Respiratory Burst Levels Research Articles

Interaction of low-level dietary supplementation of Chlorella vulgaris Beijerinck, 1890, and feeding duration on growth hormone, growth performance and serum biochemistry of red hybrid tilapia (Oreochromis mossambicus × Oreochromis niloticus).

Chlorella is one of the most widely accepted Chlorophyta used by many as livestock and aquaculture feed. Nonetheless, different studies on the overall performances of fish reported the unfavourable effect of high-level supplementations of Chlorella vulgaris. The current study determined the impact of low-level dietary supplementation of C. vulgaris alongside the different feeding durations and their interactions on the growth hormone (GH), growth performances, serum-biochemical indices, hepatic function and some immunological parameters of red hybrid tilapia. The fingerlings (mean weight: 14.25 ± 0.01 g, length: 13.5 ± 0.49 cm) were fed diets containing 0, 0.99%, 2.91% and 4.76% of C. vulgaris powder per kilogram dry diet for 90 days. GH, growth performance, serum-biochemical indices (total serum protein, albumin, globulin, glucose, aspartate aminotransferase and alanine aminotransferase) and some immunological (respiratory burst and lysozyme activities) parameters of the fish were examined after 30, 60 and 90 days of feeding. The results demonstrated that tilapia fed C. vulgaris-supplemented diets showed increased levels of respiratory burst, lysozyme, albumin and total protein, GH and growth performances (P < 0.05), and the effects were duration dependent. After the 90 days of feeding, there was no adverse effect on the hepatic function of the fish. Besides, low survivability was observed in the control group than in the group fed the experimental diets. The group fed the diet supplemented with 4.76% C. vulgaris had significantly higher (P < 0.05) lysozyme activity throughout the duration of the feeding experiment. These results indicate that C. vulgaris enhanced growth performances, GH concentration, serum-biochemistry and some immunological parameters of red hybrid tilapia.

Ambient NO2 hinders neutrophil extracellular trap formation in rats: Assessment of the role of neutrophil autophagy

NO2 has been known to impair immunity and exacerbate susceptibility to infectious diseases. However, scant notice has been taken of the effect of NO2 on neutrophils. Neutrophil extracellular traps (NETs) formation is necessary for NETosis development by neutrophils as an immune system against pathogens. By analyzing the morphology and signature components of NETs, we focused for the first time on finding that 10 ppm of NO2 exposure for 15 consecutive days can hinder the formation of NETs. Next, we used NO2 in vivo derivatives to probe the mechanism for NETs formation in vitro. Our findings showed that NO2 suppression of respiratory burst levels and mitogen-activated protein kinase (MAPK)/Phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling was related to NO2 reduction in NETs formation. Inhibition of phorbol myristate acetate (PMA)-induced NETs formation by NO2 hindered autophagy, as evidenced by increased mTOR protein expression, decreased LC3 protein expression, and reduced autophagic vesicles. By activating mTOR-mediated autophagy, rapamycin (Rapa) reduced the inhibition of PMA-induced NETs by NO2. This study will provide valuable insights into the mechanisms of immunotoxicity of NO2, new insights into the etiology of diseases linked to NETs formation, and a theoretical basis for protection against such illnesses.

Effects of different phylotypes of avian pathogenic Escherichia coli isolated from broiler chickens with colibacillosis on heterophils functional activities: an in vitro study.

Avian pathogenic Escherichia coli (APEC) is a major cause of colibacillosis and is associated with economic losses to the poultry production worldwide. Heterophils are the first line of immune defense of the avian host against invasive pathogens. In this study, APEC isolates from chickens with colibacillosis were assigned to phylogenetic groups and immunological activities of heterophils against these groups were examined. A total of 92 APEC isolates was obtained from 106 samples of diverse organs collected from chickens with colibacillosis from different farms in West Azerbaijan province, Iran. Isolates were assigned to phylogenetic groups based on the Clermont triplex PCR method, and immunological activities (including phagocytosis, respiratory burst and bacterial killing) of heterophils against these groups were examined. As results, the frequency of A, B1, B2 and D groups were 35.87, 44.57, 5.43 and 14.13%, respectively. In addition, opsonized Escherichia coli isolates belonging to B1 group significantly enhanced the level of respiratory burst (0.52 ± 0.02%) while the killing level of them was significantly lower than the other groups (29.40 ± 5.09%). There was no significant difference in phagocytic activity of heterophils against the phylogenetic groups. In conclusion, incomplete immune responses to B1 phylogenetic group maybe a principal cause of mortality by colibacillosis caused by this group. It is suggested to study heterophilic immune reaction against E. coli phylogenetic group for development of effective prevention strategy.

