Objective:To investigate and analyze the effect of ubiquitin-editing protein A20 on monocytes activity in patients with ventilator-associated pneumonia (VAP).Methods:Twenty-four VAP patients (VAP group) and twelve healthy controls (control group) were included from the First Affiliated Hospital of Zhengzhou University between February 2019 and September 2019. Peripheral blood mononuclear cells (PBMCs) and bronchial alveolar lavage fluid (BALF) (both infection site and non-infection site) were collected from VAP patients, while PBMCs were collected from healthy controls. A20 level in CD14 + monocytes were measured. CD14 + monocytes and CD4 + T cells were purified from VAP patients. CD14 + monocytes were transfected by A20 siRNA. Transfected CD14 + monocytes were directly/indirectly co-cultured with autologous CD4 + T cells. The secretion of interferon-γ (IFN-γ) and interleukin-17 (IL-17) by CD4 + T cells was investigated. Transfected CD14 + monocytes were directly/indirectly co-cultured with NCI-H889 cells. Cytotoxicity, and cytokines/granzyme B level, and tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/Fas ligand (FasL) level was assessed. Student t test or SNK-q test was used for comparison. Results:VAP group had elevated percentage of circulating CD14 +A20 + cells than control group [(66.14±19.62)% vs. (52.52±13.71)%, P<0.05], and also had increased A20 mean fluorescence intensity (MFI) than control group [(268.0±72.56) vs. (197.4±60.01), P<0.05]. The percentage of CD14 +A20 + cells in BALF from infection site was higher than from non-infection site in VAP group [(66.14±19.62)% vs. (52.52±13.71)%, P<0.05], while A20 MFI in infection site was also up-regulated compared with non-infection site [(268.0±72.56) vs. (197.4±60.01), P<0.05]. In direct contact co-culture, A20 siRNA transfected CD14 + monocytes, which were purified from peripheral blood and BALF of VAP patients, induced elevated percentage of IFN-γ and IL-17 secreting CD4 + T cells than un-transfection or control siRNA transfection ( P<0.05). However, there were no significant differences of CD4 +IFN-γ + or CD4 +IL-17 + percentages among un-transfection, control siRNA transfection, and A20 siRNA transfection ( P>0.05). A20 siRNA transfected CD14 + monocytes, which were purified from peripheral blood and BALF of VAP patients, induced increased target cell death in both direct and indirect contact co-culture than un-transfection or control siRNA transfection ( P<0.05). Tumor necrosis factor-α, IL-6, granzyme B level and TRAIL MFI was also up-regulated ( P<0.05). There was no remarkable difference of target cell death between direct and indirect contact co-culture ( P>0.05). Conclusions:A20 was increasingly expressed in monocytes of VAP patients, and might dampen the activity of monocytes.

A novel approach for the detection of influenza virus is of paramount importance for quick diagnosis and therapy. In this study monoclonal antibody (mAb)-conjugated MNPs/AuNPs were developed to detect the H1N1 virus. MNPs and AuNPs were synthesized and loaded with mAbs. The UV-vis spectra exhibited absorbance at 528 nm. XRD revealed the presence of crystalline particles with various diffraction peaks. FTIR confirmed the occurrence of capping molecules in the synthesized NPs. NP stability was evidenced by zeta measurements. The shape and size (mean size 15 nm) of the NPs were determined using SEM and transmission electron microscopy (TEM). In this study the mAb-AuNPs produced a redshift in the absorption spectrum due to plasmon coupling. The absorption increased when H1N1 concentration increased from 0 to 5.0 ng/mL, with the detection limit being 0.05 ng/mL. The sensitivity of mAb-AuNPs was greater than that of ELISA. Since the mAb-AuNP-based colorimetric immunosensor is simple, cost-effective, and rapidly detects H1N1, it has good prospects in pharmaceuticals and clinical diagnosis.

