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

Although heparin is better known as an anticoagulant, it also has several anti-inflammatory effects. Heparin is known to inhibit neutrophil adhesion, chemotaxis and oxygen free radical production. In addition, heparin is also known to act as an oxygen radical scavenger. Our hypothesis was that heparin would attenuate renal ischemia reperfusion injury. In this study, we investigated whether heparin had a protective effect on renal ischemia reperfusion injury. Sheep (n = 12) were prepared for the chronic study with venous, arterial and urinary catheters inserted. In addition, pneumatic occluders and ultrasonic flow probes were placed on renal arteries. After a 5-day recovery period, the sheep were randomized to either a heparin treatment group (400 IU/kg i.v. bolus 10 minutes before renal artery occlusion, followed by a continuous effusion 25,000 IU in 250 ml of 0.9% NaCl at 10 ml/hr, n = 6) or a control group (n = 6), which received an equivalent volume of 0.9% NaCl. All the sheep then underwent 90 minutes of bilateral renal ischemia followed by 24 hours of reperfusion. Blood urea nitrogen (BUN), serum creatinine (Scr), and creatinine clearance (CrCl) were determined at various intervals during both the ischemic and reperfusion periods. Kidney tissue samples were obtained at autopsy for histologic examination. As a result, there were significant differences in the degree of inflammation (1.50 +/- 1.24 Vs 0.50 +/- 0.79, P < 0.05) between the control and heparin treatment groups, but not in the degree of injury (2.83 +/- 0.44 Vs 2.33 +/- 0.28). In this study, heparin significantly attenuated polymorphonuclear leukocytes (PMNs) infiltration within the interstitium, but it did not affect the degree of renal damage as measured by urinary chemistries or renal tubular damage as assessed by histopathologic evaluation.
Animal ; Anticoagulants/pharmacology* ; Cell Movement/drug effects ; Female ; Heparin/pharmacology* ; Ischemia/pathology* ; Kidney/pathology ; Kidney/drug effects* ; Neutrophils/physiology ; Neutrophils/drug effects* ; Renal Circulation* ; Reperfusion Injury/pathology* ; Sheep

To investigate the effects of butyl alcohol extract of Baitouweng Decoction(BAEB) on neutrophil chemotaxis in vaginal mucosa of mice with vulvovaginal candidiasis(VVC). Seventy-two SPF female Kunming mice were randomly divided into normal control group, model group, fluconazole group, BAEB low-dose group, middle-dose group and high-dose group. Subcutaneous injection of estradiol benzoate was conducted to induce pseudo-estrus, and then 2×10~6 CFU·mL~(-1)of Candida albicans was inoculated into vaginal lumen, followed by drug treatment for 7 days. Gram staining was used to observe the morphological changes of C. albicans in vagina; vaginal fungal load was detected on agar plate. Histological changes of vaginal tissues in mice were observed by HE staining. Lactate dehydrogenase(LDH), interleukin-6(IL-6) and tumor necrosis factor(TNF-α) levels in mouse lavage fluid were detected by enzyme-linked immunosorbent assay(ELISA). Neutrophils in vaginal lavage fluid was observed and counted by using Pap smear. The levels of IL-8 and MIP-2 in vaginal mucosa were detected by ELISA. IL-8 and MIP-2 mRNA levels in vaginal mucosa of mice were detected by qRT-PCR. The results showed that as compared with the normal group, VVC model group had a large number of hyphae and a high level of fungal loadinvagina. The vaginal mucosa was completely destroyed, the number of neutrophils increased, and the protein and mRNA levels of IL-8 and MIP-2 were up-regulated. After BAEB treatment, the hyphae of the treatment group was decreased, the fungal load was decreased, the impaired mucosa showed different degrees of improvement, the inflammatory factors were decreased to varying degrees, and the protein and mRNA levels of chemokine IL-8 and MIP-2 were down-regulated. In conclusion, BAEB may be used to treat VVC by inhibiting vulvovaginal candidiasis via blocking neutrophils recruitment into vagina.
1-Butanol ; Animals ; Candida albicans ; Candidiasis, Vulvovaginal/drug therapy* ; Chemotaxis/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Female ; Mice ; Mucous Membrane/drug effects* ; Neutrophils/drug effects* ; Vagina/diagnostic imaging*

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

OBJECTIVETo testify whether absolute neutrophil count (ANC) response to preimmunosuppressive-therapy (pre-IST) granulocyte-stimulating factor (G-CSF) treatment could predict early response to IST in severe aplastic anemia (SAA).

