OBJECTIVETo investigate the influence of succinic acid on the apoptosis of polymorphonuclear neutrophil (PMN) in human peripheral blood, and to explore its role in infection.

METHODSPMNs were incubated in vitro, and its concentration was adjusted to 5 x 10(6)/mL. Then the cells were divided into normal control group and 5,10, 20, 30 mmol/L succinic acid groups according to different concentrations of succinic acid added into the medium. The supernatant of the cultures in each groups were collected to determine the superoxide content. 1 mL cell suspension was collected from 5, 20 mmol/L succinic acid groups before treatment and at 2, 4, 6, 8, 10 post-treatment hours (PTH) for the determination of caspase-3 activity and the apoptosis rate.

RESULTSThe content of superoxide in 5, 10, 20, 30 mmol/L succinic acid groups (0.437 +/- 0.056, 0.432 +/- 0.024, 0.395 +/- 0.049, 0.386 +/- 0.010) was significantly lower than that in control group (0.505 +/- 0.028, P < 0.05). The caspase-3 activity in each group increased along with the incubation time, but was in lower concentration in 5 mmol/L succinic acid group and in higher concentration in 20 mmol/L succinic acid group when compared with that in control group (P < 0.05). The apoptosis rate of PMN in control group was (6.1 +/- 1.1)% before incubation, and it reached (13.2 +/- 2.0)% at 2 PTH, and (27.7 +/- 3.7)% at 10 PTH. The apoptosis rate of PMN in 5 mmol/L succinic acid group was lower than that in control group except that at 4 PTH (P < 0.05). On the other hand, the apoptosis rate in 20 mmol/L succinic acid group (during 4-10 PTH) were obviously higher at each time points compared with the control group (P < 0.05).

CONCLUSIONLow concentration of succinic acid can suppress the apoptosis of PMN, while high concentration of succinic acid has an opposite effect. It is known that bacteria can produce succinic acid.

Apoptosis ; drug effects ; Cells, Cultured ; Humans ; Neutrophil Activation ; Neutrophils ; cytology ; drug effects ; Succinic Acid ; pharmacology

Adrenal Cortex Hormones ; therapeutic use ; Apoptosis ; drug effects ; Drug Resistance ; Humans ; Neutrophils ; cytology ; drug effects ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; Theophylline ; therapeutic use

OBJECTIVEVerotoxin-producing Escherichia coli (VTEC) strains of serotype O157 : H7 have been implicated in a wide spectrum of diseases, including blood diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To further explore the pathological role of verotoxin (VT) in HUS and other VTEC associated diseases, we investigated the effects of recombinant verotoxin 2 (rVT2) on the biological activity of neutrophils.

METHODSThe technique of flow cytometry, a fluorescent probe 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM), and the assay of reduced cytochrome c to detect superoxide production were used in this study.

RESULTSgammaVT2 significantly inhibited spontaneous apoptosis in neutrophils. Neutrophils with prolonged survival due to gammaVT2 maintained various biological functions, such as the expression of adhesion molecules (shading CD62L and raising CD11b/CD18), adherence to human umbilical vein endothelial cells (HUVECs), and generation of superoxide (O(2)(-)).

CONCLUSIONProlongation of the functional life-span of neutrophils by gammaVT2 may accelerate inflammatory responses at sites of inflammation. This may play a crucial role in neutrophil-mediated tissue injury in HUS and other VTEC-associated diseases.

Apoptosis ; drug effects ; Cell Adhesion ; Endothelium, Vascular ; cytology ; Humans ; Neutrophils ; drug effects ; physiology ; Recombinant Proteins ; toxicity ; Shiga Toxin 2 ; toxicity ; Superoxides ; metabolism

OBJECTIVETo study the effect and molecular mechanism of two haw leaf extracts, Vitexin-rhamnoside (VR) and Vitexin-glucoside (VG), and their preparation, Aoshaen injection (AI), on the polymorphonuclear leucocyte (PMN) adhesion during human umbilical vein endothelial cell (HUVEC) anoxia/reoxygenation (A/R) injury.

