergy sensitization and allergy immunotherapy development.

The present study explored the cooperative effect of both alanine (Ala) and gentamicin (Gent) on metabolic mechanisms by which exogenous Ala potentiates Gent to kill antibiotic-resistant Vibrio alginolyticus. To test this, GC-MS-based metabolomics was used to characterize Ala-, Gent- and both-induced metabolic profiles, identifying nitric oxide (NO) production pathway as the most key clue to understand metabolic mechanisms. Gent, Ala and both led to low, lower and lowest activity of total nitric oxide synthase (tNOS) and level of NO, respectively. NOS promoter L-arginine and inhibitor NG-Monomethyl-L-arginine inhibited and promoted the killing, respectively, with the elevation and decrease of NOS activity and NO level. The present study further showed that CysJ is the enzyme-producing NO in V. alginolyticus. These results indicate that the cooperative effect of Ala and Gent causes the lowest NO, which plays a key role in Ala-potentiated Gent-mediated killing.

Layer assignment, which is a part of global routing, aims to assign the flat nets to 3D distribution and decrease the via count without violating edge or via constraint. Negotiation-based method is used to obtain good result but bring too much time complexity, this work presents a net partition technology to reduce the problem complexity, and develops enhanced heuristic methods of effective layer shifting and rip-up procedure which could control the via count without multiple iteration process. Experimental results show that this method could bring inspiring improvement than LAVA.

A severe immune response in patients with coronavirus disease 2019 (COVID-19) can cause a potentially lethal unconstrained inflammatory cytokine storm, known as cytokine release syndrome (CRS). The present study provides an overview of the biology underlying CRS and how targeted inhibition of interleukin (IL)-6 signaling may improve outcomes and the survival of patients suffering from COVID-19. Preliminary clinical results have indicated that antagonism of the IL-6 receptor (IL-6R), including with the FDA-approved humanized monoclonal antibody tocilizumab, can improve the outcomes of patients with severe or critical COVID-19 while maintaining a good safety profile. The available clinical data support the expansion of clinical trials using IL-6R targeting inhibitors for severe and critical COVID-19 treatment.

Background Chronic active Epstein-Barr virus infection (CAEBV) is a rare disease, which is difficult to be differentiated from inflammatory bowel disease (IBD). To cause the attention, we present twelve cases of CAEBV in immunocompetent patients with gastrointestinal tract involvement. Methods Twelve patients who fulfilled the diagnostic criteria of CAEBV were enrolled in this retrospective study. The control group was consisted of twenty-four IBD patients with EBV-DNA value increased in peripheral blood. The clinicopathologic and endoscopic characteristics were reviewed and analyzed. Results The major clinical presentations of CAEBV patients were intermittent fever (100%), hepatomegaly/splenomegaly (58%), lymphadenopathy (50%), diarrhea (50%) and hematochezia (50%). Compared with IBD patients, the incidence of intermittent fever and increased level of ferritin were significantly higher among CAEBV patients. The median values for EBV detected in peripheral blood were significantly higher in CAEBV group (1.42*10^6 copies/μg) than in IBD group (3.2*10^3 copies/μg, p Conclusions Symptoms such as intermittent fever, increased level of ferritin and atypical endoscopic findings could be a sign for CAEBV. Early detections of EBV-DNA in serum and EBV-encoded small nuclear RNA (EBER) by in situ hybridization in intestinal tissue are essential for differential diagnosis between CAEBV and IBD.

Background Mast cells (MCs) mediate a key role in allergic diseases. Detailed studies of how the neuroleptic drug pimozide affects MC activity are lacking. The aim of this study was to investigate pimozide inhibition of immunoglobulin E (IgE)-mediated MC activation and MC-mediated allergic responses. Method MCs were stimulated with anti-dinitrophenyl (DNP) IgE antibodies and DNP-horse serum albumin (HSA) antigen (Ag), and anti-allergic pimozide effects were detected by measuring β-hexosaminidase levels. Morphological changes were observed histologically. In vivo pimozide effects were assessed in passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-sensitized active systemic anaphylaxis mouse (ASA) model experiments. Levels of phosphorylated (p-) SYK (spleen tyrosine kinase) and MAPKs (mitogen-activated protein kinases) were detected in western blots. Results We found that pimozide inhibited MC degranulation, reduced MC release of β-hexosaminidase dose-dependently in activated RBL-2H3 (IC50: 13.52 μM) and bone marrow derived MC (BMMC) (IC50: 42.42 μM), and reduced MC morphological changes. The IgE/Ag-induced migration effect was suppressed by pimozide treatment dose-dependently. Pimozide down-regulated IgE/Ag-induced phosphorylation of SYK and MAPKs in activated MCs. Moreover, pimozide attenuated allergic reactions in PCA and ASA model mice, and decreased MC populations among splenic cells. Conclusions The antipsychotic drug pimozide can suppress IgE-mediated MC activation in vitro and in vivo and should be considered for repurposing to suppress MC-mediated diseases.

