Interleukin-8 Production in Primary Cultures of Human Gastric Epithelial Cells Induced by Helicobacter pylori

Interleukin-8 (IL-8) production by the gastricmucosa is increased in Helicobacter pylori infection.Previous studies indicated that H. pylori induces IL-8synthesis in cancer cell lines, and the ability of H. pylori to stimulate IL-8 production issupposed to be associated with cag A and other cagpathogenicity island genes, including pic B gene. In thepresent study, we investigated the induction of IL-8 in primary cultures of normal human gastricepithelial cells to elucidate the IL-8 induction by wildtype strains and by the pic B knockout strain. Humangastric epithelial cells were obtained from surgically resected specimens from four patients. Three H.pylori strains (TN2F4; type 1 clinical isolate, TN2F4m1;isogenic pic B mutant of TN2F4, Tx30a; type 2 strain)were cocultured with the normal gastric epithelial cells or the transformed MKN-28. IL-8 levels inculture medium were determined by enzyme immunoassay.Human gastric epithelial cells produced IL-8 at a 10 -50times higher level than MKN-28 did when cocultured with TN2F4. The mutant TN2F4m1 induced IL-8 atsignificantly lower levels than the parent strain. Cellsfrom four patients behaved similarly on IL-8 production.The results of the present study demonstrated the induction of IL-8 in normal gastricepithelial cells, suggesting that pic B gene product mayplay an essential role in vivo.     

深低温暴露诱导的人肾小管上皮细胞自噬体数量特征分析

目的 通过构建HK2-GFP-LC3稳定细胞系,研究20℃深低温暴露诱导的自噬体数量特征,为明确自噬低温暴露响应和自噬相关药物高通量筛选提供依据.方法 构建稳定表达GFP-LC3融合蛋白的HK2-GFP-LC3细胞系,建立HK2-GFP-LC3细胞体外20℃低温暴露自噬分析模型,用荧光显微镜进行自噬体计数,进行低温暴露...     

β2-Agonist-Elevated Stress Response in Human Bronchial Epithelial Cells in Vivo and in Vitro

Recent studies report high baseline levels of stress (heat shock) proteins in bronchial epithelial cells from asthmatic individuals. The promoter of the gene encoding the 72-kDa heat shock protein has an element responsive to cAMP, which may be affected by β-agonists. This study examined stress protein levels in subjects enrolled in a segmental lung allergen challenge study to determine whether β-agonist medication could contribute to a stress response. Subjects were divided on the basis of no premedication (n= 17), metered dose inhalations of albuterol (n= 24), or placebo inhalation (n= 3) prior to bronchoscopy. Levels of the inducible stress protein Hsp72 and constitutive Hsp73 were quantitated in bronchial epithelial cells from brush biopsy of allergic nonasthmatic, allergic asthmatic, and normal individuals. Mean levels were increased significantly (p < 0.003 and p < 0.004, respectively) in those subjects who received albuterol premedication. No significant differences were found between clinical groups of individuals or for placebo inhalation vs nonpremedication. Albuterol in vitro increased the levels of Hsp72 and Hsp73 in epithelial cells from either nonpremedicated or placebo-treated donors; the Hsp72 levels correlated linearly with increased albuterol concentration (r= 0.81, p < 0.01). Therefore, β-agonists elevate or prolong an elevated stress response in epithelial cells, possibly through cAMP-mediated effects. Accepted for publication: 20 December 1996     

炎症细胞在心肌纤维化中的作用

心肌纤维化与心力衰竭、心律失常以及心源性猝死等密切相关。预防和逆转心肌纤维化是临床研究的热点之一。心肌纤维化的发病机制还未完全明确,目前认为炎症细胞与心肌纤维化的发生发展有着重要的联系,而自噬作为炎症细胞功能调控的重要因素影响心肌纤维化的转归。本文就炎症细胞及其自噬在心肌纤维化作用的最新研究进展作一综述。     

Regulation of Viral Infection-induced Airway Remodeling Cytokine Production by the TLR3-EGFR Signaling Pathway in Human Bronchial Epithelial Cells

Toll-like receptor 3 (TLR3) is involved in the virus-induced pulmonary inflammatory response, but its role in airway remodeling after viral infection is unclear. We explored the role of TLR3 in poly(I:C)-induced inflammatory cytokines and mucin 5AC (MUC5AC) production in human bronchial epithelial cells by Western blotting, RT-PCR and ELISA. The expression of TLR3, MUC5AC, Matrixmetalloproteinase (MMP9), Transforming growth factor (TGF-β1) and Vascular endothelial growth factor (VEGF) in human bronchial epithelial cells increased in a dose-dependent manner after exposure to poly(I:C), and this effect was inhibited by treatment with TLR3 siRNA. The phosphorylation of epithelial growth factor receptor (EGFR)/ERK/P38 Mitogen-activated protein kinases (MAPK) proteins increased after poly(I:C) treatment, and inhibition of this signaling pathway decreased TLR3 expression and MUC5AC and TGF-β1 production in human bronchial epithelial cells. The TLR3-EGFR signaling pathway is involved in the production of airway remodeling cytokines after virus infection. Inhibiting EGFR and its signaling pathway may be a therapeutic strategy for modifying airway remodeling.     