METHODSThe cell model of A/R injury duplicated by breaking off the oxygen supplying of HUVEC for 60 min followed with reoxygenating for 30 min (phase 1) or 240 min (phase 2) was taken as the experimental objective. The effects of testing drugs (VR, VG and AI) on PMN adhesion in the model cells were measured by enzyme immunoassay, and their effects on PMN superficial adhesion molecule CD11/CD18 expression were measured by flow cytometer respectively.

RESULTSAfter 60 min of anoxia, HUVEC was shrunk and deformed. The adhesion between PMN and HUVEC significantly revealed at phase 1 in the model group, but it was fewer in the normal cell group, and also lesser in the groups treated with various drugs. The condition of cell adhesion revealed at phase 2 was the similar to that at phase 1. All testing drugs, VR, VG and AI, showed inhibitory effect on the cell adhesion at either phase 1 or phase 2, showing a certain dose-effect relationship. The expression of CD11/ CD18 was also inhibited by the testing drugs, and a good dose-effect relation was shown by VG and AI.

CONCLUSIONAt the resting condition, there are almost no expression of CD11/CD18 molecule, but it could be enhanced by incubating PMN with supernate of A/R injured HUVEC culture, and more marked at phase 1. Adding the test drugs into the supernate could inhibit the enhancing of CD11/CD18 molecule expression and reduce the PMN-HUVEC adhesion, which may be one of the molecular mechanisms of haw leaf extracts and their preparation in protecting heart against A/R injury.

Cell Adhesion ; drug effects ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Crataegus ; chemistry ; Female ; Humans ; Hypoxia ; drug therapy ; physiopathology ; Neutrophils ; drug effects ; physiology ; Oxygen ; metabolism ; Plant Extracts ; pharmacology ; Plant Leaves ; chemistry ; Pregnancy ; Umbilical Veins ; cytology ; drug effects

AIMTo investigate the role of TNF-alpha in vascular endothelial cells injury mediated by freezing/thaw ing PMN.

METHODSFreezing/thawing cell model was founded using rat PMN isolated by dextran sedimentation technique and VEC cultured in vitro. The injury level of VEC was indicated by measuring activity of LDH in medium. The number of frozen/thawed PMN adhering to VEC was counted with Phagocytizing reactive dyes the degree of frozen/thawed PMN and VEC adhesion. Expression of LFA-1 on the surface of frozen/thawed PMN was analyzed with flow cytometry.

RESULTSTNF-alpha could obviously upregulate expression of LFA-1 on surfaced of frozen/thawed PMN. Upregulation of LFA-1 expression promoted adhesion of frozen/thawed PMN and normal VEC,and aggravated VEC injury. Monoclonal antibody against LFA-1 could partly block adhesion of frozen/thawed PMN and normal VEC,and attenuate VEC injury.

CONCLUSIONTNF-alpha can promote expression of LFA-1 on surface of frozen/thawed PMN adhering of frozen/thawed PMN to normal VEC and VEC injury increase, monoclonal antibody against LFA-1 could partly block PMN-VEC adhesion and attenuate VEC injury.

Animals ; Cell Adhesion ; Cells, Cultured ; Endothelial Cells ; drug effects ; Endothelium, Vascular ; cytology ; Freezing ; Lymphocyte Function-Associated Antigen-1 ; metabolism ; Neutrophils ; cytology ; metabolism ; Rats ; Tumor Necrosis Factor-alpha ; pharmacology

Animals ; Animals, Newborn ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Female ; Lipopolysaccharides ; toxicity ; Lung ; drug effects ; enzymology ; pathology ; Male ; Neutrophils ; cytology ; Peroxidase ; analysis ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effects of Propyl Gallate (PrG) on cellular adhesion between human To investigate the effects of Propyl Gallate (PrG) on cellular adhesion between human umbilical vein endothelial cells (HUVEC) and polymorphonuclear leukocytes (PMN) as well as the expression umbilical vein endothelial cells (HUVEC) and polymorphonuclear leukocytes (PMN) as well as the expression of intercellular adhesion molecule-1 (ICAM-1, CD54) and E-selectin (CD62E) on the VEC surface. of intercellular adhesion molecule-1 (ICAM-1, CD54) and E-selectin (CD62E) on the VEC surface.