Aim This study explored chlamydia trachomatis (CT), ureaplasma urealyticum (UU) and/or neisseria gonorrhoeae (NG) in 5893 women with urinary tract infections (UTIs) in Shanghai. Methods From January 2009 to December 2014, 5893 women with UTIs in Shanghai were selected to undergo CT, UU and NG detection. Baseline characteristics including age, education level, occupation, reproductive history, sexual behavior and contraceptive method were obtained for epidemiological analysis. Results The total CT, UU and/or NG infection rate in the urine samples of 5893 patients was 50.69% (2987/5893), while the infection rate in vaginal secretion samples was 56.22% (3313/5893). The two detection methods were consistent. Patients aged 21-30, service personnel and unemployed persons had the highest rates of CT, UU and/or NG infection, while patients with higher education levels exhibited lower rates. As the number of previous pregnancies, natural births, abortions, sexual partners and the frequency of sexual intercourse increased, the rates of CT, UU and/or NG infection were elevated. Sexual intercourse during the menstruation period, a lack of cleaning before sexual intercourse and the use of intrauterine devices could all lead to an increased rate of CT, UU and/or NG infection. Conclusions These data revealed that the rate of CT, UU and/or NG infection may be associated with age, education level, occupation, reproductive history, sexual behavior and type of contraceptive method in female patients with UTI in Shanghai.

The key parameters of vertical AlN Schottky barrier diodes (SBDs) with variable drift layer thickness (DLT) and drift layer concentration (DLC) are investigated. The specific on-resistance (R on,sp) decreased to 0.5 mΩ ⋅ cm2 and the breakdown voltage (V BR) decreased from 3.4 kV to 1.1 kV by changing the DLC from 1015 cm−3 to 3 × 1016 cm−3. The V BR increases from 1.5 kV to 3.4 kV and the R on,sp also increases to 12.64 mΩ ⋅ cm2 by increasing DLT from 4-μm to 11-μm. The V BR enhancement results from the increase of depletion region extension. The Baliga’s figure of merit (BFOM) of 3.8 GW/cm2 was obtained in the structure of 11-μm DLT and 1016 cm−3 DLC without FP. When DLT or DLC is variable, the consideration of the value of BFOM is essential. In this paper, we also present the vertical AlN SBD with a field plate (FP), which decreases the crowding of electric field in electrode edge. All the key parameters were optimized by simulating based on Silvaco-ATLAS.

Osteolytic bone lesions can be a consequence of leukemic bone infiltration or focal bone destruction by inflammatory factors released from leukemic cells. Destructive bone lesions have a negative impact on the quality of life of leukemia patients, causing unbearable pain and, in some cases, limb paralysis. However, the mechanism, by which leukemic cells produce destructive bone lesions, and the effect of therapeutics on osteolytic lesions have not been fully elucidated yet and, thus, stand to benefit from anin vivomodel. To that end, HL-60 cells were transformed by retrovirus-mediated constitutively active (CA) STAT5 expression and injected into nonobese diabetic (NOD)/SCID mice via the tail vein. After three weeks, lumbar spines were subjected to histocytometric analysis. Xenograft mice developed hind limb paralysis in 2-3 weeks, which was consistent with the consequences of spinal bone destruction by extramedullary invasion of leukemia cells. Thein vivomodel will improve the understanding and treatment of osteolytic bone lesions caused by myeloid leukemic cells.

The antipsychotic drug pimozide has been found to exhibit anticancer effects. Previously, it was demonstrated that pimozide inhibits hepatocellular carcinoma (HCC) cell growth, but its pharmacodynamic characteristics remain unclear. The aim of the present study was to investigate the reversibility and mechanism of the ability of pimozide to inhibit cell proliferation in liver cancer. Cell viability was determined by Cell Counting Kit‑8 and colony formation assay. The cell cycle distribution was analyzed by flow cytometry with Ki‑67 and PI staining. ROS production of HCC cells was detected with DCFH‑DA and inhibited with NAC treatment. Western blot assay was performed to detect the expression of related signaling molecules in HCC cells. Our results showed that pimozide promoted G0/G1 phase arrest in HCC cell lines without significant cell death. Its anti‑proliferative effects on HCC cells were reversible, consistent with involvement of cell quiescence and reactive oxygen species (ROS) production. Pimozide enhanced inhibition of HCC cell proliferation by sorafenib. In conclusion, elucidation of pimozide's reversible proliferation inhibition in liver cancer and additive activity with a well‑established anticancer drug warrants further exploration of the potential of pimozide as an adjuvant anticancer therapy.

