Peripheral Polymorphonuclear Leukocytes Research Articles

Peptides Derived from Soybean β-Conglycinin Induce the Migration of Human Peripheral Polymorphonuclear Leukocytes.

Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein β-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of β-conglycinin, induces the migration of dibutyryl cAMP (Bt2 cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt2 cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt2 cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean β-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

Effect of injectable trace mineral supplementation on peripheral polymorphonuclear leukocyte function, antioxidant enzymes, health, and performance in dairy cows in semi-arid conditions

The objective of this study was to evaluate the effect of subcutaneous injections of 15 mg/mL Cu, 5 mg/mL Se, 60 mg/mL Zn, and 10 mg/mL Mn on health, performance, polymorphonuclear leukocyte (PMNL) function, circulating glutathione peroxidase (GPx) and superoxide dismutase (SOD) concentrations, and inflammation of dairy cows undergoing the transition period in high temperature-humidity index. A total of 923 multiparous cows from 2 commercial dairy farms were randomly allocated into 1 of 2 treatment groups as follows: control and injectable trace mineral supplementation (ITMS). Cows in the ITMS group received 7 mL of subcutaneous injections at dry-off (208 ± 3 d of gestation), 260 ± 3 d of gestation, and at 35 ± 3 d in milk (DIM). Data regarding health traits, reproductive performance, milk yield, and survivability were extracted from farm database software, and animals were followed-up until 300 DIM. For a subset of 142 cows from one herd, blood samples were collected at enrollment, and at 3 ± 1, 7 ± 1, 10 ± 1, and 35 ± 3 DIM to evaluate hematology, PMNL function, GPx and SOD concentrations, and circulating haptoglobin. Logistic regression was used to assess health and pregnancy per artificial insemination at first service. Cox proportional hazards models were used to evaluate hazard of pregnancy and culling. Mixed linear regression models accounting for repeated measures were used to assess all continuous variables collected over time. Parity, twinning, and previous gestation length were considered as potential confounders. Farm was included as a random effect. The ITMS cows tended to have lower incidence of metritis and stillbirth compared with control group. However, ITMS treatment did not influence the incidence of other diseases (e.g., mastitis, retained placenta), milk yield, reproductive performance, culling, and leukocyte count. Neutrophil-to-lymphocyte ratio, PMNL phagocytosis, and oxidative burst as well as intensity of the oxidative burst were greater for ITMS-treated cows in comparison to control cows. The ITMS cows had decreased expression of the adhesion molecule L-selectin on PMNL surface. The serum concentration of GPx and SOD were not affected by ITMS treatment. In conclusion, ITMS tended to reduce the incidence of metritis and stillbirth parturition, improved PMNL function, and improved the inflammatory status of dairy cows undergoing the transition period in high temperature-humidity index conditions. However, these findings did not translate into improved milk yield, reproductive performance, and survivability.

Oxidative Stress in Patients with Advanced CKD and Renal Replacement Therapy: The Key Role of Peripheral Blood Leukocytes.

Oxidative stress plays a key role in the pathophysiology of chronic kidney disease (CKD). Most studies have investigated peripheral redox state focus on plasma, but not in different immune cells. Our study analyzed several redox state markers in plasma and isolated peripheral polymorphonuclear (PMNs) and mononuclear (MNs) leukocytes from advanced-CKD patients, also evaluating differences of hemodialysis (HD) and peritoneal dialysis (PD) procedures. Antioxidant (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH)) and oxidant parameters (xanthine oxidase (XO), oxidized glutathione (GSSG), malondialdehyde (MDA)) were assessed in plasma, PMNs and MNs from non-dialysis-dependent-CKD (NDD-CKD), HD and PD patients and healthy controls. Increased oxidative stress and damage were observed in plasma, PMNs and MNs from NDD-CKD, HD and PD patients (increased XO, GSSG and MDA; decreased SOD, CAT, GPX and GSH; altered GSSG/GSH balance). Several oxidative alterations were more exacerbated in PMNs, whereas others were only observed in MNs. Dialysis procedures had a positive effect on preserving the GSSG/GSH balance in PMNs. Interestingly, PD patients showed greater oxidative stress than HD patients, especially in MNs. The assessment of redox state parameters in PMNs and MNs could have potential use as biomarkers of the CKD progression.

Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows.

