ABSTRACT
Objective To investigate the influence of early growth response gene-1 (EGR1) on the autophagy of host cells following infection with human T cell leukemia virus type 1 (HTLV-1).MethodsA HTLV-1-positive cell line MT2 was co-cultured with HeLa cells for 24 h to construct the virus early infection model.Immunoblotting assay was used to detect the expression of HTLV-1 core protein p19 and EGR1.Luciferase reporter gene analysis was used to detect the transcriptional activity of 5′-regulatory sequence of EGR1 at different time points after co-culturing.An effective small interfering RNA (siRNA) targeting EGR1 was screened out and transfected into HeLa cells by Lipofectamine 2000.Then the transfected HeLa cells were co-cultured with the HTLV-1-positive cell line MT2 for 24 h.Immunoblotting assay was used to detect HTLV-1 core protein p19, EGR1 and autophagy-related protein LC3.Real-time PCR was performed to detect viral load.Autophagosome was analyzed by immunofluorescence after co-culturing.Results The expression of EGR1 and the transcriptional activity of pEGR1-luc gradually increased after co-culturing HeLa cells with MT2 cells for 8 h (P<0.01).The expression of EGR1 was positively correlated with host cell autophagy following HTLV-1 infection.The effective siRNA for silencing the expression of EGR1 was obtained and named as siE2.The viral load, the expression of HTLV-1 core protein p19 and the proportion of LC3B/LC3A in the co-culture model were markedly down-regulated by RNA interference with siE2, which was concomitant with a persistent decrease of intracellular autophagosome (P<0.01).Conclusion EGR1 is associated with host cell autophagy and viral replication in HTLV-1 infection.
ABSTRACT
Software for quantitative analysis of endothelial cell (EC) area, form fac-tor, intercell-plasma-membrane distance and percentage of intercellular gap area in the cellmonolayer was established in a computer image analysing system with great capacity, highspeed and high resolution. The effects of platelet activating factor (PAF) on EC morpho-logical parameters were studied. The results showed that the untreated EC monolayer hadclose contact and narrow gap among cells. After 60 min treatment with 10~(-8)mol/L PAF,the cells showed obvious retraction, with procession of different size from the plasma mem-brane and increased intercellular gap. Computerized quantitative analysis revealed thatthe treatment with PAF for 10 min increased intercellular distance and percentage of inter-cellular gap area in cell monolayer. Thirty minutes treatment decreased cell area butincreased form factor ascertaining to the retraction of EC. This is probably an importantmechanism of increased vascular permeability induced by PAF It is also suggested thatcomputerized image analysis is helpful in rapid, precise and quantitative measurement ofEC morphological changes. It provides a new method for studying the effects of endothe-lial cells in increased vascular permeability.
ABSTRACT
Platelet-activating factor(PAF) at different concentrations were added to in vitro cultured bovine=pulmonary artery endothelial cells(BPAEC) when lactate dehydrogenase(LDH) release rate, angiotensin converting enzyme(ACE) activity and malondialdehyde (MDA) content were determined. The influences of specific PAF receptor antagonist on PAF effects and the stimulating effect of PAF on leukocyte-endothelial cell adhesion were also observed. The results showed that when PAF were added to BPAECs, there were no significant change in LDH release rate, ACE activities were only slightly increased. No obvious changes in cellbound MDA, but supernatant MDA content was increased in cells treated with high concentration of PAF (10~(-6)). The PAF receptor antagonist, SRI 63-441 made the cell-bound MDA content higher than that of PAF treated cells, whereas the supernatant MDA became lower. PAF may promote endothelial-leukocyte adhesion either through its effects on endothelial cells or on leukocytes, suggesting that PAF has no obvious damaging effect on endothelial cells but may activate endothelial cells thus promoting endothelialleukocyte adhesion.
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A bolus injection of endotoxin and platelet activating factor ( PAF ) via external jugular vein in rats resulted in severe pulmonary edema, accompanied by significant increase in extravascular lung water volume, bronchoalveolar lavage fluid ( BALF ) protein concentration, BALF cell counts and polymorphonuclearcyte percentage. PAF receptor antagonist SRI63-441 showed partial protective effects on endotoxin and PAF induced lung edema and BALF cytological changes.
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Actute canine hemorrhagic pancreatitis was induced by injection of autobile 1 ml/ kg into the main pancreatic duct, and the effects of pretrcatment with phospholipase A2 inhibitor-chloroquine on pulmonary edema were observed.The results revealed that in animals with acute pancreatitis, lung index and wet to dry lung weight ratio increased significantly, and pulmonary blood volume and extravascular water volume also increased obviously.There were also increments of protein and cell contents in bronchoalveolar lavage fluid.Cell sorting showed significantly decreased percentage of macrophages, and increased percentage of PMN and type n pneumocytes.Pretreat-ment with chloroquine prevented the development of pulmonary edema and the appreciably increased percentage of PMN.This experiment suggests that in acute canine pancreatitis, phospholipase AI may play an important part in pulmonary edema, and chloroquine has potential clinical value in the treatment.