Simvastatin Increases the Ability of Roflumilast N-oxide to Inhibit Cigarette Smoke-Induced Epithelial to Mesenchymal Transition in Well-differentiated Human Bronchial Epithelial Cells in vitro

Background: Cigarette smoking contributes to epithelial-mesenchymal transition (EMT) in COPD small bronchi as part of the lung remodeling process. We recently observed that roflumilast N-oxide (RNO), the active metabolite of the PDE4 inhibitor roflumilast, prevents cigarette smoke-induced EMT in differentiated human bronchial epithelial cells. Further, statins were shown to protect renal and alveolar epithelial cells from EMT. Objectives: To analyze how RNO and simvastatin (SIM) interact on CSE-induced EMT in well-differentiated human bronchial epithelial cells (WD-HBEC) from small bronchi in vitro. Methods: WD-HBEC were stimulated with CSE (2.5%). The mesenchymal markers vimentin, collagen type I and α-SMA, the epithelial markers E-cadherin and ZO-1, as well as β-catenin were quantified by real time quantitative PCR or Western blotting. Intracellular reactive oxygen species (ROS) were measured using the H₂DCF-DA probe. GTP-Rac1 and pAkt were evaluated by Western blotting. Results: The combination of RNO at 2 nM and SIM at 100 nM was (over) additive to reverse CSE-induced EMT. CSE-induced EMT was partially mediated by the generation of ROS and the activation of the PI3K/Akt/β-catenin pathway. Both RNO at 2 nM and SIM at 100 nM partially abrogated this pathway, and its combination almost abolished ROS/ PI3K/Akt/β-catenin signaling and therefore EMT. Conclusions: The PDE4 inhibitor roflumilast N-oxide acts (over)additively with simvastatin to prevent CSE-induced EMT in WD-HBEC in vitro.     

Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air–Liquid Interface Cultured Human Primary Bronchial Epithelial Cells

The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory, and antioxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. First, we show that resveratrol and pterostilbene antiviral activity in African green monkey kidney cells. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly five rounds of replication, demonstrating the long-lasting effect of these compounds. Second, as the upper respiratory tract represents the initial site of SARS-CoV-2 replication, we also assessed antiviral activity in air–liquid interface (ALI) cultured human primary bronchial epithelial cells, isolated from healthy volunteers. Resveratrol and pterostilbene showed a strong antiviral effect in these cells up to 48 h post-infection. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to inhibit SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether these drugs are advantageous for COVID-19 treatment.     

Lung Inflammatory Responses and Hyperinflation Induced by an Intratracheal Exposure to Lipopolysaccharide in Rats

Exposure of the respiratory tract to lipopolysaccharide (LPS) induces acute local inflammation and tissue injury associated with the various deliveries of LPS. To determine potential association of local inflammatory responses with respiratory tract dysfunction, infiltration of inflammatory cells, production of inflammatory mediators, lung hyperinflation and edema were measured in Wister rats 2, 4, and 24 h after an intratracheal administration of LPS at different doses (5, 50, 500 and 5000 g/ml/kg). Lung hyperinflation determined by an increased excised lung gas volume was significantly increased 2 and 4 h after LPS instillation and lung edema occurred from 2 h onward. Peak BAL levels of TNF appeared at 2 h, MCP-1 at 4 h, and IL-6 at 2 and 4 h, while BAL levels of IL-1 were increased during 24 h after the intratracheal instillation of LPS. Neutrophilia in BAL fluid was noted from 2 h post-challenge. Our results demonstrate a clear dose-related change in the lung weight at 4 and 24 h, in the BAL levels of MCP-1 at 4 h, and IL-6 and IL-1 at 2 and 4 h. It seems important to understand polymorphisms of LPS-induced lung hyperinflation and inflammation. Lung hyperinflation and inflammation may be independent during the development of acute lung injury.     

