Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2(.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.
Anions/*metabolism ; Female ; Humans ; Neutrophils/enzymology/*metabolism/parasitology ; Peroxidase/metabolism ; Superoxides/*metabolism ; Trichomonas Infections/enzymology/*metabolism/parasitology ; Trichomonas vaginalis/*isolation & purification/physiology

In this study, the main antioxidant enzymes (AOE) of glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) were identified, and the influence of sex and age in healthy human polymorphonuclear leukocytes (PMNL) was determined. The SOD, GPX, CAT and MPO activities were investigated in intestinal parasite negative human PMNL from 109 healthy subjects aged from 6 to 70 years (55 males and 54 females) using simple and sensitive enzyme assays. Blood cells, such as eosinophils, platelets, neutrophils, monocytes, and macrophages also synthesize antioxidant enzymes (AOE). They constitute an important proportion and are also the major participants in a number of pathological conditions that suggest the involvement of AOE. A linear effect of age on SOD activity (p < 0.05) both in males and females was found. A similar effect with GPX activity (p < 0.05) was observed in males only. This showed that the activities of all these enzymes increase with age. In addition, SOD activity was significantly higher in females than males between the age of 19 and 70 years (p < 0.001). This analysis also showed that there is a negative correlation between the CAT-GPX (p < 0.05) activities and positive correlations between MPO-GPX (p < 0.05) activities only in females. No correlation among the other enzyme activities was found in either sex group. This study showed the activities of antioxidant enzyme activities and the correlations of these enyzmes activities with each other in healthy human PMNLs were age- and sex-dependent. This information may assisit in understanding the importance of antioxidant enzymes in the physiological and pathological conditions associated with PMNL.
Adolescent ; Adult ; Aged ; Aging/*metabolism ; Child ; Female ; Human ; Male ; Middle Aged ; Neutrophils/*enzymology ; Oxidoreductases/*metabolism ; Reference Values ; *Sex Characteristics

OBJECTIVETo evaluate the variety of non-myeloperoxidase-mediated system activity of neutrophils in newborns during bacterial infection and the effect of cord plasma on the activation of non-myeloperoxidase-mediated system.

METHODSAn infection model with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and a non-infection model with phorbol-12-myristate-13-acetate (PMA) were established to investigate the activation of non-myeloperoxidase-mediated system in neutrophils. According to the intensity of fluorescence, the activation of non-myeloperoxidase-mediated system of neutrophils was detected by flow cytometry (FCM). The blood cells and plasma were separated from cord blood and adult blood and cross-mixed in order to investigate the opsonic activity.

RESULTSIn the non-infection model, the activation of non-myeloperoxidase-mediated system with PMA stimulation in cord blood was lower compared with that in adult blood, the statistical difference was significant (t = 3.378, P < 0.01). In the infection model, the activations of non-myeloperoxidase-mediated system in cord blood were also lower compared with those in adult blood, while the statistical difference could only be found in the model with E. coli stimulation (t = 12.150, P < 0.001). Furthermore the experiments demonstrated that cord plasma could deeply depress the non-myeloperoxidase-mediated system activity with E. coli stimulation. On the contrary, adult plasma could successfully recruit the potential of non-myeloperoxidase-mediated system activity of neutrophils in newborns.

CONCLUSIONThe function of neonatal neutrophils might not developed very well. As a stimulant, E. coli failed to induce the non-myeloperoxidase-mediated system activity in neonates, which might be related to the lower level of immunoglobulins in cord blood. This result indicated that immunoglobulins played a more important modulating role in bacterial killing during gram-negative bacterial infections.

Escherichia coli ; immunology ; Fetal Blood ; immunology ; Flow Cytometry ; Humans ; Infant, Newborn ; Neutrophils ; enzymology ; immunology ; Peroxidase ; metabolism ; Staphylococcus aureus ; immunology

OBJECTIVETo investigate the changes of neutrophil myeloperoxidase (MPO) blood concentration gradient between the systemic circulation and the coronary circulation among patients with acute coronary syndrome and its clinical value.

METHODSFifty patients underwent coronary angiography, which including 10 patients in AMI group, 20 patients in UA group, 10 patients in SA group and 10 subjects served as control. The levels of MPO and hs-CRP were measured in the serum of blood collected from femoral vein, aortic artery root and coronary sinus.