ABSTRACT
We established a method to study vascular permeability in vitro on perfused endothelial cell monolayer cultured on micropore filter membrane. It can be used to determine filtration coefficient (Kf) and osmotic reflection coefficient (?) to proteins. Hanks balanced salt solution (HBSS) or 5 g/L albumin in HBSS was used to perfuse confluent endothelial monolayer. Control Kf values were 10.1 ?0.75 and 3.6?0.75?l . min-1 . cm-2 . kPa-1 (n = 3, x?sx) respectively for HBSS and albumin HBSS perfusion, suggesting that albumin may decrease endothelial monolayer permeability to water and small molecules. After exposure of endothelial monolayer to 10-8 mol/L platelet-activating factor (PAF) for 30 min, Kf values increased to 193.1% and 133.3% respectively for HBSS and albumin HBSS perfusion. Protein clearance rate (?l. min-1 . cm-2:) and osmotic reflection coefficient of control endothelial monolayer were 8.0?3.22 and 0. 37?0.09 respectively. In PAF treated endothelial monolayer,they were 12.2 ? 2.95ul min-1 cm 2 and 0.18?0.06, revealing increased permeability to albumin. Computer-assisted image processing demonstrated that PAF treatment decreased cell area while increased cell form factor and intercellular space. The results sug-gest that endothelial cells retracted, rounded and it may be an important mechanism in PAF-induced increased vascular permeability.
ABSTRACT
The effects of hydrogen peroxide (H2O2) on endothelial-polymorphonuclear cells (EC-PMN) adhesion and their mechanisms wsre studied in cultured bovine pulmonary artery endothelial monolayers in vitro. H2O2 at various concentrations (10-1, 10-2, 10-3mol/L respectively) stimulated EC dependent PMN adhesion, of which l02mol/L H2O2 was the most potent one, increasing adhesion to 2.3 times that of the control. Pretreatment of PMNs with SRI 63-441, a platelet-activating factor (PAF) receptor antagonist, had no effect on H2O2 induced EC-PMN adhesion. Pretreatment of ECs with SRI 63-441 before H2O2 exposure significantly decreased PMN adherence to ECs. Pretreatment of ECs with phospholipase A2 inhibitor p-bromophenacyl-bromide or cahnodulin antagonist chlorpromazine and aildum ion chelate EGTA obviously decreased H2O2 induced increment of EC-PMN adhesion. These results suggest that H2O2 may activate ECs, causing the inflow of extracellular calcium or the release of calcium from intracellular deposits. Increased intracellular Ca2+ may bind with calmodulin to activate phospholipase A2 thus initiating PAF synthesis and promoting EC-PMN adhesion.
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Intravenous administration of E. coli endotoxin (2mg/kg) caused a persistent increase in platelet activating factor (PAF) contents in arterial blood and bronchoalveolar fluid (BALF). Meanwhile phospholipase A2(PLA2) activity in serum and BALF was elevated and mean arterial blood pressure declined. The total cell number, expecially polymorphonuclear leukocytes and lymphocytes, and protein concentration in BALF were markedly increased 6 h after endotoxin injection. Lung index and extravascular lung water content of endotoxin-treated animals were significantly highter than those of controls. Pretreatment with PAF receptor antagonist SRI 63-441 blocked the increase in PLA, activity and attenuated endotoxin-induced hypotension and acute lung injury. The results suggest that PAF mediates endotoxin-induced lung injury, and leukocyte activation by PAF and the subsequent release of oxygen metabolites and lysoenzymes are important intermediate mechanisms leading to high permeability pulmonary edema.
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A method for isolation and primary culture of guinea pig alveolar type Ⅱ epithelial cells is reported. The injurious effects of phospholipase A2 (PLA2) on the cultured cells were studied. Trypsinizing solution was instilled into the bronchoalveolar space. The lungs were digested, sectioned, vortexed and filtered. The yield of original cell mixture was 172.3?106 cells/guinea pig, of which 33.6% were type Ⅱcells. At 48 h of culture following short term selective attachment 7.9?106 cells were obtained, of which 66.1% were type Ⅱ ceils. The purity could further be raised to 76.2% (63.9% -92%) if pretreated with ethyienediarnine tetraacetic acid (EDTA) before trypsinization to remove attached cells. The characteristics of cultured cells were observed under phase contrast microscopy, light microscopy and electron microscopy. After exposure of the cells to 300U/ml PLA2 for 4h at 37℃, detachment and lactic dehydrogenase (LDH) release of the cultured cells increased significantly. It is suggested that PLA, may damage and promote detachment of cultured type Ⅱ cells and it may play an important part in the development of some diseases.
ABSTRACT
Twenty mongrel dogs were used and randomly divided into three groups: sham-operated controls, dogs with induced acute hemorrhagic pancreatitis (AHP) and those treated with chloroquine (Ch1) before AHP was induced (AHP+Ch1). AHP was induced by injcction of auto-bile 1 ml/kg into the main pancreatic duct. The results revealed that in AHP, bronchoalveolar lavage fluid (BALF) cell count increased significantly, particularly the alveolar typeⅡ cells and polymorphonuclear leukocytes (PMNs). BALF and cell total phospholipid content (TPL) decreased significantly, of which lysophos-phatidylcholine (LPC) increased, phosphatidylcholine (PC) and dipalmitoly phosphatidylcholine (DPPC) decreased. Lung homogenate showed no change in TPL, but had increased percentage of LPC and decreased percentage of PC and DPPC. Surface activity of BALF and its extracted phospholipid decreased significantly in AHP. Pretreatment with chloroquine can obviously prevent some of the changes in BALF cell types and in percent composition of phospholipid, but had no impact on PS activity. It is suggested that PLA2 participates in developments of pulmonary surfactant system defects and chloroquine has potential value in the treatment of acute lung injury that follows acute pancreatits.