pEGFP-C3/SUMF2重组真核表达载体的构建及在人支气管上皮细胞中的表达

目的构建与增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）融合的人硫酸酯酶修饰因子2（SUMF2）真核表达载体。方法用RT-PCR技术从人支气管上皮细胞（human bronchial epithelial cells,HBEC）获得SUMF2的cDNA模板,PCR方法扩增人SUMF2基因。用EcoRⅠ和BamHⅠ双酶切扩增人SUMF2基因及质粒pEGFP-C3;将2种酶切产物按常规方法连接、转化大肠杆菌Top10;挑取菌落培养,提取质粒,酶切鉴定,测序;将测序正确的重组载体用脂质体法转染HBEC,荧光倒置显微镜观察。提取转染细胞的总蛋白进行Western blot检测。结果扩增出1条约857bp的片段,与预期的SUMF2大小相符。酶切结果显示重组质粒pEGFP-C3/SUMF2被切成2条片段,其中1条为pEGFP-C3载体,另1条为目的片段。经测序鉴定,序列与GenBank（NM₀15411.2）中的序列高度同源。荧光倒置显微镜观察显示,人SUMF2基因主要在内质网表达。Western blot结果显示在相对分子质量为37×103处有一蛋白条带,与预期大小相符。结论成功构建重组表达载体pEGFP-C3/SUMF2,为进一步研究SUMF2对IL-13的作用奠定了实验基础。     

鸡螺杆菌诱导胃上皮细胞白细胞介素8转录及病理意义

目的：观察鸡螺杆菌（H.pullorum）诱导胃上皮细胞白细胞介素（IL）8转录并探讨其免疫致病机制。方法：分离培养H.pullorum菌株及构建带有IL－8报告基因的人胃癌细胞系L5F11，用液体闪烁计数仪测定荧光素酶（IL－8转录）活性。结果：所有10株H.pullorum诱导荧光素酶活性较幽门螺杆菌(H.pylori)野生型cag致病岛阴性菌株G50明显增强（P＜0．05－0．001）。结论：H.pullorum 通过诱导上皮细胞IL－8转录而参与其免疫致病过程。     

CREB3 Plays an Important Role in HPSE-Facilitated HSV-1 Release in Human Corneal Epithelial Cells

Herpes simplex virus type-1 (HSV-1) exploits several host factors to enhance its replication and release from infected cells. It induces the production of host enzyme heparanase (HPSE) to aid in egress. While the mechanism by which HPSE assists in viral release is well-characterized, other host factors that are recruited along with HPSE for viral release are less well understood. In this study, we identify cyclic-AMP-responsive element-binding protein3 (CREB3) as a key player in HPSE-facilitated HSV-1 egress. When CREB3 is transiently upregulated in human corneal epithelial cells, HSV-1 release from the infected cells is correspondingly enhanced. This activity is linked to HPSE expression such that HPSE-transfected corneal epithelial (HCE) cells more highly express CREB3 than wild-type cells while the cells knocked out for HPSE show very little CREB3 expression. CREB3-transfected HCE cells showed significantly higher export of HPSE upon infection than wild-type cells. Our data suggests that coat protein complex II (COPII), which mediates HPSE trafficking, is also upregulated via a CREB3-dependent pathway during HSV-1 infection. Finally, the co-transfection of CREB3 and HPSE in HCE cells shows the highest viral release compared to either treatment alone, establishing CREB3 as a key player in HPSE-facilitated HSV-1 egress.     

槲皮素诱导人肝癌HepG_2细胞凋亡的实验研究

目的探讨槲皮素诱导HepG2细胞凋亡的作用机制。方法应用透射电镜观察药物作用组及对照组细胞形态学变化;AnnexinV荧光染色和流式细胞仪检测药物作用组及对照组HepG2细胞凋亡和死亡率,以及逆转录聚合酶链反应(RT-PCR)法检测药物作用组及对照组凋亡相关基因fas的变化。结果槲皮素对HepG2细胞的生长有明显的抑制作用,并诱导肿瘤细胞发生凋亡,凋亡细胞表现为细胞固缩、核染色质碎裂,流式细胞仪检测凋亡率为13.2%,细胞停在G1和G2期。AnnexinV标记的方法检测凋亡时发现,坏死与凋亡共存。在槲皮素诱导HepG2细胞凋亡过程中,凋亡相关基因fas转录水平比用药前增强。结论诱导凋亡为槲皮素抑癌的机制之一,槲皮素诱导HepG2细胞凋亡可能与fas基因表达有关。     

Altered Ultrastructure of Lactating Rat Mammary Epithelial Cells Induced by Chronic Ethanol Ingestion

The ethanol and acetaldehyde uptake by the lactating rat mammary gland as well as their effects on this gland at the ultrastructural level have been studied. The extraction of acetaldehyde was greater than that of ethanol both after chronic and acute ethanol treatment. Chronic ethanol administration resulted in a loss of the mammary cell polarization, in a reduction of the Golgi dictyosomal elements and in several abnormalities at the level of casein maturation and secretion, whereas lipid synthesis and secretion did not seem to be affected. Normal spherical casein micelles took on a filament-like structure and casein vesicles appeared fused together forming macrovesicles. All these alterations were specific of ethanol and/or acetaldehyde action and were not due to the associated malnutrition, as deduced from the lack of visible effects in the nutritional control group.     