Background and purpose Mast cells are important in allergic reactions. Here, we assessed the anti-allergic effects of the anti-cancer drug tozasertib specifically regarding regulatory effects on mast cell activation. Experimental approach Tozasertib effects on mast cell degranulation were determined by measuring β-hexosaminidase and histamine release and by assessing morphological changes in RBL-2H3 and mouse bone marrow-derived mast cells (BMMCs) stimulated with mouse anti-dinitrophenyl (DNP)-IgE/DNP-human serum albumin or human LAD2 cells activated with phorbol-12-myristate 13-acetate plus calcium ionophore (PMACI). Western blots were performed to detect the expression of molecules involved in NF-κB, MAPK, and Aurora kinase signalling. in vivo anti-allergic effects of tozasertib were determined in the murine IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) models. Key results Tozasertib treatment decreased high-affinity IgE receptor (FceRI) or PMACI-mediated degranulation in RBL-2H3 cells and in BMMCs or LAD2 cells as shown by β-hexosaminidase or histamine levels. Similarly, tozasertib prevented morphological changes in mast cells, such as particle release and F-actin reorganization. In addition, tozasertib markedly decreased expression of phosphorylated (p)-NF-κB p65, p-Erk1/2, p-p38, and p-Aurora A/B, indicating that tozasertib can inhibit the signalling pathway mediating mast cell activation. Tozasertib attenuated IgE/Ag-induced PCA dose-dependently, as shown by reduced Evans blue staining. Similarly, tozasertib reduced body temperature levels and serum histamine levels in OVA-challenged ASA mice. Conclusion and implications The Aurora kinase inhibitor tozasertib suppressed mast cell activation in vitro and in vivo. Tozasertib may be a potential drug, targeting mast cell activation, to treat allergic diseases or mastocytosis.

Background Mast cell activation causes degranulation and release of cytokines, thereby promoting inflammation. The aim of this study was to investigate the inhibitory effect of CDK4/6 inhibition on mast cell activation in vitro and in vivo. Methods RBL-2H3 rat basophilic leukemia cells (BLCs) and mouse bone marrow-derived mast cells (BMMCs) were sensitized with anti-dinitrophenol (DNP) immunoglobulin (Ig)E antibodies, stimulated with DNP-human serum albumin (HSA) antigens, and treated with the CDK4/6 inhibitor palbociclib. Histological stains were applied to reveal cytomorphological changes. Murine IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) models were used to examine palbociclib effects on allergic reactions in vivo. Western blots were performed to detect the expression of cell signaling molecules associated with mast cell activation. Results Activated BLCs and BMMCs released copious granule-related mediators (histamine and β-hexosaminidase), which was reduced by palbociclib in a concentration-dependent manner. Palbociclib inhibited expression of the mast cell activation marker CD63 in activated BLCs and inhibited granule release (visualized with toluidine blue staining) while preventing morphological changes, (elongated shape maintained) and filamentous actin (F-actin) reorganization. Palbociclib suppressed molecular Lyn and/or mitogen-activated protein kinase (MAPK) signaling associated with mast cell activation in stimulated BLCs and attenuated allergic reactions in PCA mice dose dependently. Palbociclib attenuated body temperature reduction and diminished serum histamine levels in ovalbumin OVA-challenged ASA mice. Conclusion Palbociclib suppresses IgE-mediated mast cell activation in vitro and in vivo, suggesting that it may be developed into a therapy for mast cell-mediated allergic diseases via inhibition of mast cell degranulation.

Background The identification of house dust mite (HDM) allergens and epitopes is important for allergy diagnosis and treatment. We sought to identify the Dermatophagoides pteronyssinus group 24 allergen (Der p 24) and to identify its immunodominant IgE epitope(s). Methods Der p 24 cDNA was cloned and expressed in a pET expression system. The IgE binding activity of purified recombinant (r)Der p 24 was evaluated by western blotting. Truncated Der p 24 proteins and overlapping synthetic polypeptides were subjected to IgE binding assays. Balb/c mice were immunized to investigate IgE epitope induction of IgE production. IgE binding of the 32 N-terminal residues of Der p 24 was compared to other Der p epitopes in enzyme-linked immunosorbent assays and dot blot assays. Human skin prick tests (SPTs) were performed. Results We cloned and expressed Der p 24 cDNA (GenBank accession no. KP893174.1). HDM allergic sera bound rDer p 24 in vitro and 5/10 HDM allergic patients (50%) had positive SPT reactions to rDer p 24. The immunodominant IgE epitope of Der p 24 was localized to the N-terminal 32-residue region, which produced a high specific IgE antibody titer in vivo and promoted mast cell β-hexosaminidase release. The IgE binding activity this N-terminal epitope of Der p 24 was stronger than that of Der p 1 or Der p 2 IgE epitopes. Conclusions We identified Der p 24 as a major HDM allergen with strong IgE binding activity via an immunodominant IgE epitope in the N-terminal 32-residue region, which triggers IgE production in vivo. The identified Der p 24 epitope may support HDM allergy diagnosis and treatment.