METHODSClinical data and hematologic response of 125 SAA patients treated with antithymocyte globulin (r-ATG) combined with cyclosporine were retrospectively analyzed. Correlation of ANC response to pre-IST G-CSF treatment and early response to IST were statistically analyzed, and receiver operating characteristic (ROC) curve was used to estimate the value of increased ANC (∆ANC) in predicting early IST response.

RESULTSThe hematologic response (HR) rate to IST in ANC reponded patients was significantly higher than non-responded group (3-month HR 49.0% vs 28.9%, P=0.023; 6-month HR 61.2% vs 40.8%, P=0.026). With ∆ANC≥0.5×10⁹/L as cutoff level, the best point to predict early IST response was 10 days after G-CSF (d 10). Response of ANC to pre-IST G-CSF treatment at d 10 was among the independent factors of predicting 3-month (P=0.004), but not for 6-month response to IST. The overall 5-year survival rate was 92.8% and 69.5% in ANC responded and non-responed groups, respectively (P=0.025).

CONCLUSIONResponding to pre-IST G-CSF treatment reflected the residual bone marrow hematopoiesis, and could act as a convenient and practical predictor to early IST response as well as long-term survival in SAA.

Anemia, Aplastic ; drug therapy ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Humans ; Immunosuppressive Agents ; therapeutic use ; Neutrophils ; drug effects

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

To investigate the effects of cigarette smoking in different manners on acute lung injury in rats.The commercially available cigarettes with tar of 1,5, 11 mg were smoked in Canada depth smoking (health canada method, HCM) manner, and those with tar of 11 mg were also smoked in international standard (ISO) smoking manner. Rats were fixed and exposed to mainstream in a manner of nose-mouth exposure. After 28 days, the bronchoalveolar lavage fluids from left lung were collected for counting and classification of inflammatory cells and determination of pro-inflammatory cytokines IL-1β and TNF-α. The right lungs were subjected to histological examination and determination of myeloperoxidase (MPO) and superoxide dismutase (SOD) activities and glutathione, reactive oxygen species (ROS) and malondialdehyde (MDA) levels.In both HCM and ISO manners, the degree of lung injury was closely related to the tar content of cigarettes, and significant decrease in the body weight of rats was observed after smoking for one week. In a HCM manner, smoking with cigarette of 11 mg tar resulted in robust infiltration of macrophages, lymphocytes and neutrophils into lungs, significant increase in IL-1β and TNF-α levels and MPO activities, and significant decrease in GSH levels and SOD activities and increase in ROS and MDA levels (all<0.05). Smoking with cigarette of 5 mg tar led to moderate increase in IL-1β and TNF-α levels, and MPO activities (all<0.05), and moderate decrease in GSH levels and SOD activities and increase of ROS and MDA levels (all<0.05). However, smoking with cigarette of 1 mg tar affected neither inflammatory cell infiltration nor IL-1β and TNF-α levels.Cigarette smoking in nose-mouth exposure manner can induce acute lung injury in rats; and the degree of lung injury is closely related to the content of tar and other hazards in cigarettes.
Acute Lung Injury ; etiology ; pathology ; physiopathology ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Chemotaxis, Leukocyte ; drug effects ; Glutathione ; analysis ; drug effects ; Interleukin-1beta ; analysis ; drug effects ; Lung ; chemistry ; pathology ; Lymphocytes ; drug effects ; pathology ; Macrophages ; drug effects ; pathology ; Male ; Malondialdehyde ; analysis ; Neutrophil Infiltration ; drug effects ; Neutrophils ; drug effects ; pathology ; Peroxidase ; analysis ; drug effects ; Rats ; Reactive Oxygen Species ; analysis ; Smoking ; adverse effects ; Superoxide Dismutase ; analysis ; drug effects ; Tobacco Products ; adverse effects ; classification ; Tumor Necrosis Factor-alpha ; analysis ; drug effects ; Weight Loss ; drug effects