Simple SummaryDairy cows experience immune suppression around calving, which can result in the development of uterine diseases. White blood cells, and more specifically polymorphonuclear leukocytes (PMN), are important components of the immune system. Although it is regarded as a common research approach to study PMN isolated from blood, it would be interesting to know more about the viability and functionality of uterine PMN. We developed a method to isolate PMN from the bovine postpartum uterus to perform viability and functionality tests. We also evaluated whether we could identify uterine PMN using a specific antibody. Uterine cells were recovered using an adapted medical brush, and PMN were successfully isolated and identified. The percentage of viable intra-uterine PMN in postpartum cows (9–37 days in milk) roughly ranged from 10 to 80%, indicating that the viability of the uterine PMN is highly dynamic. We could also identify PMN that ingested labeled particles, which lets us conclude that uterine PMN are functional. Using the presently described methods, further research can be performed to unravel the role of uterine PMN viability and functionality in bovine uterine health.Postpartum dairy cows experience impaired peripheral polymorphonuclear leukocyte (PMN) functionality, which has been associated with reproductive tract inflammatory diseases. However, it has not been elucidated yet whether endometrial PMN functionality is (equally) impaired. We developed a method for endometrial PMN isolation and flow cytometric assessment of their viability and functionality. We also evaluated PMN immunolabeling, using a specific bovine granulocyte marker, CH138A. Blood and endometrial cytobrush samples were collected in duplicate from seventeen clinically healthy Holstein-Friesian cows between 9 and 37 days in milk. The proportion of viable, apoptotic, and necrotic PMN in endometrial samples roughly ranged from 10 to 80%, indicating highly dynamic endometrial PMN populations in the postpartum uteri. Endometrial PMN functionality testing revealed that PMN immunolabeling increased the accuracy, although this protocol might influence the median fluorescence intensity of the sample. Phagocytosis seemed the most stable and reliable endometrial PMN function and could be assessed satisfactorily without prior CH138A immunolabeling. However, the interpretation of oxidative burst and intracellular proteolysis tests remains challenging. The correlation between peripheral and endometrial PMN functionality was poor. Further research is warranted to unravel the role of uterine PMN viability and functionality in bovine uterine health.

In vitro effects of nonesterified fatty acids and β-hydroxybutyric acid on inflammatory cytokine expression in bovine peripheral blood leukocytes

Negative energy balance (NEB) occurs in dairy cows during the transition period, during which time the blood concentrations of nonesterified fatty acids (NEFA) and metabolic ketones such as β-hydroxybutyric acid (BHBA) are elevated. These increased levels may disrupt immune functions and are regarded as risk factors associated with postparturient inflammatory disorders. Therefore, the aim of this study was to investigate the effects of NEFAs and BHBA on inflammatory cytokine expression in bovine peripheral leukocytes in vitro. Peripheral blood mononuclear cells (PBMCs) and peripheral polymorphonuclear leukocytes (PMNLs) were collected from 16 Holstein cows and treated for 4 h with NEFA (0.1, 0.6, and 1.5 mM) or BHBA (5 and 10 mM), alone or combination with lipopolysaccharides (LPS, 1 µg/mL). The expression levels of the inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and IL-10 were then determined. The results indicated that PBMCs and PMNLs responded to NEFA but tolerated BHBA. NEFA treatment dose-dependently induced the expression of TNF-α, IL-1β, IL-8, and IL-10 in PBMCs and that of IL-1β, IL-6, IL-8, and IL-10 in PMNLs. Combination treatment of LPS with NEFA further increased the levels of IL-10 expression in PBMCs and IL-1β and IL-6 expression in PMNLs. Taken together, these findings suggest that NEFA and BHBA enhance the expression of inflammatory cytokines in PBMCs and PMNLs and may disrupt the immune regulation of peripheral leukocytes, leading to an increased risk of inflammatory disorders. Preventing increases in blood NEFA concentration may thus help to reduce the risk of inflammatory disorders in dairy cows during the transition period. Highlights In cows with NEB, during the transition period, NEFA but not BHBA induced proinflammatory cytokine expression in PBMCs and PMNLs. NEFA enhanced LPS-induced proinflammatory cytokine expression in both PBMCs and PMNLs.

Immunological dysfunction in chronic arsenic exposure: From subclinical condition to skin cancer.