Hydrogen sulfide upregulates miR-16-5p targeting PiK3R1 and RAF1 to inhibit neutrophil extracellular trap formation in chickens.

Hydrogen sulfide (H2S) is a toxic air pollutant that causes immune damage. Recent studies have found that neutrophil extracellular trap (NET) formation is one way in which neutrophils exert immune functions. In addition, the formation of NETs is also related to thrombosis and autoimmune diseases. Recent studies have shown that miRNAs are involved in the regulation of a variety of pathophysiological processes. Here, we investigated the role of H2S in regulating the formation of NETs by affecting miR-16-5p. Our study established an in vitro H2S exposure model for neutrophils using phorbol-myristate-acetate (PMA) to induce NET formation. We observed the morphological changes of cells with scanning electron microscopy and fluorescence microscopy. Then, the content of extracellular DNA and the expression of MPO and NE in each group were detected. The results showed that H2S inhibited the formation of NETs. The expression of miR-16-5p and its target genes PiK3R1 and RAF1 was then measured by qRT-PCR. H2S upregulated miR-16-5p and inhibited expression of the target genes PiK3R1 and RAF1, and it subsequently inhibited the Pi3K/AKT and ERK pathways and decreased respiratory burst levels. Furthermore, H2S attenuated inositol 1,4,5-trisphosphate receptor (IP3R)-mediated endoplasmic reticulum calcium outflow as well as autophagy caused by PMA. This study enriches H2S immunotoxicity research and provides a possible solution for the treatment of NET-related diseases.

EVALUACIÓN DE LA TOXICIDAD DEL CLORPIRIFÓS EN ALEVINOS DE CACHAMA BLANCA (Piaractus brachypomus)

Pesticides have become an important environmental problem due to their indiscriminate and lack of control use, which ends up affecting drastically non-target organisms, including aquatic species. The acute and chronic toxicity of chlorpyrifos was determined in fingerlings of red-bellied pacu ( Piaractus brachypomus ), and it was established the value of the median lethal concentration (LC 50 ) at 96 h, which was evaluated in 88 fish exposed to chlorpyrifos concentrations from 0.015 to 1.5 μg/L. The value established for CL 50 was 0.11 μg/L (p < 0.05). The chronic toxicity test to sublethal doses (0.5, 0.1 and 0.001 x of the LC 50 dose) was performed in 60 animals per treatment plus a witness group. Animals were exposed for 50 days, followed by 50 days without exposure, to evaluate both respiratory burst activity and plasma bactericidal activity in a challenge with Aeromonas hydrophila . Lower levels of respiratory burst were found in the exposed group to 0.0011 μg/L in 20 and 50 days, which increased in the exposed group to 0.055 μg/L at day 90. In addition, the bactericidal activity of plasma showed a decrease until day 40 in exposed animals compared to the witness. Also, in a trial of subchronic exposure without recovery to sublethal doses of chlorpyrifos (0.011 μg/L), lesions were found in gills, liver, kidney and brain. This study has demonstrated the high sensitivity of this species to chlorpyrifos, postulating P. brachypomus as a bioindicator of water pollution.

Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA

BackgroundA recent study found that CD177 served as a receptor of membrane-bound proteinase-3 (mPR3) in a subset of neutrophils. Furthermore, CD177 has been identified as a high-affinity heterophilic binding partner for the endothelial cell platelet endothelial cell adhesion molecule-1 (PECAM-1). The current study aimed to investigate whether the interaction between PECAM-1 and CD177 could influence mPR3 expression as well as PR3-antineutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation and glomerular endothelial cell (GEnC) injury.MethodsThe effect of interaction between CD177 and PECAM-1 on mPR3 expression was explored by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The effect of PECAM-1 on neutrophil activation and GEnC injury induced by PR3-ANCA-positive immunoglobulin (Ig)Gs was evaluated by dihydrorhodamine (DHR) assay and ELISA. CD177-negative neutrophils were selected by magnetic cell sorting (MACS), and the inhibitory effect of PECAM-1 on CD177-negative and mixed neutrophils was explored by measuring neutrophil degranulation.ResultsThe level of specific interaction between CD177 and PECAM-1 was elevated with increasing CD177 concentration. The expression of mPR3 significantly decreased in neutrophils preincubated with PECAM-1 in a dose-dependent manner. Consistently, the levels of respiratory burst and degranulation induced by PR3-ANCA-positive IgGs in recombinant human tumor necrosis factor-alpha (TNF-α)-primed neutrophils was significantly reduced by preincubation with PECAM-1 (440.6 ± 123.0 vs. 511.4 ± 95.5, p < 0.05; and 3155.0 ± 1733.0 ng/ml vs. 5903.0 ± 717.5 ng/ml, p < 0.05, respectively). In CD177-negative neutrophils incubated with PR3-ANCA-positive IgGs, the level of degranulation was not significantly changed by preincubation with PECAM-1. However, in mixed neutrophils, PECAM-1 significantly decreased the level of degranulation induced by PR3-ANCA-positive IgGs (1015.9 ± 229.2% vs. 1725.2 ± 412.4%, p < 0.01). Furthermore, with preincubation of TNF-α-primed neutrophils with PECAM-1, the level of soluble intercellular cell adhesion molecule-1 (sICAM-1), a marker of endothelial cell activation and injury, in the supernatant of GEnCs treated with primed neutrophils plus PR3-ANCA-positive IgGs was significantly attenuated (112.7 ± 24.2 pg/ml vs. 167.5 ± 27.7 pg/ml, p < 0.05).ConclusionsPECAM-1 can decrease the level of mPR3 expression on neutrophils, resulting in attenuation of neutrophil activation and subsequent GEnC injury induced by PR3-ANCA-positive IgGs.

Studies on the effects of LPS, ß-glucan and metabolic inhibitors on the respiratory burst and gene expression in Atlantic salmon macrophages.

Reactive oxygen species (ROS) production in macrophage-like cells is induced as an antimicrobial defence against invading pathogens. In this study, we have explored how different stimuli and metabolic inhibitors affect the level of respiratory burst in Atlantic salmon (Salmo salar L.) head kidney macrophage-like cells. Cells stimulated invitro by bacterial lipopolysaccharide (LPS) and ß-glucan showed increased production of ROS compared to unstimulated cells. Both stimulation and costimulation by curdlan (ß-glucan) induced a higher production of ROS compared to stimulation and costimulation by LPS. Metabolic inhibitors co-incubated with the stimulants did not, in most cases, perturb the level of ROS generation in the salmon macrophage-like cells. The NAD+ content as well as the NAD+ /NADH ratio increased in curdlan and LPS+curdlan-stimulated cells compared to control cells, which indicated increased metabolic activity in the stimulated cells. Supporting these findings, gene analysis using real-time quantitative PCR showed that arginase-1 and IL-1ß genes were highly expressed in the stimulated cells.

Administration of Rhodiola kirilowii Extracts during Mouse Pregnancy and Lactation Stimulates Innate but Not Adaptive Immunity of the Offspring.

