Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

ABSTRACT Background: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p 0.001) or indirectly (p 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.

Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)

Cytological endometritis in dairy cows: diagnostic threshold, risk factors, and impact on reproductive performance

We determined the threshold proportion of polymorphonuclear leukocytes (PMNs) for a diagnosis of cytological endometritis (CEM), the risk factors for this condition, and its impact on reproductive performance in dairy cows. Uterine cytology was performed on 407 Holstein cows 4 weeks postpartum to determine the proportions of endometrial cells and PMNs. A receiver operator characteristics curve was used to determine the threshold above which the PMN proportion affected the likelihood of cows conceiving by 200 days postpartum. The optimal threshold was ≥ 14% PMN (sensitivity, 31.3%; specificity, 81.7%; p < 0.05). The farm identity, retained placenta (odds ratio [OR] = 1.87), and septicemic metritis (OR = 3.07) were risk factors for CEM (p < 0.05). Cows with CEM were less likely to resume cyclicity (OR = 0.58) and to conceive by 200 days postpartum (hazard ratio = 0.58). Cows with CEM tended (p < 0.1) to be less likely to become pregnant after their first insemination (OR = 0.65) and to require a greater number of inseminations per conception (2.3 vs. 2.2). In conclusion, a PMN threshold of 14% defined the presence of CEM at 4 weeks postpartum. The farm, retained placenta, and septicemic metritis were risk factors for CEM, which reduces subsequent reproductive performance.

Clinical value of neutrophil gelatinase -associated lipocalin and lactate dehydrogenase detection for screening of bacterial peritonitis in patients with ascites / 中国基层医药

Objective To build a simple,automatable detection method for diagnosing bacterial peritonitis. Methods Neutrophil gelatinase -associated lipocalin(NGAL),lactate dehydrogenase(LDH),proteins and glucose were assessed in peritoneal fluids from patients with nonmalignant ascites,along with nucleated cell count and differ-entiation.Results One hundred and eleven specimens were analyzed,26 of which with PMN count≥250 ×106 /L, thus compatible with bacterial peritonitis.The median concentration of LDH and NGAL was 3.4 and 3.7 -fold higher in samples with PMN≥250 ×106 /L.The concentration of proteins was also higher in samples with ≥250 ×106 PMN /L, whereas that of glucose was lower.The PMN count significantly correlated with peritoneal fluid values of LDH(r =0.859),NGAL(r =0.774)and proteins(r =0.268),but not with glucose(r =-0.069).The area under ROC curve was 0.88 for LDH,0.89 for NGAL and 0.94 for their combination(both tests positive),whereas that of proteins and glucose was 0.80 and 0.71,respectively.Sensitivities and specificities were 0.81 and 0.87 for LDH≥227U /L, 0.96 and 0.75 for NGAL≥120μg/L,0.77 and 0.95 for their combination.The agreement with PMN count was 0.86 for LDH,0.80 for NGAL,and 0.91 for their combination.Conclusion Assessment of NGAL in peritoneal flu-ids,especially in combination with LDH,may be a reliable approach for screening of bacterial peritonitis in patients with nonmalignant ascites.

Increased expression of myeloperoxidase,CD11b and IL-8 in rat asthma model / 基础医学与临床

Objective To observe expression of myeloperoxidase(MPO),CD11b and IL-8 and to studay the functions and possible mechanism of polymorphonuclear leukocyte(PMN) in rat asthma.Methods The rats were randomly divided into two groups.Blood PMN were isolated and purified,MPO were detected by immunohistochemistry techniques,IL-8 was detected by ELISA,blood PMN CD11b were detected by flow cytometry(MFI).The total cell numbers and differential cell numbers in BALF were counted.Results MPO in bronchial wall and blood PMN,CD11b and IL-8 in blood and BALF expressions in asthma group were significantly increased than those in control group(P

Effect of Amrinone, a Selective Inhibitor of Phosphodiesterase III, on PMNs-induced Cardiac Dysfunction in Ischemia/reperfusion