Exposure to arsenic is a global health issue. Long-term arsenic exposure may associate with various cancers and many other pathological effects. Over 100 million people worldwide are exposed to arsenic particularly in countries such as Bangladesh, Chile, China, India, Mexico, Taiwan and the USA. Drinking of water contaminated with arsenic is the major route of human exposure. Skin lesions are considered to be the most common adverse effects associated with chronic arsenic exposure. Skin lesions usually develop with the latency period spanning more than 20 years from first exposure. Arsenic-induced Bowen's disease, the most frequently encountered carcinoma in situ resulting from chronic arsenic exposure, is characterized by multiple and recrudescent lesions. Long-term arsenic exposure results in impaired immunity in susceptible individuals. In the prenatal stage, enhanced placental inflammatory responses and reduced placental T cells by arsenic may result in decreased thymic size and functions in newborns. In childhood, arsenic exposure may reduce peripheral CD4+ cells and interleukin-2 secretion which leads to susceptibility to opportunistic infections. There was an impairment of macrophage function and oxidative DNA damage of peripheral polymorphonuclear leukocytes in adults with skin lesions. In arsenic-induced Bowen's disease lesions, a decrease in the number and functions of Langerhans cells and, in parallel, a selective CD4+ cell apoptosis was noticed. These findings provide scientific evidence for understanding the phenomenon of arsenic-induced immune escape in the early stage of carcinogenesis and a reasonable explanation for multiple and recrudescent arsenic cancers in the skin.

Free circulating active elastase contributes to chronic inflammation in patients on hemodialysis.

Atherosclerosis and cardiovascular complications are prevalent among patients undergoing chronic hemodialysis (HD). In this population, peripheral polymorphonuclear leukocytes (PMNLs) are primed, releasing proinflammatory mediators such as elastase. Elastase is normally inhibited by a specific inhibitor, avoiding undesirable degradation of cellular and extracellular components. This study tested the hypothesis that in states of noninfectious inflammation, elastase is released by PMNLs and acts in an uncontrolled manner to inflict vascular damage. Blood was collected from patients undergoing HD and healthy controls (HC). PMNL intracellular and surface expressions of elastase were determined by quantitative real-time PCR, Western blotting, and flow cytometry. The elastase activity was evaluated using a fluorescent substrate. The levels of serum α1-antitrypsin (α1-AT), the natural elastase inhibitor, were determined by Western blot. Free active elastase was elevated in HD sera, whereas the levels of α1-AT were decreased compared with HC. The levels of the intracellular elastase enzyme and its activity were lower in HD PMNLs despite similar expression levels of elastase mRNA. Elastase binding to PMNL cell surface was higher in HD compared with HC. The increased circulating levels of free active elastase released from primed HD PMNLs together with the higher cell surface-bound enzymes and the lower levels of α1-AT result in the higher elastase activity in HD sera. This exacerbated elastase activity could lead to the endothelial dysfunction, as hypothesized. In addition, it suggests that free circulating elastase can serve as a new biomarker and therapeutic target to reduce inflammation and vascular complications in patients on hemodialysis.

Primed polymorphonuclear leukocytes from hemodialysis patients enhance monocyte transendothelial migration

Increased counts and priming of peripheral polymorphonuclear leukocytes (PMNLs) are associated with future or ongoing atherosclerosis; however, the role of PMNLs in enhancing monocyte transendothelial migration is still unclear. Our aims were to examine endothelial and monocyte activation, transmigration, and posttransmigration activation induced ex vivo by in vivo primed PMNLs and the effect of antioxidants on the activation. A unique ex vivo coculture system of three cell types was developed in this study, enabling interactions among the following: primary human umbilical vein endothelial cells (HUVECs), monocytes (THP-1 cell line), and in vivo primed PMNLs from hemodialysis (HD) patients and healthy control (HC) subjects. The interactions among these cells were examined, and an intervention with superoxide dismutase and catalase was performed. Preexposed HUVECs to HD/HC PMNLs showed a significant monocyte transmigration yield, 120-170% above HCs. Monocyte exposure to HD PMNLs induced pre- and posttransmigration activation. When the three cell types were cocultivated at the same time, monocyte chemoattractant protein-1 protein levels released from HUVECs, and activation markers on HUVECs [CD54 and chemokine (C-X3-C motif) ligand 1] and monocytes [chemokine (C-X3-C) receptor 1 and chemokine (C-C motif) receptor 2] were increased. Monocyte transmigration yield decreased to 70% (compared with HC subjects) due to adherence and accumulation of monocytes to HUVECs. When superoxide dismutase and catalase were used, reduced HUVEC and monocyte activation markers brought the transmigration yields to control levels and abolished accumulation of monocytes, emphasizing the role of superoxide in this process. We conclude that peripheral primed PMNLs play a pivotal role in enhancing monocyte transendotelial migration, the hallmark of the atherosclerotic process. Primed PMNLs can be used as a mediator and a biomarker of atherosclerosis even before plaque formation.NEW & NOTEWORTHY Primed polymorphonuclear leukocytes are key mediators in monocyte transendothelial migration, a new understanding of the initiation of endothelial dysfunction and monocyte activation, transmigration, and accumulation in the subendothelial layer.