The use of antibiotics during pregnancy and lactation is associated with an increased risk of developmental disorders. One of the natural medicinal plants—Rhodiola kirilowii, widely used as an immunostimulant in adults—might be a good alternative to antibiotic treatment. The aim of present study was to assess whether daily oral administration of 20 mg/kg of Rhodiola kirilowii aqueous (RKW) or 50% hydroalcoholic (RKW-A) extracts affected hematological and immunological parameters of 6-week-old mouse progeny. There was no significant change in hematological parameters of blood with the exception of hemoglobin, which was significantly higher (about 4%) in RKW group. Offspring of mothers fed Rhodiola kirilowii extracts had increased percentage of granulocytes and decreased percentage of lymphocytes. These changes correlated with decreased percentage of CD3+/CD4+ T-cells (RKW and RKW-A), decrease of CD8+ cells, and increase percentage of NK cells in RKW group. In addition, both types of Rhodiola kirilowii extracts stimulated granulocyte phagocytosis and increased level of respiratory burst. In conclusion, the long-term supplementation of mouse mothers during pregnancy and lactation with RKW or RKW-A extracts affects the immune system of their progeny. These results should be taken into consideration before administration of Rhodiola kirilowii to pregnant and lactating women.

High mobility group box 1 contributes to anti-neutrophil cytoplasmic antibody-induced neutrophils activation through receptor for advanced glycation end products (RAGE) and Toll-like receptor 4.

IntroductionHigh mobility group box-1 (HMGB1), a typical damage-associated molecular pattern (DAMP) protein, is associated with inflammatory conditions and tissue damage. Our recent study found that circulating HMGB1 levels could reflect the disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The current study aimed to investigate whether HMGB1 participated in ANCA-induced neutrophil activation, which is one of the most important pathogenic aspects in the development of AAV.MethodsThe various effects of HMGB1 in ANCA-induced neutrophil activation were measured. Antagonists for relevant receptors and signaling molecules were employed.ResultsANCA antigens translocation on neutrophils primed with HMGB1 was significantly higher than non-primed neutrophils. The levels of respiratory burst and degranulation increased significantly in HMGB1-primed neutrophils activated with ANCA-positive IgG, as compared with non-primed neutrophils. Furthermore, blocking Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE), rather than TLR2, resulted in a significant decrease in HMGB1-induced ANCA antigens translocation, respiratory burst and degranulation. Similar effects were also found when blocking MyD88 and NF-κB.ConclusionsHMGB1 could prime neutrophils by increasing ANCA antigens translocation, and the primed neutrophils could be further induced by ANCA, resulting in the respiratory burst and degranulation. This process is TLR4- and RAGE-dependent through the MyD88/NF-κB pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0587-4) contains supplementary material, which is available to authorized users.

Aspergillus fumigatus diffusates suppress polymorphonuclear neutrophil phagocytic functions and respiratory burst levels in hematopoietic stem cell transplantation patients.

Invasive aspergillosis (IA) is a severe infection that commonly occurs in immunocompromised patients after hematopoietic stem cell transplantation (HSCT). The present study explores the effect of Aspergillus fumigatus diffusates (AfDs) on phagocytic function and superoxide anion (O2(-)) burst levels in polymorphonuclear neutrophils (PMNs) from post-HSCT patients. A. fumigatus conidia with or without AfD were used to stimulate the PMN from healthy donor or HSCT patient for two hours. PMN morphology was visualized by scanning electron microscopy. The levels of respiratory burst O2(-) produced by the PMNs were determined by flow cytometry. PMN phagocytic rates and phagocytic indexes were observed and calculated using periodic acid-Schiff (PAS) staining under a light-field microscope. No difference was found between the PMN phagocytic rates, phagocytic indexes, or O2(-) respiratory burst levels in health donor PMNs following treatments of A. fumigatus conidia with or without AfD. However, significant inhibition of these indices was seen in the PMNs from HSCT patients following treatment of A. fumigatus conidia plus AfD, compared to that with conidium treatment alone (P < 0.05). Therefore, AfD significantly inhibited the phagocytic function of PMNs from HSCT patients, potentially through inhibition of intracellular respiratory burst levels during phagocytosis. This suggests that the reason underlying the greater susceptibility of HSCT patients to aspergillosis might be the existence of AfD in vivo during infection. Further research on the mechanisms by which AfD affects the phagocytic function of PMNs from HSCT patients is therefore of great significance for the prevention of IA.

Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was ∼10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was ∼1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only ∼1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface.