RESULTSCompared with the control, concentrations of LDL in the AMI, UA and SA groups were significantly increased, while the latter three groups did not differ from each other. In the UA patients, the in-gate percentage of MPO decreased in the coronary sinus compared with that in the root of aortic artery (P < 0.01); the in-gate percentage of MPO decreased through coronary circulation more than through systemic circulation (P < 0.001); the average fluorescent intensity of MPO and the concentrations of hs-CRP showed no difference between samples from the coronary sinus and that from the root of aortic artery. In the AMI patients, the average fluorescent intensity of MPO in the coronary sinus was weakened compared with that in the root of aortic artery (P < 0.05); it decreased through coronary circulation more than through systemic circulation (P < 0.001); neither the in-gate percentage of MPO nor the concentrations of hs-CRP showed significant difference between samples from the coronary sinus and that from the root of aortic artery. In the control and SA groups, samples from the femoral vein, the root of aortic artery, and the coronary sinus did not show differences at the serum level of MPO and hs-CRP. In the UA group, the in-gate percentage of MPO correlated positively with the concentration of hs-CRP (r = 0.78, P < 0.01), and with the level of LDL as well (r = 0.52, P < 0.05); In the AMI group, the average fluorescent intensity of MPO correlated negatively with the concentration of hs-CRP (r = -0.80, P < 0.01), and showed no correlation with the level of LDL (r = 0.22, P > 0.05).

CONCLUSIONSMPO is a better marker for inflammation of the local plaques. It may be one of the mechanisms that MPO induces the transforming from LDL to ox-LDL in plaques vulnerability.

Acute Coronary Syndrome ; blood ; metabolism ; physiopathology ; Aged ; Biomarkers ; blood ; Case-Control Studies ; Coronary Circulation ; Female ; Humans ; Male ; Middle Aged ; Neutrophils ; enzymology ; Peroxidase ; blood

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 prepare a purified recombinant human neutrophil elastase (HNE) using genetic engineering technology, and pave the way for the preparation of the antibody to HNE and establishment of semen HNE detection methods.

METHODSHNE mRNA was obtained from human peripheral blood granulocytes with specific HNE primers, and the cDNA of HNE was cloned into the plasmid pGEX-2T to derive a recombinant plasmid pGEX-2T/HNE. After PCR identification, double-enzyme digestion and gene sequencing, the recombinant plasmid was transferred into competent Escherichia coli DH5alpha and further induced to express the recombinant fusion protein GST/HNE by isopropyl beta-D-thiosulfate galactosidase (IPTG). The recombinant fusion protein was cleaved by thrombin and further purified with glutathione agarose beads to obtain purified recombinant HNE.

RESULTSThe recombinant plasmid pGEX-2T/HNE was successfully prepared and transferred into E. coli DH5o; the expression of the recombinant fusion protein GST/ HNE was successfully induced by IPTG at 18 degrees C overnight; and the purified recombinant protein HNE was successfully obtained by thrombin cleavage and purification of glutathione agarose beads.

CONCLUSIONThe acquirement of purified recombinant HNE has prepared the ground for the preparation of the antibody to HNE and establishment of semen HNE detection methods.

DNA Primers ; Genetic Engineering ; Humans ; Leukocyte Elastase ; biosynthesis ; genetics ; Neutrophils ; enzymology ; Polymerase Chain Reaction ; methods ; RNA, Messenger ; genetics ; Recombinant Proteins ; biosynthesis

OBJECTIVEIn this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions.

METHODSNeutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot.

RESULTSIn co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05).

CONCLUSIONSCoculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

Animals ; Base Sequence ; Bronchi ; cytology ; enzymology ; metabolism ; Cattle ; Cell Adhesion Molecules ; metabolism ; Cell Line ; Coculture Techniques ; DNA Primers ; Down-Regulation ; drug effects ; Epithelial Cells ; enzymology ; metabolism ; Isoflavones ; pharmacology ; NF-kappa B ; metabolism ; Neutrophils ; enzymology ; metabolism ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; p38 Mitogen-Activated Protein Kinases ; metabolism