Effects of intrauterine infusion of Escherichia coli lipopolysaccharide on uterine mRNA gene expression and peripheral polymorphonuclear leukocytes in Jersey cows diagnosed with purulent vaginal discharge

The objectives of the current experiment were to investigate the effects of intrauterine treatment of cows with purulent vaginal discharge (PVD) using lipopolysaccharide (LPS) from Escherichia coli on uterine mRNA expression of genes related to inflammatory responses, peripheral polymorphonuclear leukocyte (PMN) function, hematological parameters, and blood concentrations of cortisol, haptoglobin, and progesterone (P4). Jersey cows (n = 3,084) were examined for PVD at 35 ± 6 d postpartum using the Metricheck device (Simcro, Hamilton, New Zealand). At examination, 310 cows had PVD (10.1%), but to ensure proper collection and processing of samples, 267 cows were used in this experiment. Cows were balanced for lactation number, body condition score, and milk yield, and randomly assigned to the control treatment [intrauterine infusion of 20 mL of phosphate-buffered saline (PBS); n = 87] or to receive intrauterine infusion of 20 mL of PBS containing 150 µg (LPS150; n = 91) or 300 µg (LPS300; n = 89) of E. coli LPS. Uteri were biopsied in a subgroup of cows at 6 h after infusion and in another subgroup of cows at 24 h after infusion. Peripheral PMN expression of adhesion molecules (L-selectin and MAC-1) and phagocytosis and oxidative burst were evaluated at 0, 2, and 6 h after infusion. Blood sampled 0, 2, 6, 24, 48, and 168 h after infusion was used for complete hemogram and to determine concentrations of cortisol, haptoglobin, and P4. Treatment did not affect uterine mRNA expression of adhesion molecules [endothelial leukocyte adhesion molecule (E-selectin), intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)], cytokines (tumor necrosis factor-α, IL-1β, IL-6, IL-8, and IL-10), and toll-like receptor-4. Treatment did not affect PMN expression of L-selectin, but intensity of expression of MAC-1 was higher for LPS150 cows than PBS cows, and tended to be higher in LPS150 than LPS300 cows. Furthermore, a greater percentage of PMN from LPS300 cows were positive for phagocytosis and oxidative burst compared with PBS and LPS150 cows. No effects were observed of treatment on hematological parameters and concentrations of cortisol, haptoglobin, and P4. These observations suggest that intrauterine infusion of E. coli LPS moderately stimulates peripheral PMN function, but further research is needed to better understand the immunomodulatory effects of LPS in the uterus of cows with PVD.

Sexual differences and cellular diversity of rat peripheral polymorphonuclear leukocytes monitored with a monoclonal antibody, RP-3

Sexual differences and cellular diversity of rat peripheral polymorphonuclear leukocytes monitored with a monoclonal antibody, RP-3

Maternal Undernutrition Programs Tissue-Specific Epigenetic Changes in the Glucocorticoid Receptor in Adult Offspring