C-reactive protein enhances the respiratory burst of neutrophils-induced by antineutrophil cytoplasmic antibody

Serum C-reactive protein (CRP) was one of the useful biomarkers for evaluating the disease activity in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Cumulating studies proved that CRP was pathogenic in a variety of diseases. In the current study, the in vitro effects of CRP to prime neutrophils for ANCA-induced respiratory burst were investigated with flow cytometry. Without TNF-α in the reactive system, ANCA could only induce a slight level of respiratory burst of neutrophils. CRP could enhance the respiratory burst of neutrophils induced by ANCA against myeloperoxidse [mean fluorescence intensity (MFI, 68.45±16.87 vs. 58.65±15.09, P<0.05) or by ANCA against proteinase 3 (MFI, 79.51±15.90 vs. 61.73±14.89, P<0.05). Although CRP (50μg/mL, incubating for 30min) could not active neutrophils alone, after incubation with neutrophils for 10min, CRP (50μg/mL) could increase the expression of membrane proteinase 3 of neutrophils (MFI, 365.27±143.50 vs. 235.32±124.65, P<0.05). Heat-treated CRP could not enhance the levels of neutrophils respiratory burst induced by ANCA or increase the expression of membrane proteinase 3 of neutrophils. So CRP can prime neutrophils and enhance the respiratory burst induced by ANCA and might be pathogenic in AAV.

C-Reactive Protein Plays a Role in Antibody-Mediated Platelet Destruction

Abstract 526IgG-mediated disorders like fetal or neonatal alloimmune thrombocytopenia (FNAIT) and the autoimmune acute immune thrombocytopenia (ITP), are caused by platelet-IgG-antibodies targeting platelets, resulting in Fcγ-receptor (FcγR)-mediated phagocytosis of platelets and eventually in thrombocytopenia. Markers to predict severity of these diseases are not yet available. Platelet-antibodies levels are related with outcome, however lack acceptable accuracy to predict disease severity.We hypothesized that a bioassay measuring antibody-mediated platelet destruction might correlate with clinical outcome and therefore we developed an in vitro respiratory burst assay, using polymorphonuclear neutrophils (PMN). We observed that platelets opsonized with antibodies purified from sera of alloimmunized women did not result in strong antibody-mediated phagocyte responses unless the reaction occurred in the presence of patient serum (p<0.001) or normal human serum (NHS, p<0.001), also when recombinant human antibodies (IgG1 anti-HPA-1a) were used (p<0.001). This indicated that a serum-derived factor was required for antibody-mediated phagocytic activity by PMN. Serum heat inactivation excluded the role of complement, but the effect was dependent on Ca2+, and the level or the activity of this factor varied between different NHS (n=14). We therefore postulated that C-Reactive Protein (CRP), a Ca2+-dependent acute phase protein, variably present in NHS and upregulated during infections, could be this factor. CRP-neutralization using pneumococcal cell-wall polysaccharide and depletion of CRP from serum using phosphorylcholine beads reduced the phagocytic activity towards IgG-opsonized platelets (p<0.01 and p<0.001, respectively) while addition of CRP at physiological concentrations to serum enhanced it (p<0.05). Furthermore, the level of respiratory burst towards opsonized platelets in presence of various NHS correlated positively with the serum CRP levels (n=14, R2=0.45, p=0.0084). Fluorescently labelled CRP bound to IgG-opsonized platelets in a Ca2+-dependent manner (p<0.001). We hypothesized that a ligand for CRP, phosphatidylcholine, might be exposed on platelets after activation of platelet-FcγRIIa. However, the phagocytosis-enhancing effect did not appear to be mediated through platelet-FcγRIIa, as FcγRII-blocking antibodies did not decrease the level of phagocytosis. Also when using anti-D opsonized red blood cells (RBCs), which are FcγR-negative, we observed that the phagocytic activity was higher in the presence of serum (p<0.001). Using radioactively-labelled CRP, we found CRP to only bind to the IgG-opsonized on surfaces, independently of FcγR, as this effect was apparent on RBCs (p<0.05). Importantly, the concentration of CRP in sera from neonates with severe FNAIT was significantly elevated compared to healthy cord blood samples (4.21 mg/L vs. 0.05 mg/L, n=21, p=0.0008), and also in sera from children with acute ITP compared to healthy children (1.98 mg/L vs. 0.26 mg/L, n=19, p=0.02), indicating that CRP may be a contributing factor for platelet clearance in the presence of anti-platelet antibodies. To also test this hypothesis in vivo, we utilized our previously established mouse model (Teeling et al. Blood. 2001; 98:1095-1099). We administered 0.75 μg of rat-anti-platelet antibody causing mean platelet counts to drop from 1050 × 109/L to 645 × 109/L in 16 hours (n=7, p=0.0001). When 200 μg CRP was co-administrated, the mean platelet counts dropped further to 345 × 109/L (n=7, p=0.0060), while administration of CRP alone had no effect on platelet counts, demonstrating that CRP potentiates the degree of thrombocytopenia induced by an anti-platelet antibodies in vivo.These findings support a novel fundamental mechanism in which CRP can aggravate IgG-mediated phagocyte responses in FNAIT and acute ITP and perhaps also in other IgG-mediated responses in both health and disease. This involvement of CRP may possibly explain the lack of clinical correlation between severity and anti-platelet antibody titers and the frequent occurrence of acute ITP upon infection. We conclude that CRP functions as a previously unknown pathogenic co-factor in phagocyte responses towards IgG-opsonized platelets through a direct binding to IgG, thereby contributing to platelet destruction. Disclosures:No relevant conflicts of interest to declare.