Ischemia followed by reperfusion in the presence of polymorphonuclear leukocytes (PMNs) results in a marked cardiac contractile dysfunction. Amrinone, a specific inhibitor of phosphodiesterase 3, has an antioxidant activity against PMNs. Therefore, we hypothesized that amrinone could attenuate PMNs-induced cardiac dysfunction by suppression of reactive oxygen species (ROS) produced fby PMNs. In the present study, we examined the effects of amrinone on isolated ischemic (20 min) and reperfused (45 min) rat hearts perfused with PMNs. Amrinone at 25microM, given to hearts during the first 5 min of reperfusion, significantly improved coronary flow, left ventricular developed pressure (P< 0.001), and the maximal rate of development of left ventricular developed pressure (P< 0.001), compared with ischemic/reperfused hearts perfused with PMNs in the absence of amrinone. In addition, amrinone significantly reduced myeloperoxidase activity by 50.8%, indicating decreased PMNs infiltration (p< 0.001). Superoxide radical and hydrogen peroxide production were also significantly reduced in fMLP- and PMA-stimulated PMNs pretreated with amrinone. Hydroxyl radical was scavenged by amrinone. fMLP-induced elevation of [Ca2+]i was also inhibited by amrinone. These results provide evidence that amrinone can significantly attenuate PMN-induced cardiac contractile dysfunction in the ischemic/ reperfused rat heart via attenuation of PMNs infiltration into the myocardium and suppression of ROS release by PMNs.

Effect of Defibrotide on Rat Reflux Esophagitis

This study was aimed at evaluating the effect of defibrotide on the development of the surgically induced reflux esophagitis, on gastric secretion, lipid peroxidation, polymorphonuclear leukocytes (PMNs) accumulation, polymorphonuclear leukocytes adherence, superoxide anion and hydrogen peroxide production in PMNs, scavenge of hydroxyl radical and hydrogen peroxide, cytokine (interleukin-1beta, tumor necrosis factor-alpha) production in blood, and intracellular calcium mobilization in PMNs. Defibrotide did not inhibit the gastric secretion and not change the gastric pH. Treatment of esophagitis rats with defibrotide inhibited lipid peroxidation, and myeloperoxidase (MPO) in the esophagus in comparison with untreated rats. Defibrotide significantly decreased the PMN adherence to superior mesenteric artery endothelium in a dose-dependent manner. Superoxide anion and hydrogen peroxide production in 1microM formylmethionylleucylphenylalanine (fMLP) - or 0.1microgram/ml N-phorbol 12- myristate 13-acetate (PMA) -activated PMNs was inhibited by defibrotide in a dose-dependent fashion. Defibrotide effectively scavenged the hydrogen peroxide but did not scavenge the hydroxyl radical. Treatment of esophagitis rats with defibrotide inhibited interleukin-1beta production in the blood in comparison with untreated rats, but tumor necrosis factor-alpha production was not affected by defibrotide. The fMLP-induced elevation of intracellular calcium in PMNs was inhibited by defibrotide. The results of this study suggest that defibrotide may have partly beneficial protective effects against reflux esophagitis by the inhibition lipid peroxidation, PMNs accumulation, PMNs adherence to endothelium, reactive oxygen species production in PMNs, inflammatory cytokine production (i.e. interleukin-1beta), and intracellular calcium mobilization in PMNs in rats.

Effects of Hyaluronic Acid on the Polymorphonuclear Leukocyte (PMN) Release of Active Oxygen and Protection of Bovine Corneal Endothelial Cells from Activated PMNs

The goal of this study was to evaluate the function of hyaluronic acid (HA) on the active oxygen release from polymorphonuclear leukocytes (PMNs) and the protective effect of bovine corneal endothelial cells (BCEC) from activated PMNs. We used HA with three different molecular weights (MW 700, 000, 2, 000, 000, and 4, 000, 000) and five different concentrations (0, 0.1, 1, 2, and 3 mg/ml). We evaluated the amount of released superoxide from activated PMNs by using dismutase-inhibitable ferricytochrome C reduction. To compare the property and protective effect of HA with those of other viscoelastic substances, we used the same concentration of methylcellulose. HA suppressed superoxide release from PMNs and protected BCEC from activated PMNs in a dose-dependent, rather than a molecular weight-dependent, manner. The effect of HA reached almost a plateau at concentration above 2 mg/ml. However, methylcellulose, another viscoelastic substance, showed a similar effect. Therefore, it seems that the suppression of superoxide released from PMNs is not a property that is unique to HA, but is a general property of viscoelastic substances. Our results indicate that the action mechanism of HA proceeds not only through cell surface HA-receptor. We think that HA also acts as a physical barrier and/or a scavenger of superoxide.