Recent studies have documented that Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) pathway can modulate the apoptotic program in a myocardial ischemia/reperfusion (I/R) model. To date, however, limited studies have examined the role of JAK3 on myocardial I/R injury. Here, we investigated the potential effects of pharmacological JAK3 inhibition with JANEX-1 in a myocardial I/R model. Mice were subjected to 45 min of ischemia followed by varying periods of reperfusion. JANEX-1 was injected 1 h before ischemia by intraperitoneal injection. Treatment with JANEX-1 significantly decreased plasma creatine kinase and lactate dehydrogenase activities, reduced infarct size, reversed I/R-induced functional deterioration of the myocardium and reduced myocardial apoptosis. Histological analysis revealed an increase in neutrophil and macrophage infiltration within the infarcted area, which was markedly reduced by JANEX-1 treatment. In parallel, in in vitro studies where neutrophils and macrophages were treated with JANEX-1 or isolated from JAK3 knockout mice, there was an impairment in the migration potential toward interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), respectively. Of note, however, JANEX-1 did not affect the expression of IL-8 and MCP-1 in the myocardium. The pharmacological inhibition of JAK3 might represent an effective approach to reduce inflammation-mediated apoptotic damage initiated by myocardial I/R injury.
Animals ; Apoptosis/drug effects ; Cell Movement/drug effects ; Chemokines/pharmacology ; Heart Function Tests/drug effects ; Inflammation/pathology ; Janus Kinase 3/*antagonists & inhibitors/metabolism ; Macrophages/drug effects/metabolism/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Myocardial Reperfusion Injury/drug therapy/*enzymology/physiopathology/*prevention & control ; Myocardium/enzymology/pathology ; Myocytes, Cardiac/drug effects/metabolism/pathology ; Neutrophils/drug effects/metabolism/pathology ; Quinazolines/pharmacology/therapeutic use

An activation domain in p67(phox) (residues 199-210) is critical for regulating NADPH oxidase activity in cell-free system [10] To determine the steady state reduction of FAD, thioacetamide-FAD was reconstituted in gp91(phox), and the fluorescence of its oxidised form was monitored. Omission of p67(phox) decreased the steady state reduction of the FAD from 28% to 4%, but omission of p47(phox) had little effect. A series of the truncated forms of p67(phox) were expressed in E.coli to determine the domain in p67(phox) which is essential for regulating the steady state of FAD reduction. The minimal length of p67(phox) for for regulating the steady state of FAD reduction is shown to be 1-210 using a series of truncation mutants which indicates that the region 199-210 is also important for regulating electron flow within flavocytochrome b(558). The deletion of this domain not only decreased the superoxide generation but also decreased the steady state of FAD reduction. Therefore, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.
Amino Acid Sequence ; Base Sequence ; Cell Membrane/metabolism ; Cell-Free System ; DNA Primers ; Flavin-Adenine Dinucleotide/*metabolism ; Humans ; Kinetics ; Membrane Glycoproteins/*metabolism ; Molecular Sequence Data ; NADH Dehydrogenase/metabolism ; *NADPH Oxidase ; Neutrophils/enzymology/metabolism ; Oxidation-Reduction ; Peptide Fragments/chemistry ; Phosphoproteins/*chemistry/*metabolism ; Polymerase Chain Reaction ; Sequence Deletion

PURPOSE: Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of pulmonary fibrosis. To understand the role of MMP-2 and MMP-9 in pulmonary fibrosis, we evaluated the sequential dynamic change and different cellular sources of the 2 MMPs along the time course and their differential expression in the bronchoalveolar lavage (BAL) fluid and in the lung parenchyma of the bleomycin-induced pulmonary fibrosis models in rats. MATERIALS AND METHODS: The level of MMPs in BAL fluid of 54 bleomycin-treated rats was assessed by zymography from 1 to 28 days after intratracheal bleomycin instillation. The level of MMPs in lung parenchyma was evaluated by immunohistochemistry. RESULTS: MMP-2 and MMP-9 were markedly increased in both the BAL fluid and in the lung parenchyma of the bleomycin-treated rats, especially in the early phase with the peak on the 4th day. The levels of both MMPs in the BAL fluid correlated generally well to those in lung parenchyma, although the level of MMP-9 in BAL fluid was higher than MMP-2. In the lung parenchyma, the 2 MMPs, in early stage, were predominantly expressed in the inflammatory cells. In late stage, type II pneumocytes and alveolar epithelial cells at the periphery of the fibrotic foci retained MMP expression, which was more prominent in the cells showing features of cellular injury and/or repair. CONCLUSION: In bleomycin-induced pulmonary fibrosis, MMP-2 and MMP-9 may play important roles, especially in the early phase. In the late stage, the MMP-2 and MMP-9 may play a role in the process of repair.
Animals ; Antibiotics, Antineoplastic/toxicity ; Bleomycin/toxicity ; Bronchioles/*enzymology/pathology ; Bronchoalveolar Lavage Fluid/cytology/immunology ; Disease Models, Animal ; Enzyme Activation ; Gelatin ; Immunohistochemistry ; Male ; Matrix Metalloproteinase 2/*metabolism ; Matrix Metalloproteinase 9/*metabolism ; Neutrophils/pathology ; Pulmonary Fibrosis/chemically induced/*metabolism/*pathology ; Rats ; Rats, Sprague-Dawley