High potential to reverse the inhibition of myeloperoxidase by ceruloplasmin of anti-myeloperoxidase autoantibodies of IgG3 subclass

Among the four subclasses, anti-neutrophil cytoplasmic antibodies (ANCAs) against myeloperoxidase (MPO) IgG, IgG3 subclass was associated with severe disease. This study aimed to investigate some aspects of the in vitro pathogenic potential of MPO-ANCA of IgG3 subclass. IgG from plasma of 10 consecutive patients with positive MPO-ANCA was divided into the IgG poor in IgG3 and the IgG rich in IgG3 with protein A. Then, the ability to influence the oxidation activity of MPO, the ability to induce respiratory burst of neutrophils, and the ability to reverse the binding between ceruloplasmin (CP) and MPO were compared between the two fractions. The avidity of the MPO-ANCA-positive IgG rich in IgG3 was higher than that of the MPO-ANCA-positive IgG poor in IgG3 in 7 out of 10 patients. At the same antigen-binding level, the level of respiratory burst of neutrophils induced by the MPO-ANCA-positive IgG rich in IgG3 was significantly higher than that induced by the MPO-ANCA-positive IgG poor in IgG3 (1429.70 ± 137.85 vs. 1321.70 ± 244.51, P = 0.049, expressed by the mean fluorescence intensity); the binding level between MPO and CP after adding the MPO-ANCA-positive IgG rich in IgG3 was significantly lower than after adding the MPO-ANCA-positive IgG poor in IgG3 (0.599 ± 0.214 vs. 0.777 ± 0.353, P = 0.010, expressed by A 405); there was no significant difference in the MPO activity between adding the MPO-ANCA-positive IgG poor in IgG3 and rich in IgG3 (P = 0.217). In conclusion, MPO-ANCA of IgG3 subclass might have stronger ability to reverse the inhibition of MPO by CP than other subclasses.

Oxidative metabolism of lung macrophages exposed to sodium arsenite

Arsenic pollution has become increasingly severe. It occurs as the result of geological processes and different human activities. Arsenic toxicity at the respiratory level occurs mainly by inhalation of products of coal combustion. The aim of this study was to evaluate sodium arsenite (As 3+) toxicity in murine alveolar macrophages (AMs) in vitro and its association with the alterations in cell metabolism. No changes in viability, apoptosis or cell area were detected in AMs treated with As 3+ concentrations up to 2 μM for 24–96 h. A marked decrease in these end-points was observed for As 3+ concentrations ranging from 2.5 μM to 10 μM. Regarding the dynamics of the endo-exocytic process triggered by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell incorporation, no variations were detected for As 3+ concentrations lower than 2 μM while higher concentrations markedly modified this response. MTT specific activity, as a measure of cell metabolic activity, was not modified irrespective of the As 3+ concentration assayed. However, nitroblue tetrazolium (NBT) specific activity, as a measure of superoxide anion generation, is responsive but only to low As 3+ doses. Although this study focuses on lung macrophages, the effects of As 3+ described herein may also apply to the response of macrophages residing in other organs. Arsenite modifies the metabolic and the oxidative status of AMs in vitro. When macrophages are in an As 3+ rich medium, they exhibit a reduction in respiratory burst levels and lose their intrinsic capacity to respond to toxicants.

