Danielsenhowe5439

Objective To compare the consistency of immunohistochemical staining between the two commercial secondary antibodies. Methods Eighteen common immunohistochemical primary antibodies were selected and positive and negative controls were set up according to the recommendations from the AD Hoc Committee of International Experts. Under the same experimental conditions, the DAKO automatic immunohistochemical staining platform was used to test two different secondary antibodies for immunohistochemical staining. The standard group for the secondary antibody was provided by the DAKO polymer system (DAKO EnVision FLEX, High pH), and the experimental group for the secondary antibody was provided by the Power-StainTM kit (Power-StainTM 1.0 Poly HRP DAB Kit for Mouse+Rabbit). Subsequently, the images were captured. A single-blind, positioning, qualitative and semi-quantitative scoring criterion was used for describing the positive stains by the experienced pathologist. Absorbance corrected values, measured area values and positive integral absorbance were detected by the digital pathology quantitative measurement in the same areas from the two groups. Then, the mean absorbance was calculated. Results The stains of all the samples from the two groups showed accurate location and consistent qualitative evaluation. No significant differences were found between the two groups in all the semi-quantitative scoring, including stain intensity, positive stain percentages and mean absorbance. Conclusion The two commercial secondary antibodies have strong consistency in the immunohistochemical staining.Objective To investigate the role of phosphatidylinositol 3-kinase (PI3K) isoforms in type III receptor tyrosine kinase KIT mutation-mediated signaling and cell proliferation. Methods The wild-type KIT and the common KIT mutations V560D and W557K558del in gastrointestinal stromal tumors (GIST) were stably expressed in BaF3 cells. The cells were treated with PI3K isoforms PI3Kα, PI3Kβ and PI3Kδ specific inhibitors or pan PI3K inhibitor. The activation of KIT and its downstream signals was detected by immunoprecipitation and Western blot analysis. GIST-T1 cells were treated with the same drug, and the activation of KIT and its downstream signals was also detected by immunoprecipitation and Western blot analysis, and cell proliferation and apoptosis were detected by MTT assay and flow cytometry, respectively. Results Compared with the controls, in BaF3 cells expressing wild-type KIT and its mutants, the activation of KIT and its downstream signaling molecules AKT and ERK was inhibited the most by PI3Kδ specific inhibitor, followed by the specific inhibitors of PI3Kα and PI3Kβ subtype. In GIST-T1 cells, the activation of KIT and its downstream signals was inhibited the most by PI3Kβ specific inhibitor, followed by PI3Kδ and PI3Kα specific inhibitors. Conclusion In BaF3 cells, PI3Kδ subtype plays a major role in KIT activation and its downstream signal transduction, while in GIST-T1 cells, PI3Kβ subtype plays a major role in KIT activation and its downstream signal transduction. These results indicate that PI3K isoforms play different roles in KIT mutation-mediated cell transformation depending on the host cells.Objective To investigate the effect of ephrin type-A receptor 2 (EphA2) on the expression of inflammatory cytokines in airway epithelial cells induced by house dust mite extract (HDM) and the underlying mechanism. Methods The cell model of EphA2 knockdown was established by transfection of EphA2 siRNA into airway cell line 16HBE cells. After the 16HBE cells were stimulated with HDM, the mRNA levels of EphA2, interleukin 6 (IL-6) and IL-8 were determined by real-time quantitative PCR (qPCR), and the protein levels of IL-6, IL-8, IL-17A, IL-17F and tumor necrosis factor-α (TNF-α) were measured by cytometric bead array (CBA). Western blotting was used to analyze the protein expression of EphA2, phosphorylated EphA2 (p-EphA2), signal transducer and activator of transcription (STAT3), phosphorylated STAT3 (p-STAT3), p38 mitogen-activated protein kinases (p38 MAPK), phosphorylated p38 MAPK (p-p38 MAPK), nuclear factor κ-B p65 (NF-κB p65) and phosphorylated NF-κB p65 (p-NF-κB p65). Then, in the 16HEB cells stimulated by STAT3 inhibitor Stattic or p38 MAPK inhibitor SB203580 in combination with HDM, the mRNA and protein expression levels of IL-6 and IL-8 were detected by qPCR and CBA. Results Knockdown of EphA2 significantly inhibited the expression of IL-6 and IL-8 in HDM-induced 16HBE, and reduced the total protein and phosphorylated levels of STAT3 and p38 MAPK, but had no significant effect on the total protein and phosphorylated levels of NF-κB p65. After stattic inhibited the expression and activation of STAT3, the mRNA and protein levels of IL-6 and IL-8 significantly decreased in HDM-induced 16HBE cells. Interestingly, while SB203580 inhibited the activation of p38 MAPK signaling pathway, it only inhibited the mRNA levels of IL-6 and IL-8 in HDM-induced 16HBE cells, but had no effect on their protein levels. Conclusion HDM can induce the expression of IL-6 and IL-8 in 16HBE cells to participate in airway inflammation by activating the EphA2-STAT3/p38 MAPK pathway.Objective To study the therapeutic effect of rapamycin (RAPA) on experimental autoimmune myasthenia gravis (EAMG) rats and to explore the related immune mechanisms. Methods The mouse-derived acetylcholine receptor alpha subunit 97-116 peptide (R97-116) was used to immunize Lewis rats to establish an EAMG rat model. The rats were randomly divided into three groups complete Freund's adjuvant control group (CFA group), EAMG model control group, and RAPA treatment group [1 mg/(kg.d)]. The Lennon muscle strength scoring scale was used to evaluate rats' clinical symptoms in each group once every two days, and their body mass was recorded. selleck kinase inhibitor ELISA was performed to detect the level of anti-R97-116 antibodies in the peripheral blood of rats. Flow cytometry was used to detect the numbers of Th17 cells and regulatory T cells (Tregs) in rat splenocytes. Splenocytes were stimulated with 5 μg/mL concanavalin A (ConA), 10 μg/mL R97-116 and RPMI1640 medium, and the proliferation activity of rat splenocytes was tested by CCK-8 assay.