METHODSA human VEC inflammation model was induced by tumor necrosis factor alpha (TNF-alpha). VECs were pre- A human VEC inflammation model was induced by tumor necrosis factor alpha (TNF-alpha). VECs were preincubated with varying concentrations of PrG (0.001-5 mmol/L) or 1 per thousand DMSO (v:v) or 10 mmol/L acetylsalicylic incubated with varying concentrations of PrG (0.001-5 mmol/L) or 1 per thousand DMSO (v:v) or 10 mmol/L acetylsalicylic acid (ASA) for 1 h, and then were stimulated with 10 ng/mL TNF-alpha for 6 h. Rose bengal vital staining method acid (ASA) for 1 h, and then were stimulated with 10 ng/mL TNF-alpha for 6 h. Rose bengal vital staining method was used to measure the adherence rate of PMN to VEC, while flow cytometry was used to determine the was used to measure the adherence rate of PMN to VEC, while flow cytometry was used to determine the expression of CD54 and CD62E on the VEC surface. expression of CD54 and CD62E on the VEC surface.

RESULTSAfter 6 h of incubation with TNF-alpha, the adherence After 6 h of incubation with TNF-alpha, the adherence of PMN to HUVECs as well as the percentage of fluorescence-positive cells and mean fluorescence intensity of PMN to HUVECs as well as the percentage of fluorescence-positive cells and mean fluorescence intensity (MFI) of surface CD54 and CD62E in HUVECs increased significantly ( (MFI) of surface CD54 and CD62E in HUVECs increased significantly (P<0.01). Pre-treatment of HUVECs with <0.01). Pre-treatment of HUVECs with PrG (0.1-5 mmol/L) significantly suppressed the adherence of PMN to VECs induced by TNF-alpha (PrG (0.1-5 mmol/L) significantly suppressed the adherence of PMN to VECs induced by TNF-alpha (P<0.05). PrG <0.05). PrG (1-5 mmol/L) inhibited the VEC surface expression of CD62E and CD54 in a dose-dependent way ( (1-5 mmol/L) inhibited the VEC surface expression of CD62E and CD54 in a dose-dependent way (P<0.05). PrG <0.05). PrG at lower concentrations (0.001-0.1 mmol/L) showed no effect on CD54 expression, while it showed a slightly at lower concentrations (0.001-0.1 mmol/L) showed no effect on CD54 expression, while it showed a slightly increasing trend in CD62E expression (increasing trend in CD62E expression (P>0.05). ASA at 10 mmol/L had no obvious effect on the positive rate of >0.05). ASA at 10 mmol/L had no obvious effect on the positive rate of CD62E and CD54. CD62E and CD54.

CONCLUSIONSHigh concentrations of PrG (0.1-5 mmol/L) exert its inhibitory effect on cellular High concentrations of PrG (0.1-5 mmol/L) exert its inhibitory effect on cellular adherence of PMN to HUVECs, and its mechanism may be related to inhibiting surface expression of CD54 and adherence of PMN to HUVECs, and its mechanism may be related to inhibiting surface expression of CD54 and CD62E in HUVECs. Its action concentration was lower than that of ASA. CD62E in HUVECs. Its action concentration was lower than that of ASA.

Cell Adhesion ; drug effects ; Endothelial Cells ; cytology ; drug effects ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; metabolism ; Fluorescence ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Neutrophils ; cytology ; drug effects ; Propyl Gallate ; chemistry ; pharmacology ; Staining and Labeling ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo explore the detoxication and tissue generation effect of Astragalus (As) in wound healing and its relation with inflammatory reaction, through observing the effect of Astragalus polysaccharides (AP) on neutrophil-endothelial cell adhesion and expression of related adhesive molecules.

METHODSHuman polymorphonuclear leucocyte (PMN) or human umbilical vein endothelial cell (HUVEC) was treated separately with AP, AP plus interleukin 1 (IL-1) and tumor necrosis factor (TNF) to study the effect of AP on PMN adhesion with HUVEC by rose bengal staining, and that on expression of superficial adhesive factor by means of Cell-ELISA and APAAP method.

RESULTSWhen AP acted on HUVEC, it could significantly promote the adhesion of HUVEC with PMN, while when AP acted on PMN, the adhesion would not increase. When HUVEC was treated by AP plus IL-1, the IL-1 induced PMN adhesion with HUVEC could be strengthened, and the expression of HUVEC superficial adhesive factor ICAM-1 induced by IL-1 and TNF was strengthened also, but when PMN treated with AP, it showed no effect on the expression of adhesive factor CD18.