Increased Reactive Oxygen Species Production by Peripheral Blood Mononuclear Leukocytes, not by Polymorphonuclear Leukocytes, in Atopic Dermatitis / 소아알레르기및호흡기학회지

PURPOSE: Reactive oxygen species (ROS) are known as a potential mediators that sustain chronic inflammation in atopic dermatitis (AD). To determine the role of peripheral blood mononuclear leukocytes (MO) and polymorphonuclear leukocytes (PMN) in prolonged inflammation, ROS generation of those cells in AD was examined. METHODS: Seventeen AD patients and 10 healthy controls were enrolled. MO and PMN were stimulated with the reagents: phobol ester (PMA), adenosine triphosphate (ATP), and chemotactic peptide (f-MLP). ROS levels were measured using chemiluminescence assay. RESULTS: In AD, chemiluminescence response of unstimulated MO was higher than that of normal controls. MO from AD patients produced 1.58-1.80 higher ROS for up to 30 minutes than the controls. When the cells were treated with the reagents (PMA, ATP, f-MLP), all the stimuli enhanced chemiluminescence activity of MO. When MO were treated with PMA, the ratio of ROS produced by MO of patients to that of the controls decreased. When the cells were treated with either ATP or f-MLP, the quantity of ROS generated by MO from the controls was greater than the controls. PMN from both AD patients and the controls generated ROS for 30 min similarly. As treated with the reagents, PMN from AD patients produced a smaller ROS than the controls. CONCLUSION: These results indicate MO but not PMN from AD patients were primed and ready for activation in vivo, and a reduced function of PMN from AD patients was observed. In conclusion, enhanced respiratory burst activity of MO is implicated in the prolonged inflammation of AD.

A Murine Model of Toluene Diisocyanate-induced Contact Hypersensitivity

BACKGROUND: Toluene diisocyanate (TDI) can cause contact allergy and occupational asthma, but the mechanism underlying sensitization to this chemical compound remains controversal. Also the correlation of mast cell with contact hypersensitivity (CHS) and the role of mast cell in the TDI-induced CHS is unknown. This issue was investigated by administrating TDI on the skin of genetically mast cell-deficient WBB6F1/J-Kit(W)/ Kit(W-v) (W/W(V)) and congenic normal WBB6F1/J-Kit +/+ (+/+) mice. METHODS: To development of animal model of TDI-induced CHS and to investigate the correlation of mast cell with CHS and the role of mast cell in the TDI-induced CHS, W/W(V) and +/+ mice were sensitized with TDI on the back skin at day 1 and day 8, and then challenged with 1% TDI on the ear at day 15. At 1, 2, 4, 8, and 24 hours after 1% TDI challenge, the ear thicknesses were measured. It was investigated the histologic changes of dermis in the ear of W/W(V) and +/+ mice at 24 hours after 1% TDI challenge. RESULTS: TDI induced a significant ear swelling response in W/W(V) and +/+ mice. TDI induced the significant infiltrations of polymorphonuclear leukocytes and eosinophils in W/W(V) and +/+ mice, but not of mast cells in normal mice. And TDI increased a characteristic extent of mast cell degranulation in normal mice. There were no significant differences in the ear swelling and the infiltrations of polymorphonuclear leukocytes and eosinophils of normal versus W/W(V) mice, either at baseline or after TDI-induced CHS. CONCLUSION: From the above results, TDI can be used as a murine CHS model, and the mast cells may not be essential in TDI-induced CHS.

