Nitric oxide-induced membrane tubulovesicular extensions (cytonemes) of human neutrophils catch and hold Salmonella enterica serovar Typhimurium at a distance from the cell surface

Nitric oxide (NO) plays an important role in host defense against bacterial infections such as salmonellosis. NO and 4-bromophenacyl bromide (BPB) induce the formation of long tubulovesicular extensions (TVE, cytonemes, membrane tethers) from human neutrophils. These TVE serve as cellular sensory and adhesive organelles. In the present study, we demonstrated that in the presence of the NO donor, diethylamine NONOate or BPB human neutrophils bound and aggregated Salmonella enterica serovar Typhimurium bacteria extracellularly by TVE. In contrast, inhibition of NO-synthase activity by N ^ω-nitro- l -arginine methyl ester stimulated neutrophil phagocytosis (ingestion) of bacteria. Neutrophil TVE consisted of membrane-covered cytoplasm as was shown by the fluorescent cytoplasmic dye 2',7'-bis(2carboxyethyl)-5,(6)-carboxyfluorescein, and the fluorescent lipid, BODIPY-labeled sulfatide. Disruption and shedding of TVE were accompanied by the appearance of specific invaginations (porosomes) on neutrophil cell bodies. These invaginations corresponded to the variations in diameter of TVE (160–240 nm). We hypothesized that TVE represented protrusions of neutrophil exocytotic trafficking through special structures on the neutrophil surface. In conclusion, we propose a novel mechanism by which NO-induced TVE formation enables neutrophils to bind and aggregate bacteria at a distance.     

The effect of selenium sources and supplementation on neutrophil functions in dairy cows

Selenium (Se), an essential micronutrient, is believed to enhance neutrophil functions. This study aimed to compare the effects of supplemented organic (Sel-Plex®) and inorganic (sodium selenite) Se on neutrophil functions in high-producing dairy cows, during the periparturient period. Twenty-five Holstein cows were randomly allocated to five dietary treatments as follows: control diet (basal diet without Se supplementation), IN 0.3 (basal diet supplemented with inorganic Se at 0.3 mg/kg dry matter (DM)), IN 0.5 (inorganic Se at 0.5 mg/kg DM), OR 0.3 (organic Se at 0.3 mg/kg DM) and OR 0.5 (organic Se at 0.5 mg/kg DM). Some evaluated parameters included neutrophil functions and plasma Se concentrations in cows and plasma Se concentrations in calves. Neutrophil phagocytosis did not significantly differ among the five groups. However, organic Se supplementation significantly increased (P < 0.01) the respiratory burst of neutrophils when compared to cows fed IN 0.3 and the control diet. In comparison to inorganic Se, neutrophil apoptosis was decreased (P < 0.01) when cows were fed organic Se or the control diets. These effects of organic Se on respiratory burst activities and apoptosis of neutrophils were in a dose-dependent manner. Calf plasma Se concentrations were higher (P < 0.05) when cows were fed OR 0.5 and IN 0.5.     

Thyroid hormone regulation of cell migration and oxidative metabolism in polymorphonuclear leukocytes: clinical evidence in thyroidectomized subjects on thyroxine replacement therapy

Migration and superoxide anion (O2-) generation were studied in polymorphonuclear leukocytes (PMNs) from 14 athyreotic patients, previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice: in hypothyroidism (visit 1) and after TSH-suppressive L-T4 replacement therapy (visit 2). Random migration and N-formyl-Met-Leu-Phe (fMLP) 0.1-microM induced chemotaxis were similar in cells from patients at both visit 1 and visit 2 and from healthy controls. On the contrary, resting O2- generation in cells from patients was significantly lower than control values, both at visit 1 and 2. At visit 1, fMLP 0.1 muM-induced O2- generation was significantly lower than control values, while phorbol-myristate acetate (PMA) 100-ng/ml induced O2- generation was similar in cells from patients and from controls. At visit 2 both responses increased, resulting in fMLP-induced O2- generation superimposable to control values and PMA-induced O2- generation significantly higher with respect to both visit 1 and cells from controls. In vitro exposure of PMNs from healthy subjects to L-T4 did not affect O2- generation in resting cells, and significantly increased that induced by fMLP or PMA only at high, supra-physiological concentrations. Neither TSH nor T3 had significant effects at any of the concentrations tested. The present results document the existence of a correlation between thyroid status and oxidative metabolism of human PMNs, which is however unlikely to depend upon a direct action of thyroid hormones on these cells.     