Epidemiological data indicate that an adverse maternal environment during pregnancy predisposes offspring to metabolic syndrome with increased obesity, and type 2 diabetes. The mechanisms are still unclear although epigenetic modifications are implicated and the hypothalamus is a likely target. We hypothesized that maternal undernutrition (UN) around conception in sheep would lead to epigenetic changes in hypothalamic neurons regulating energy balance in the offspring, up to 5 years after the maternal insult. We found striking evidence of decreased glucocorticoid receptor (GR) promoter methylation, decreased histone lysine 27 trimethylation, and increased histone H3 lysine 9 acetylation in hypothalami from male and female adult offspring of UN mothers. These findings are entirely compatible with the increased GR mRNA and protein observed in the hypothalami. The increased GR predicted the decreased hypothalamic proopiomelanocortin expression and increased obesity that we observed in the 5-year-old adult males. The epigenetic and expression changes in GR were specific to the hypothalamus. Hippocampal GR mRNA and protein were decreased in UN offspring, whereas pituitary GR was altered in a sex-specific manner. In peripheral polymorphonuclear leukocytes there were no changes in GR methylation or protein, indicating that this epigenetic analysis did not predict changes in the brain. Overall, these results suggest that moderate changes in maternal nutrition, around the time of conception, signal life-long and tissue-specific epigenetic alterations in a key gene regulating energy balance in the hypothalamus.

Peripheral polymorphonuclear leukocyte activation as a systemic inflammatory response in ischemic stroke

Stroke is one of the leading causes of mortality and morbidity in the world. The activation of polymorphonuclear leukocytes (PMNL) plays an important role in the inflammatory response after ischemic stroke. However, in the current literature, there are few studies discussing the process and role of peripheral PMNL activation. Here, we give a comprehensive description of peripheral PMNL activation after ischemic stroke and discuss their potential roles in the process of ischemic injury. Based on our analysis, peripheral PMNL activation is supposed to be attributed to systemic inflammatory response to cerebral ischemic insult, not reflecting the activity of PMNL in local ischemic brain. Inhibiting peripheral PMNL activation in stroke animals has been effective in reducing infarction and improving behavioral outcome; thus, the same approach of inhibiting peripheral PMNL activation is a promising therapeutic strategy for stroke patients.

Lercanidipine effect on polymorphonuclear leukocyte-related inflammation and insulin resistance in essential hypertension patients

Inflammation, insulin resistance and oxidative stress (OS), are among the mechanisms that have been implicated in pathogenesis of essential hypertension (EH). Peripheral polymorphonuclear leukocytes (PMNLs) are primed in EH patients, releasing uncontrolled superoxide anion contributing to OS in these patients. PMNL priming correlates with insulin resistance and with PMNL intracellular calcium ([Ca2+]i). Recent studies have attributed to the anti-hypertensive drug lercanidipine, a third generation calcium-channel blocker, and additional anti-ischemic and anti-oxidative characteristics. To evaluate the possible non-traditional effect of two months of lercanidipine treatment on insulin resistance and on PMNL-related inflammation in EH patients. Non-smoking EH patients with untreated mild to moderate high blood pressure (BP) were included. Low-graded inflammation was reflected by WBC and PMNL counts and by PMNL apoptosis. Systemic inflammation was measured by plasma fibrinogen, C-reactive protein (CRP) and albumin levels. Fasting serum insulin levels served as a marker of insulin resistance. Two months of lercanidipine treatment showed significant decrease in BP, WBC and PMNL counts, and also in PMNL apoptosis, CRP and serum insulin levels and significant increase in serum albumin levels. Rates of superoxide release from PMNLs, WBC and PMNL counts and insulin levels positively correlated with mean arterial blood pressure values. We imply that use of lercanidipine can be favored in EH patients due to its combined anti-PMNL priming and anti-inflammatory effects, in addition to its anti-hypertensive characteristics. Key words: Essential hypertension, lercanidipine, low-graded inflammation, primed polymorphonuclear leukocytes, oxidative stress.

Lercanidipine effect on polymorphonuclear leukocyte-related inflammation and insulin resistance in essential hypertension patients.