Comparison of blood and milk non-specific immune parameters in heifers after calving in relation to udder health

A practical protocol to study udder immune status in field conditions was planned with the aim to assess different non-specific immune parameters in milk samples from dairy heifers during the periparturient period. Five herds located in northern Italy were selected and overall 39 heifers were enrolled in the trial. Milk samples were taken at 7, 14, 21, 28, 45, 60, and 75 days after calving. The parameters assessed were N-acetyl-beta-glucosaminidase (NAGase), lysozyme, respiratory burst (RB), somatic cell counts (SCC) and serum protein profile. SCC and NAGase were higher in the first sampling after calving, while lysozyme showed large variations during the observation period without a definite trend. The levels of RB observed in the first two weeks after calving, even if lower, were not statistically different from the values observed in samples taken over the following weeks. This study confirmed that the levels of immune components in milk are different from what is observed at blood level in the same cow. A significant decrease in RB in milk polymorphonuclear leukocytes (PMN) post-calving was not observed; milk PMN from healthy cows showed low RB levels, while the values from infected quarters were significantly higher. Significant differences between healthy and infected animals were also observed for milk NAG, lactoglobulin and albumin. These data suggest that udder immune response could be influenced both by the cow immune status and by external factors such as pathogens and management. Therefore, the reduction in immune defences, particularly in heifers, is not unavoidable and methods to boost PMN activity should be explored.

Decreased phosphorylation of protein kinase B and extracellular signal-regulated kinase in neutrophils from patients with myelodysplasia

AbstractNeutrophils from patients with myelodysplastic syndrome (MDS) show a disturbed differentiation pattern and are generally dysfunctional. To study these defects in more detail, we investigated reactive-oxygen species (ROS) production and F-actin polymerization in neutrophils from MDS patients and healthy controls and the involvement of N-formyl-L-methionyl-L-lucyl-L-phenylaline (fMLP) and granulocyte macrophage–colony-stimulating factor (GM-CSF)–stimulated signal transduction pathways. Following fMLP stimulation, similar levels of respiratory burst, F-actin polymerization, and activation of the small GTPase Rac2 were demonstrated in MDS and normal neutrophils. However, GM-CSF and G-CSF priming of ROS production were significantly decreased in MDS patients. We subsequently investigated the signal transduction pathways involved in ROS generation and demonstrated that fMLP-stimulated ROS production was inhibited by the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the MAPK/ERK kinase (MEK) inhibitor U0126. In contrast, ROS production induced by fMLP stimulation of GM-CSF–primed cells was inhibited by LY294002 and U0126. This coincides with enhanced protein kinase B (PKB/Akt) phosphorylation that was PI3K dependent and enhanced extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation that was PI3K independent. We demonstrated higher protein levels of the PI3K subunit p110 in neutrophils from MDS patients and found that though the fMLP-induced phosphorylation of PKB/Akt and ERK1/2 could also be enhanced by pretreatment with GM-CSF in these patients, the degree and kinetics of PKB/Akt and ERK1/2 phosphorylation were significantly disturbed. These defects were observed despite a normal GM-CSF–induced signal transducer and activator of transcription 5 (STAT5) phosphorylation. Our results indicate that the reduced priming of neutrophil ROS production in MDS patients might be caused by a disturbed convergence of the fMLP and GM-CSF signaling routes.