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

Neutrophils play a major role in host defense against microbial infection. There are some clues indicate that neutrophils may also play a role in the pathophysiology of the airway obstruction in chronic asthma. We studied the roles of intracellular calcium and GTP gamma S in the regulation of neutrophils exocytosis using pipette perfusion and membrane capacitance measurement technique in whole cell patch clamp configuration. The results showed that the membrane capacitance increase induced by calcium revealed a biphasic process. The first phase occurred when the calcium level was between 0.2-14 micromol/L with a plateau amplitude of 1.23 pF and a calcium EC50 of 1.1 micromol/L. This phase might correspond to the release of the tertiary granules. The second phase occurred when the calcium concentration was between 20-70 micromol/L with a plateau increment of 6.36 pF, the calcium EC50 being about 33 micromol/L. This phase might represent the release of the primary and secondary granules. Intracellular calcium also simultaneously increased the exocytotic rate and the eventual extent in neutrophils. On the other hand, GTP gamma S can increase the exocytotic rate in a dose-dependent manner but had no effect on the eventual extent of membrane capacitance increment (>6 pF) if the cell was stimulated for a long period (>20 min). GTP gamma S (ranging from 20 to 100 micromol/L) induced the neutrophils to release all four types of the granules at very low intracellular calcium level.
Calcium ; metabolism ; Cell Degranulation ; drug effects ; Exocytosis ; drug effects ; GTP-Binding Proteins ; metabolism ; physiology ; Guanosine Triphosphate ; analogs & derivatives ; pharmacology ; Humans ; Neutrophils ; metabolism ; physiology ; Patch-Clamp Techniques

OBJECTIVETo investigate the effect of one of the acute-phase proteins, fibrinogen, on the release of IL-1beta and -8 by human peripheral polymorphonuclear leukocytes (PMN) and the possible role of fibrinogen during the destruction of periodontium.

METHODSPeripheral PMN were isolated by discontinuous density gradient centrifuging technique. The freshly isolated PMN were suspended in Hank's balanced saline solution (1 x 10(9)/L) supplemented with 0.5% BSA and 0.1% glucose. The levels of IL-1beta and -8 in the supernatants produced by cultured cells upon the addition of human fibrinogen at different concentrations were measured by ELISA technique.

RESULTSIncubated with human fibrinogen at 2 g/L or 10 g/L for different time periods, human peripheral PMN released significantly greater amount of IL-1beta [(10.41 +/- 0.37) - (35.86 +/- 0.30) ng/L or (22.81 +/- 0.45) - (57.77 +/- 2.08) ng/L] and IL-8 [(93.90 +/- 13.95) - (2045.66 +/- 53.03) ng/L or (115.02 +/- 10.61) - (3858.69 +/- 25.65) ng/L] than PMN without the stimulation of fibrinogen (IL-1beta, P < 0.001, and IL-8, P < or = 0.016). The higher concentration of fibrinogen or the longer treatment time, the higher levels of IL-1beta and -8 were released by PMN (P < 0.001).

CONCLUSIONSFibrinogen induced the secretion of pro-inflammatory cytokines IL-1beta and -8 by PMN and may be involved in magnification of the inflammatory response of periodontium and bone resorption.

Cells, Cultured ; Fibrinogen ; pharmacology ; Humans ; Interleukin-1beta ; metabolism ; Interleukin-8 ; metabolism ; Middle Aged ; Neutrophils ; drug effects ; secretion