Phenol Toxicity and Conjugation in Human Colonic Epithelial Cells

Background: Colonic epithelial cells are exposed to a range of potentially harmful luminal factors, including phenols, but it is unresolved whether these compounds impair the integrity of the epithelium. The aim of this study was to describe the effect of phenol exposure on human colonic epithelial cells in vitro and the conjugation pathways involved in detoxification. Methods: Primary human colonic epithelial cell cultures or HT-29 cell cultures were exposed to paracetamol, dinitrophenol or phenol (0.1-5 mM) for 24 h. Cell viability was measured using the methyltetrazoleum test. Phenol conjugation products released from cell cultures were identified by high-pressure liquid chromatography. Phenol glucuronidase (PGD) and sulphotransferase (PST) enzyme activities were measured in isolated cell homogenates. Results: Paracetamol, dinitrophenol and phenol ( &#83 1.25 mM) significantly impaired the viability of primary colonic epithelial cell cultures. No differences between cell cultures from ulcerative colitis and control patients were observed. Paracetamol (5 mM) also induced significant cell damage in HT-29 cells. Glucuronidation was the preferred conjugation pathway in both cell models, despite the presence of PGD and PST activity. Conclusion: Phenols have a direct toxic effect on human colonic epithelial cells in vitro, which supports the view that dietary fermentation metabolites may be involved in the modulation of chronic bowel inflammation.     

Antigen Uptake and Trafficking in Human Intestinal Epithelial Cells

Primary intestinal epithelial cells, human colonic adenocarcinoma cell lines (DLD-1, Caco-2, and HT-29), and monocytes were used as model systems to study antigen uptake, antigen-presenting cell properties, as well as the kinetics of antigen uptake in intestinal epithelial cells (IEC). Intracellular staining of fluoresceinated tetanus toxoid was not evident in the IEC until after 30 min of incubation at 37°C, whereas in monocytes intracellular punctate staining of fluoresceinated tetanus toxoid was evident after 5 mins. In polarized Caco-2 cells antigen could be internalized at both the apical and basolateral surfaces with polarized transport. When analyzed by electron microscopy, gold-labeled tetanus toxoid was internalized and found within endosomes and multivesicular bodies, but not within the lysosomal compartments by 60 min. By 2 hrs, gold-labeled tetanus toxoid was evident in the secondary lysosomes. These results demonstrate that tetanus toxoid follows an endocytic pathway in intestinal epithelial cells and that the kinetics of antigen uptake is slower than that of conventional antigen-presenting cells.     

Pleural Mesothelial Cells Mediate Inflammatory and Profibrotic Responses in Talc-induced Pleurodesis

Intrapleural talc is used to produce pleurodesis in malignant pleural effusions. Prior in vivo studies have documented an acute inflammatory response to talc in the pleural space but the cellular source of cytokines has not been identified. The aim of this study was to investigate the acute response of rabbit pleural mesothelial cells challenged with talc used for pleurodesis and compare it to prior studies of the response to talc in the rabbit pleural space. Cultured rabbit pleural mesothelial cells (PMC) were exposed to talc (25 μg/cm²) for 6, 24, or 48 h and assessed for viability, necrosis, and apoptosis by flow cytometry, Trypan Blue exclusion, and immunocytochemistry, and for the production of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and transforming growth factor-β₁ (TGF-β₁) by ELISA. More than 50% of the PMC remained viable 48 h after talc stimulation. The PMC that were nonviable were identified as either apoptotic or necrotic, with roughly 20% in each category over the 48 h. At 6 h, the IL-8, VEGF, and TGF-β₁ levels produced by talc-exposed PMC increased significantly and remained elevated for up to 48 h. These cytokine levels rose at similar times and at the same or higher levels than have been measured in the rabbit pleural space in prior studies. We report that viable, talc-exposed, pleural mesothelial cells may actively mediate the primary inflammatory pleural response in talc-induced pleurodesis.