Respiratory Burst in Neutrophils from Asthmatic Patients

Background: The role of oxygen radicals has been implicated in disease processes of asthma. We have previously shown that specific allergens were able to activate respiratory burst by neutrophils from allergic patients sensitized to allergens of the same type as those which produce clinical allergy.Objectives: In this study, we attempted to evaluate the production of respiratory burst by an anti-IgE Ab in neutrophils from asthmatic allergic patients (with and without immunotherapy treatment) and in neutrophils from healthy subjects.Method: Neutrophils were stimulated by 10 µg/mL of anti-IgE Ab for 15 min at 37°C. The production of respiratory burst from neutrophils was assayed by luminol-amplified chemiluminescence method.Results: The respiratory burst was significantly higher in neutrophils from non-IT-asthmatic patients than in neutrophils from both healthy (p<0.001) and IT-asthmatic (p<0.001) groups. The IT-asthmatic group presented levels of respiratory burst approximately equal to those from non-allergic subjects (p=0.426).Conclusions: We conclude that neutrophils obtained from allergic asthmatic patients have an increased propensity to generate respiratory bursts, in comparison with neutrophils from healthy subjects. Immunotherapy actively modifies the respiratory burst by neutrophils from allergic asthmatic patients.

Dietary organic chromium supplementation and its effect on the immune response of rainbow trout (Oncorhynchus mykiss)

Recently, there has been an increasing interest in the potential effect of dietary chromium on the health of fish, particularly with respect to their metabolism and growth. Information as to the role of this mineral on their immune response, is limited however, so the aim of this study was to assess the effects of dietary chromium yeast supplementation on the immune response of rainbow trout (Oncorhynchus mykiss).Juvenile rainbow trout (56g average weight) were fed three semipurified diets containing different levels of chromium (1540, 2340 and 4110ppb), obtained by supplementing a basal diet with 800 or 2570ppb chromium yeast, for 6 weeks. After this, time differences in their immune response were examined.A positive influence was observed on serum lysozyme activity at this time in fish maintained on the high chromium diet. The respiratory burst of head- kidney macrophages was also examined, and statistical differences were found in the level of respiratory burst elicited by macrophages from both groups of fish fed supplemented chromium after 3 and 6 weeks of feeding (absorbance at 3 weeks: 0·118, 0·166, 0·151 and 6 weeks 0·114, 0·168, 0·151 for the 1540, 2340 and 4110ppb groups). Macrophages of fish receiving diets supplemented with chromium also had a greater ability to phagocytose yeast after 6 weeks than the control fish (40·5, 48 and 48·5% macrophages phagocytic in the 1540, 2340 and 4110ppb groups, respectively).The results of the study show that chromium yeast is able to modulate the immune response of rainbow trout, and this effect appears to be both dose- and time-dependent.

The production of a lymphokine (macrophage activating factor) by rainbow trout, Oncorhynchus mykiss (Walbaum), leucocytes stimulated with the extracellular products of Mycobacterium sp.

The production of macrophage activation factor (MAF) by rainbow trout, Oncorhynchus mykiss (Walbaum), head kidney leucocytes was examined after culturing in vitro with extracellular products (ECP) collected from Mycobacterium sp. Cultures of leucocytes were prepared from naive fish, or fish previously vaccinated with either the ECP or with formalin killed whole cell preparations (WC) of the bacterium. The cells were then incubated with the ECP in vitro and the ability of their supernatants to activate macrophages assessed. Macrophages from control fish were incubated with the supernatants, and their ability to reduce nitroblue tetrazolium (NBT) measured as an indicator of macrophage activation. Incubation of head kidney macrophages from naive fish directly with 1, 10 or 100 μg mL–1 of ECP for 48 h significantly enhanced macrophage activation compared with control macrophages. Vaccination of fish with either ECP or WC had no significant effect on the respiratory burst of control macrophages 4 weeks post‐vaccination. By the eighth week, however, absorbance levels of respiratory burst reflecting both the primary (cells from vaccinated fish cultured in vitro with PBS) and the secondary (cells cultured in vitro with ECP) MAF responses of fish vaccinated with ECP and WC, had peaked and these were significantly different from the non‐vaccinated controls. This activity had fallen to levels similar to control fish by week 12 for fish vaccinated with WC.