IntroductionInflammation, insulin resistance, and oxidative stress (OS) are among the mechanisms that have been implicated in the pathogenesis of essential hypertension (EH). Peripheral polymorphonuclear leukocytes (PMNLs) are primed in EH patients, releasing uncontrolled superoxide anions contributing to OS in these patients. PMNL priming correlates with insulin resistance and PMNL intracellular calcium ([Ca2+]i). Recent studies have attributed additional anti-ischemic and antioxidative characteristics to the antihypertensive drug, lercanidipine, a third-generation calcium-channel blocker. The purpose of this study was to evaluate the possible nontraditional effect of 2 months of lercanidipine treatment on insulin resistance and on PMNL-related inflammation in EH patients.MethodsNon-smoking EH patients with untreated mild-to-moderate high blood pressure (BP) were included. Low-grade inflammation was reflected by PMNL apoptosis and by white blood cell (WBC) and PMNL counts. Systemic inflammation was measured by plasma fibrinogen, C-reactive protein (CRP), and transferrin and albumin levels. Fasting serum insulin levels served as a marker of insulin resistance.ResultsTwo months of lercanidipine treatment showed a significant decrease in BP, WBC, and PMNL counts, PMNL apoptosis, CRP, and serum insulin levels, and a significant increase in serum albumin levels. Rates of superoxide release from PMNLs, WBC and PMNL counts, and insulin levels positively correlated with mean arterial BP values.ConclusionThe use of lercanidipine can be favorable in EH patients due to its combined anti-PMNL priming and anti-inflammatory effects, in addition to its antihypertensive characteristics.

Effects of statins on oxidative stress and primed polymorphonuclear leukocytes in hyperlipidemic patients

Inflammation and oxidative stress are among the factors that have been implicated in the pathogenesis of hyperlipidemia. In metabolic syndrome and hyperlipidemic patients, peripheral polymorphonuclear leukocytes (PMNL) are primed and they release uncontrolled superoxide that contributes to oxidative stress and inflammation. Recent studies have demonstrated that the anti-hyperlipidemic drug, Atrovastatin effects improvement in endothelial function, exhibits anti-oxidative characteristics and reduces lipid markers of oxidation. To evaluate possible nontraditional effects of treatment with Atrovastatin on PMNL priming, oxidative stress and inflammation in hyperlipidemia, 50 non-smoking hyperlipidemic patients were treated for 6 months with Atrovastatin and compared to age and gender-matched healthy controls. PMNL priming was assessed by the rate of superoxide release from separated, phorbol ester-stimulated PMNL and by PMNL-CD11b levels. Inflammation was reflected by blood inflammatory markers including albumin, transferrin, C-reactive protein (CRP) and fibrinogen levels, white blood cells (WBC), PMNL counts and PMNL apoptosis. Atrovastatin treatment showed a reduction in PMNL priming, PMNL apoptosis, fibrinogen and CRP levels concomitant with decreased lipid levels. Atrovastatin may be preferred for hyperlipidemic patients owing to its combined anti-PMNL priming and anti-inflammatory effects in addition to its anti-atherogenic effects.

Effects and Mechanisms of Action of Polyene Macrolide Antibiotic Nystatin on Babesia gibsoni In Vitro

Nystatin is a membrane-active polyene macrolide antibiotic and a channel-forming ionophore. Nystatin exhibits in vitro activity against Babesia gibsoni infecting normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes. The calculated IC(50) value of nystatin against B. gibsoni infecting LK erythrocytes was 31.96 µg/ml. The anti-babesial activity of nystatin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations (HK). Moreover, when the parasites were harbored in canine HK erythrocytes, which contained high potassium and low sodium concentrations as a result of high Na-K-ATPase activity, the in vitro anti-babesial activities of nystatin also disappeared, apparently due to protection by HK erythrocytes. This suggested that nystatin could show in vitro anti-babesial activity against B. gibsoni by its ionophorous activity, the same as other ionophores such as valinomycin. Subsequently, the effects of nystatin on the host cells were observed. Nystatin could not modify the intracellular concentrations of potassium, sodium, adenosine triphosphate, or glucose in either LK or HK erythrocytes, although it caused weak hemolysis in HK erythrocytes. In addition, nystatin did not affect the survival of canine peripheral polymorphonuclear leukocytes. In conclusion, nystatin destroyed B. gibsoni by ionophorous activity but did not affect either canine erythrocytes or leukocytes in vitro.