Isoxazol-5(2H)-one: a new scaffold for potent human neutrophil elastase (HNE) inhibitors

Human neutrophil elastase (HNE) is an important target for the development of novel and selective inhibitors to treat inflammatory diseases, especially pulmonary pathologies. Here, we report the synthesis, structure–activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with an isoxazol-5(2H)-one scaffold. The most potent compound (2o) had a good balance between HNE inhibitory activity (IC₅₀ value =20?nM) and chemical stability in aqueous buffer (t_1/2=8.9?h). Analysis of reaction kinetics revealed that the most potent isoxazolone derivatives were reversible competitive inhibitors of HNE. Furthermore, since compounds 2o and 2s contain two carbonyl groups (2-N-CO and 5-CO) as possible points of attack for Ser195, the amino acid of the active site responsible for the nucleophilic attack, docking studies allowed us to clarify the different roles played by these groups.     

Mycoplasma pneumoniae infection induces a neutrophil-derived antimicrobial peptide, cathelin-related antimicrobial peptide

In innate immunity, cationic antimicrobial peptides including cathelin-related antimicrobial peptide (CRAMP) are known to play critical roles in protecting the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between CRAMP and mycoplasmas. In the present study, the antimicrobial activity of CRAMP against M. pneumoniae and the expression of CRAMP in bronchoalveolar lavage fluid (BALF) of M. pneumoniae-infected mice was examined. CRAMP at 10-20 μg/mL reduced the growth of two strains of M. pneumoniae by 100 to 1000-fold. The amount of CRAMP in the BALF of M. pneumoniae-infected mice was 20～25 ng/mL by ELISA. The presence of mature CRAMP in BALF was observed by Western blotting. Neutrophils in BALF showed a fair amount of CRAMP in their cytoplasm by immunofluorescence. Furthermore, the addition of M. pneumoniae resulted in the release of a large amount of CRAMP from neutrophils induced by thioglycolate. These results suggest that CRAMP from neutrophils may play an important role in protection against M. pneumoniae infection.     

Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α₁-antitrypsin (α₁AT) deficiency cases. It leads to increased polymerization of the serpin α₁AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α₁AT and analyzed their fluorescence properties. α₁AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α₁AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α₁AT compared to M α₁AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B.     

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.     

A 14 000-year record of paleoenvironmental change in the western basin of China's third largest lake,Lake Taihu

Effect of leaf extract of Ocimum sanctum on (i) the specific and non-specific immune responses and (ii) disease resistance against Aeromonas hydrophila was investigated in Oreochromis mossambicus. Sheep red blood cells (SRBC) and Heat aggregated bovine serum albumin (HA-BSA) were used as antigens for specific and non-specific immune response studies, respectively. Antigens were administered through intraperitoneal route. Anti-SRBC antibody titres were determined by direct haemagglutination and anti-bacterial antibodies were determined by bacterial agglutination. Nitroblue tetrazolium (NBT) assay was used to determine neutrophil activity. Disease resistance was measured in terms of relative percent survival (RPS). The immunostimulatory effect of the leaf extract of O. sanctum, when administered through intraperitoneal and oral routes was obvious. Leaf extract of O. sanctum, when administered intraperitoneally, stimulated both antibody response and neutrophil activity. Dietary intake of O. sanctum also enhanced the antibody response and disease resistance against A. hydrophila. Possibility of using O. sanctum as immunostimulant in the maintenance of finfish health in intensive freshwater aquaculture is suggested.     

PIP3在中性粒细胞信号通路中的作用及生成调节

趋化性是中性粒细胞参与机体对抗病原体的一个基本的细胞反应。中性粒细胞的趋化过程中涉及一系列信号通路来调节其运动性和极性。信号分子磷酸酰肌醇三磷酸及其参与的信号通路在中性粒细胞趋化过程中起着重要的作用,其自身的生成也受到一系列复杂因素的调节。