Objective:To analyze the effect of CD100 to monocyte cytotoxicity in non-small cell lung cancer (NSCLC) patients.Methods:Thirty-five NSCLC patients and thirteen healthy controls were included from Zhengzhou Central Hospital between March 2018 and September 2018. Peripheral blood mononuclear cells (PBMC) and bronchial alveolar lavage fluid (BALF) (both tumor site and non-tumor site) was collected from NSCLC patients, while PBMC was collected from healthy controls. Monocytes were purified from PBMC and BALF. Membrane-bound CD100 (mCD100) and CD72 expression on monocytes was measured by flow cytometry. Monocytes from NSCLC patients were stimulated with recombinant human CD100, anti-CD72, matrix metalloproteinase 14(MMP14), or anti-CD100, and were co-cultured with NCI-H1882 cells for 48 h. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), granzyme A, granzyme B level in the supernatants, CD16 expression on monocytes, and percentage of target cell death was assessed. Student t test or paired t test was used for comparison. Results:There were no significant differences of peripheral CD14 + mCD100 + percentage, CD14 + CD72 + percentage, CD100 mean fluorescence intensity (MFI), CD72 MFI between NSCLC patients and healthy controls ( P>0.05). CD14 + mCD100 + percentage, CD14 + CD72 + percentage, CD100 MFI, CD72 MFI was remarkably elevated in tumor site compared with in non-tumor site in NSCLC patients ( P<0.05). There was no remarkable difference of peripheral monocytes-induced NCI-H1882 cell death between NSCLC group and control group [(13.95±3.16)% vs (13.22±2.40)%, P=0.451]. Lung-resident monocytes-induced NCI-H1882 cell death was reduced in tumor site when compared with non-tumor site [(11.61±2.81)% vs (14.19±3.57)%, P=0.008 7]. TNF-α, IL-1β, granzyme A, granzyme B level was also decreased in the supernatants of monocytes from tumor site compared with non-tumor site in NSCLC patients( P<0.05). However, there was no statistical difference of CD16 level between two groups( P=0.666). Recombinant human CD100 stimulation promoted NCI-H1882 cell death induced by monocytes from tumor site when compared with unstimulated cells ( P<0.000 1). TNF-α, IL-1β, granzyme A, granzyme B level was also increased ( P<0.05). However, Monocytes, which were pretreated with anti-CD72, induced decreased NCI-H1882 cell death and TNF-α, IL-1β, granzyme A, granzyme B secretion in response to recombinant human CD100 stimulation ( P<0.05). Recombinant human MMP14 stimulation decreased CD14 + mCD100 + percentage and increased soluble CD100 (sCD100) level. NCI-H1882 cell death and TNF-α, IL-1β, granzyme A, granzyme B level was elevated when compared with unstimulated cells ( P<0.05). Anti-CD100 administration decreased sCD100 level. NCI-H1882 cell death and TNF-α, IL-1β, granzyme A, granzyme B level was elevated when compared with MMP14 stimulated cells ( P<0.05). Conclusions:CD100 shedding was insufficient in tumor infiltrating monocytes in NSCLC patients, leading to decreased cytotoxicity. MMP14 might elevate cytotoxicity of tumor infiltrating monocytes via promoting CD100 shedding and sCD100 formation.

目的 探讨白介素(IL)-13对IL-1受体相关激酶M(IRAK-M)敲除哮喘小鼠巨噬细胞活化及Toll样受体/白介素1受体(TLR/IL-1R)途径的影响.方法 实验分组:对照组、IRAK-M敲除组、哮喘组、IRAK-M敲除+哮喘组、哮喘+抗IL-13抗体组、IRAK-M敲除+哮喘+抗IL-13抗体组.收集肺泡灌洗液(BALF)并对细胞总数、嗜酸粒细胞、中性粒细胞、淋巴细胞、巨噬细胞计数;酶联免疫吸附(ELISA)检测BALF中IL-13、干扰素-γ(IFN-γ)、IL-4表达水平;实时荧光定量PCR(qRT-PCR)检测肺组织中甘露糖受体(MR)、精氨酸酶1(Arg1)、诱导性一氧化氮合酶(iNOS)mRNA水平;蛋白免疫印迹检测肺组织中IRAK-M、IL-13、TLR4、核转录因子kappaB (NF-κB)蛋白水平.结果 与对照组相比,哮喘组各细胞数,BALF中IL-13、IL-4水平,肺组织中MR、Arg1、iNOS mRNA水平及IL-13、IRAK-M、TLR4、胞核NF-κB蛋白水平升高,差异有统计学意义(P＜0.05);BALF中IFN-γ水平,肺组织中胞浆NF-κB蛋白水平降低,差异有统计学意义(P＜0.05).分别与IRAK-M敲除+哮喘组、哮喘+抗IL-13抗体组相比,IRAK-M敲除+哮喘+抗IL-13抗体组各细胞数,BALF中IL-13、IL-4水平,肺组织中MR、Arg1、iNOS mRNA水平及IL-13、IRAK-M、TLR4、胞核NF-κB蛋白水平降低,差异有统计学意义(P＜0.05);BALF中IFN-y水平,肺组织中胞浆NF-κB蛋白水平升高,差异有统计学意义(P＜0.05).结论 抗IL-13抗体与IRAK-M敲除功能类似,均可缓解哮喘小鼠巨噬细胞活化状况.