Intensification of oxidative stress and inflammation in type 2 diabetes despite antihyperglycemic treatment

IntroductionThe metabolic deregulation associated with diabetes mellitus (DM) causes secondary pathophysiologic changes in multiple organ systems. Endothelial injury is induced by oxidative stress (OS) and inflammation. We have previously shown that DM type 2 patients are exposed to increased OS and inflammation contributed in part by primed peripheral polymorphonuclear leukocytes (PMNLs).AimsTo characterize the effect of oral medication on PMNL priming, on PMNL-related and on systemic inflammation, in correlation to changed diabetes parameters in patient with newly diagnosed type 2 DM.MethodsPMNLs were separated from DM patient's prior and following treatment with either metformin (Glucophage), or Thiazolidinedione (rosiglitazone) and from healthy control subjects (HC). Rate of superoxide release from phorbol ester-stimulated PMNLs and CD11b on PMNLs assessed PMNL priming. White blood cells (WBC) and PMNL counts and apoptosis reflected PMNL-related inflammation. CRP, fibrinogen, transferrin and albumin blood levels reflected systemic inflammation.ResultsBoth metformin and rosiglitazone treatments reduced significantly the high levels of glucose and HbA1c, and slightly improved lipid profile during 2 months. PMNL priming parameters, higher compared to HC, increased after 2 months of metformin treatment. Rosiglitazone treatment decreased PMNL priming. ALP, higher in DM, significantly decreased following 2 months of both treatments. Systemic inflammation markers (fibrinogen, CRP), higher in DM, decreased following both treatments. Transferrin and albumin were similar to HC. PMNL-related inflammation markers were higher in DM; however, only PMNL apoptosis decreased after both treatments. Monocyte counts, higher in DM compared to HC, decreased following both treatments. Serum insulin levels, higher in DM compared to HC, decreased following both treatments. PMNL-related priming and inflammation parameters positively correlated with HbA1c.ConclusionThe present research adds new facet in evaluating anti-hyperglycemic treatment in type 2 DM patients. Despite sufficient glycemic control using both treatments, some PMNL-related parameters deteriorated. Thus, anti hyperglycemic treatment should be favored due to its combined anti-PMNL priming and anti-inflammatory effect, in addition to its anti-hyperglycemic characteristics, according to the correlation among these parameters. Such combined treatment may reduce morbidity and mortality common in DM patients.

Effect of fibrinogen on the secretion of interleukin-1beta and - 8 by polymorphonuclear leukocytes

To 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. Peripheral 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. Incubated 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). Fibrinogen 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.

Benidipine, an anti-hypertensive drug, inhibits reactive oxygen species production in polymorphonuclear leukocytes and oxidative stress in salt-loaded stroke-prone spontaneously hypertensive rats

Oxidative stress is associated with exacerbation of renal injuries in hypertension. In clinical studies benidipine hydrochloride (benidipine), a dihydropyridine calcium channel blocker with antioxidant activity, reduced oxidative stress. However, the mechanism of suppression of oxidative stress remains to be fully characterized. Reactive oxygen species production by polymorphonuclear leukocyte plays important pathological roles in hypertension. Therefore, we examined the effects of benidipine both on reactive oxygen species production of human polymorphonuclear leukocytes and oxidative stress of an animal model. Human peripheral polymorphonuclear leukocytes or polymorphonuclear leukocyte-like differentiated HL-60 cells were used to examine effects of benidipine (0.1–30 μM) on formyl-Met-Leu-Phe-induced reactive oxygen species production, calcium mobilization, NADPH oxidase activation and phosphorylation of protein kinase C substrates. High-salt (8% NaCl) loaded stroke-prone spontaneously hypertensive rats were treated with or without benidipine (1, 3, 10 mg/kg/day) for 2 weeks, and thiobarbituric acid reactive substances, a plasma oxidative stress marker, and renal expression of oxidative stress-induced genes were measured. Benidipine concentration-dependently suppressed formyl-Met-Leu-Phe-induced reactive oxygen species production in polymorphonuclear leukocytes more potently than other calcium channel blockers such as amlodipine, azelnidipine, nitrendipine and nifedipine. Benidipine partially inhibited all of intracellular Ca 2+ elevation, protein kinase C activation and NADPH oxidase activation. Salt loading in stroke-prone spontaneously hypertensive rats augmented plasma thiobarbituric acid reactive substances levels; renal dysfunction; and renal expression of transforming growth factor-β, collagen I and collagen III mRNAs; which were attenuated by benidipine treatment. These results indicate that benidipine prevents the polymorphonuclear leukocyte-derived reactive oxygen species production, which is due at least in part to its antioxidant action and inhibition of Ca 2+/protein kinase C/NADPH oxidase signaling. The attenuation of reactive oxygen species production might contribute to the drug's reduction of oxidative stress and renal injuries in hypertension.

Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury

Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10(6)) from HD patients or from healthy normal controls were cocultivated with HUVEC (10(5)) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.