A Murine Model of Toluene Diisocyanate-induced Contact Hypersensitivity

BACKGROUND: Toluene diisocyanate (TDI) can cause contact allergy and occupational asthma, but the mechanism underlying sensitization to this chemical compound remains controversal. Also the correlation of mast cell with contact hypersensitivity (CHS) and the role of mast cell in the TDI-induced CHS is unknown. This issue was investigated by administrating TDI on the skin of genetically mast cell-deficient WBB6F1/J-Kit(W)/ Kit(W-v) (W/W(V)) and congenic normal WBB6F1/J-Kit +/+ (+/+) mice. METHODS: To development of animal model of TDI-induced CHS and to investigate the correlation of mast cell with CHS and the role of mast cell in the TDI-induced CHS, W/W(V) and +/+ mice were sensitized with TDI on the back skin at day 1 and day 8, and then challenged with 1% TDI on the ear at day 15. At 1, 2, 4, 8, and 24 hours after 1% TDI challenge, the ear thicknesses were measured. It was investigated the histologic changes of dermis in the ear of W/W(V) and +/+ mice at 24 hours after 1% TDI challenge. RESULTS: TDI induced a significant ear swelling response in W/W(V) and +/+ mice. TDI induced the significant infiltrations of polymorphonuclear leukocytes and eosinophils in W/W(V) and +/+ mice, but not of mast cells in normal mice. And TDI increased a characteristic extent of mast cell degranulation in normal mice. There were no significant differences in the ear swelling and the infiltrations of polymorphonuclear leukocytes and eosinophils of normal versus W/W(V) mice, either at baseline or after TDI-induced CHS. CONCLUSION: From the above results, TDI can be used as a murine CHS model, and the mast cells may not be essential in TDI-induced CHS.

The Effect of A Polypeptide Factor Released by Polymorphonuclear Leukocytes (PMNs Factor)on Rabbit Corneal Epithelial Stem Cells

In case of limbitis, cytokines secreted by PMNs might inhibit the function of corneal epithelial stem cells located at the limbal basal cell layer. The purpose of this study was to examine whether PMNs factor affected stem cells (SC). PMNs obtained from the peritoneal cavity of rabbits after 0.2% glycogen stimulation incubated in DMEM at 37 degrees C for 24 hours. PMNs factor was obtained from the medium using dialysis. The death or inhibition of growth of rabbit corneal epithelial cells (CE) by PMNs factor was studied in vitro. PMNs factor and PBS as a control were injected into the limbal region for clinical evaluation and histopathologic study. ID 50 of PMNs factor for cultured CE was equal to the amount of polypeptide produced by 1.29 x 0 (7) PMNs. Cultured rabbit CE shrank and began to die 24 hours after exposure to PMNs factor. Abnormal findings were observed 5 days and 8 months after injection of PMNs factor at the limbus by light and electron microscopy. Clouding, defect, and vascularization of CE were noted several months after injection of PMNs factor. PMNs in limbitis may damage corneal epithelial stem cells and cause epithelial instability of the cornea.

Appearance of Polymorphonuclear Leukocytes Following Corneal Scratching Wound by Impression Cytology

The linear scratching wound was made gently on the corneal epithelium of rabbit with 21 gauge needle under an operating microscope. Impression cytology was performed at 30 minutes, 1, 2, 3 and 4 hour and 1, 2, 3 and 4 day after 0.5% tetracaine drops under an operating microscope. The filter paper was stained with hematoxylin and eosin. At 30 minute post-scratching, a few polymorphonuclear leukocytes appeared on the scratched cornea at 3 eyes (30%). At 3 and 4 hour, numerous polymorphonuclear leukocytes with corneal epithelial cells appeared on the scratched conea. By 3 day, no inflammatory cells were shown on the filter paper in all eyes. These findings suggest that the polymorphonuclear leukocytes could infiltrate on the corneal lesion at 30 minute post-scratching and the inflammatory cells might act even on the minute corneal lesion such as corneal erosion.

Prolongation of human polymorphonuclear leukocyte function by IL-1? / 中国药理学通报

AIM Interleukin 1?(IL-1?), which is a pivotal cytokine in inflammatory response, can inhibit human polymorphonuclear leukocyte (PMN) apoptosis,which can enhance PMN-mediated cytoxicity. Therefore we research the effect of IL-1? on the change of PMN functions was studied. METHODS Functional capacity of PMN was assessed by spreading, polarization (shape change), superoxide anion production and phagocytosis. RESULTS IL-1? can prolong PMN function by the inhibition of PMN apoptosis. CONCLUSION IL-1? inhibits PMN apoptosis, resulting in a larger population of surviving PMN with a greater capacity for phagocytosis and superoxide production. The later potentially facilitates PMN mediated tissue injury and may be one of mechanisms by which IL-1? contributes to organ dysfunction.