CD100 is an immune semaphorin constitutively expressed on T-cells. Matrix metalloproteinase (MMP) is an important mediator of membrane-bound CD100 (mCD100) cleavage to generate soluble CD100 (sCD100), which has immunoregulatory activity in immune cell responses. The aim of the study was to investigate the level and role of sCD100 and mCD100 in modulating CD8+ T-cell function in non-small cell lung cancer (NSCLC). sCD100 and MMP-14 levels in the serum and bronchoalveolar lavage fluid (BALF), and mCD100 expression on peripheral and lung-resident CD8+ T-cells were analysed in NSCLC patients. The ability to induce sCD100 and the effect of MMP-14 on mCD100 shedding for the regulation of non-cytolytic and cytolytic functions of CD8+ T-cells were also analysed in direct and indirect contact co-culture systems. NSCLC patients had lower serum sCD100 and higher mCD100 levels on CD8+ T-cells compared with healthy controls. BALF from the tumour site also had decreased sCD100 and increased mCD100 on CD8+ T-cells compared with the non-tumour site. Recombinant CD100 stimulation enhanced non-cytolytic and cytolytic functions of CD8+ T-cells from NSCLC patients, whereas blockade of CD100 receptor CD72 attenuated CD8+ T-cell activity. NSCLC patients had lower MMP-14 in the serum and in BALF from the tumour site. Recombinant MMP-14 mediated mCD100 shedding from CD8+ T-cell membrane, and led to promotion of CD8+ T-cell response in NSCLC patients. Overall, decreased MMP-14 resulted in insufficient CD100 shedding, leading to suppression of peripheral and lung-resident CD8+ T-cell activity in NSCLC.

BACKGROUND/AIMS:Interleukin (IL)-35 has immunosuppressive functions in autoimmune diseases, infectious diseases, and certain cancers. However, few studies have focused on its immunoregulatory activity in non-small cell lung cancer (NSCLC). Thus, we investigated the role of IL-35 in the pathogenesis of this disease.METHODS:A total of 66 NSCLC patients and 21 healthy individuals were enrolled. IL-35 expression in peripheral blood and bronchoalveolar lavage fluid (BALF) was measured. The modulatory functions of IL-35 on purified CD4+ and CD8+ T cells from NSCLC patients were investigated in direct and indirect coculture systems with NSCLC cell lines.RESULTS:IL-35 expression was significantly increased in BALF from the tumor site, but not in the peripheral blood of NSCLC patients. IL-35 did not affect the bioactivity including proliferation, cytokine production, cell cycle, and cellular invasion of NSCLC cells. It suppressed responses from type 1 T helper (Th1) and Th17 cells but elevated the regulatory T cell response in cultured CD4+ T cells from NSCLC patients, and reduced cytokine-mediated CD4+ T cells cytotoxicity to NSCLC cells. Moreover, IL-35 also inhibited cytotoxic gene expression in CD8+ T cells from NSCLC, reducing their cytolytic and noncytolytic functions.CONCLUSION:The results of this study suggest that IL-35 contributes to the dysfunction/exhaustion of T cells and limited antitumor immune responses in NSCLC.