CONCLUSIONAP promotes the adhesion between neutrophil and endothelial cell by way of promoting the expression of superficial I-CAM-1 on surface of endothelial cells, so as to improve the inflammatory reaction in the wound healing course, it possibly is one of the biological bases of the detoxication and tissue generation effects of AP.

Astragalus membranaceus ; chemistry ; Cell Adhesion ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; biosynthesis ; Interleukin-1 ; biosynthesis ; Neutrophils ; cytology ; metabolism ; Polysaccharides ; pharmacology ; Umbilical Veins ; cytology ; Wound Healing ; drug effects

OBJECTIVETo explore the activation of polymorphonuclear cells (PMNs) and vascular endothelial cells co-cultured with and stimulated by lipopolysaccharide.

METHODSPMNs in concentration of 2 x 10(6)/ml isolated from healthy volunteers by Percoll gradient were added to monolayer of ECV-304 cells grown to confluency, then different groups were prepared according to final concentration of lipopolysaccharide. The morphological change was observed under invert microscope. The changes in TNFalpha and IL-6 levels of the supernatant of the cultured cells were determined at 4, 8, 12 and 24 hours after culturation.

RESULTSThe TNFalpha production of cultured pure ECV-304 exhibited no remarkable change when stimulated by different concentrations of LPS. But the TNFalpha production of the ECV-304 increased significantly when co-cultured with PMNs at 4 hr and stimulated by LPS in concentration of 10 micro g/ml, and increased at 8 hours and lasted up to 24 hours of culturation in higher levels (P < 0.05). The IL-6 production of cultured pure ECV-304 increased obviously along with the increase of LPS concentration, and it showed no change when PMNs co-cultured with ECV-304. While the IL-6 level in the supernatant of co-cultured ECV-304 with PMNs increased sharply when stimulated by both low (100 ng/ml) and high (1 micro g/ml) concentrations of LPS and maintained at high levels up to 24 hours of culturation. The higher the concentration of LPS was, the quicker the IL-6 level increased.

CONCLUSIONCo-cultured PMNs and endothelial cells could be activated and activation state could be maitained by low concentrations of LPS.

Adult ; Cell Line ; Cells, Cultured ; Coculture Techniques ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; pharmacology ; Neutrophils ; cytology ; drug effects ; metabolism ; Time Factors ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo determine the inhibitory effects of BIO-1211, a very late antigen-4 (vla-4) antagonist, on bronchoconstriction and neutrophil adhesion in rats.

METHODSFor evaluating ovalbumin-induced bronchoconstriction in the sensitized rats, the changes in lung resistance (RL) and lung dynamic compliance (C(dyn)) were determined after antigen challenge. Neutrophils from the rats were used to determine fibronectin and serum-induced cell adhesion. The effect of BIO-1211 on wheezing was determined after inhalation of histamine and acetylcholine in guinea pigs.

RESULTBIO-1211 aerosol at 1, 3 and 10 mg/ml significantly inhibited the changes in lung resistance and lung dynamic compliance after antigen challenge in the sensitized rats in a dose-dependent manner. BIO-1211 at 25, 50, 100 and 200 microgram/ml inhibited the fibronectin-induced neutrophil adhesion by 23.5%, 24.6%, 61.4% and 58.1%, respectively, and serum-induced adhesion by 29.9%, 35.9%, 35.3% and 15.4%, respectively. Inhalation of 10 mg/ml BIO-1211 did not show any protection against histamine and acetylcholine-induced bronchoconstriction.

CONCLUSIONBIO-1211 inhibits bronchoconstriction and neutrophil adhesion, which may be associated with its effect against bronchoconstriction in rats.

Administration, Inhalation ; Animals ; Asthma ; drug therapy ; physiopathology ; Bronchoconstriction ; drug effects ; physiology ; Bronchodilator Agents ; administration & dosage ; pharmacology ; Cell Adhesion ; drug effects ; Female ; Guinea Pigs ; Male ; Neutrophils ; cytology ; drug effects ; Oligopeptides ; administration & dosage ; pharmacology ; Rats