A water-soluble polysaccharide, designated as KOMAP, was isolated and purified from the alkaline extract of king oyster mushroom, which was composed of glucose (Glc), mannose (Man) and arabinose (Ara) in a molar ratio of 6.2:2.1:2.0. It had an average molecular weight of 2.1 x 10(4) Da. GC MS analysis revealed that KOMAP was a linear structure of the polymer with a backbone composed of beta-1,4-linked glucopyranosyl and beta-1,3,6-linked mannopyranosyl units, which was terminated with alpha-1-linked arabinofuranosyl unit at C-6 position of beta-1,3,6-linked mannopyranosyl residue along the main chain in the ratio of 3.1:1. The results in the animal experiment showed that 50, 100 and 200 mg/mL of KOMAP not only inhibited the tumor growth, but also increased relative thymus and spleen indices, LPS- or ConA-induced lymphocytes proliferation, and serum cytokine IL-2,TNF-alpha, and IFN-gamma levels, as well as the activities of NK cells and CTLs in spleen of Renca tumor-bearing mice. In summary, our data indicate that the KOMAP exerts effective immunoregulatory and anti-tumor activities in vivo. (C) 2014 Elsevier Ltd. All rights reserved.

TO THE EDITOR—We read the meta-analysis by Bastos et al recently published in Clinical Infectious Diseases [1]. Because of our interest in the association of drug susceptibility testing (DST) results for first-and second-line drugs with treatment outcomes in patients with multidrug-resistant (MDR) tuberculosis and extensively drug-resistant (XDR) tuberculosis, we carefully read the entire article. In this study, the investigators first performed a meta-analysis of 31 cohort studies of patients with MDR and XDR tuberculosis to assess the relationship between treatment outcomes and results of culture-based DST for pyrazinamide, ethambutol, and the second-line drugs. They reached an important conclusion that DST for eth-ambutol, pyrazinamide, and second-line

In this study, a water-soluble longan seed polysaccharide (WLSP), with a molecular weight of 57 kDa, was isolated from longan seed. Gas chromatography (GC) analysis showed that WLSP was composed mainly of rhamnose (Rha), mannose (Man), arabinose (Ara), galactose (Gal), and glucose (Glc), with molar ratios of 2.4:1.5:2.3:5.6:6.5. The result in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that WLSP showed a dose-dependent antiproliferative effect on the proliferation of A549 human lung cancer cells, which is consistent with the amount of lactate dehydrogenase (LDH) release from A549 cells. Prompted by this antiproliferative effect, we further examined its antiproliferative mechanism and in vivo anticancer effect. Our results showed that WLSP had the ability to cause cell cycle arrest in G1 phase, activation of caspases 3 and 9, and cleavage of poly[ADP (ribose)] polymerase (PARP) in A549 cells. The result of this in vivo study showed that WLSP could suppress the growth of xenograft A549 tumors and induce apoptosis. Taken together, these results indicate that WLSP exert an anticancer effect in vitro and in vivo and may be useful for the prevention of lung tumorigenesis.

Objective To discuss the effects and mechanism of MAGEA10 expression in human lung cancer A549 cells influenced by Viili polysaccharides.Methods MTT was used to study the survival impact of non small cell lung cancer(NSCLC) A549 with viili polysaccharides in different concentration at 24 h,48 h and 72 h.qRT-PCR was used to detect the relative expression changes of MAGEA10 mRNA with Viili polysaccharides in different concentration of 10 mg/L,25 mg/L and 50 mg/L compared with those of the normal A549 cells.And Western blot was adopted to detect the antigen expression of MAGEA10 after stimulation of A549 with Viili polysaccharides in different concentration of 10 mg/L,25 mg/L and 50 mg/L.Through the expression of MAGEA10 mRNA and antigen,we discussed the effects and possible mechanism of MAGEA10 expression made by Viili polysaccharides.Results(1) Viili polysaccharides inhibited cell proliferation of A549 and its concentration was negatively related to cell survival within 0 mg/L-50 mg/L and the result was most remarkable at 48 h.(2) Compared with the blank group,the relative expression of MAGEA10 mRNA elevated at the concentration of 50 mg/L.(3) Compared with the blank group,the protein expression of MAGEA10 elevated,at the concentration of 50 mg/L.Conclusion Viili inhibited NSCLC A549 cell proliferation,but the increase of the gene transcription and translation levels of MAGEA10 in A549 cell needs further exploration for those two parts might be interdependent.This study aims to indicate that viili is possible to promote non specific immunity,and increase possibility of cancer cell recognition by CTLs,and possibly initiate apoptotic process of cancer cells if same epitope of MAGEA10 and corresponding TCR encounters.