It is generally accepted that intracellular killing of microorganisms by production of reactive oxygen species (ROS) in the phagosome of the neutrophil is an important arm of innate defense. ...High-producing dairy cows are prone to periparturient metabolic and infectious diseases. Both myeloperoxidase (MPO) activity and ROS production decrease the day of parturition. Several studies have demonstrated changes in the expression of genes involved in, for example, metabolism and defense in the circulating neutrophil during peripartum. In this study, we wanted to further characterize the periparturient neutrophil in terms of its oxidative killing capacity by analyzing the oxidative burst at 3 levels. First, the ROS phenotype was evaluated using chemiluminescence. The cows (sampled within 24 h after parturition and at 135 d in milk) showed a significantly slower production of ROS at parturition. Both primiparous (n = 13) and multiparous (n = 12) cows were included in this study, but parity did not affect the kinetics of ROS production. Second, the expression of 11 genes involved in ROS production was measured in the same cows: cytochrome b-245 α and β chain (CYBA, CYBB; coding for membrane-bound constituents of NADPH oxidase); neutrophil cytosolic factors 1, 2, and 4 (NCF1, NCF2, and NCF4); Rac family small GTPase 1 and 2 (RAC1 and RAC2; coding for regulatory proteins of NADPH oxidase); superoxide dismutase 2 (SOD2); catalase (CAT); myeloperoxidase (MPO; coding for enzymes involved in metabolizing downstream ROS); and spleen-associated tyrosine kinase (SYK; involved in signaling). During peripartum, a shift in expression in the oxidative killing pathway was observed, characterized by a downregulation of MPO and a simultaneous upregulation of the genes coding for NADPH oxidase. Third, as total DNA methylation is known to change during pregnancy, we investigated whether the observed differences were due to different methylation patterns. Promotor regions initiate transcription of particular genes; therefore, we analyzed the methylation status in annotated CpG islands of MPO and SOD2, 2 genes with a significant difference in expression between both lactation stages. The differences in methylation of these CpG islands were nonsignificant. High-throughput techniques may be necessary to obtain more detailed information on the total DNA methylation dynamics in bovine neutrophils and increase our understanding of how gene expression is controlled in neutrophils.
Intramammary infections of dairy cows with Gram-positive bacteria such as Staphylococcus aureus (major cause of mastitis) have received a lot of attention because of their major economic impact on ...the dairy farm through production losses induced by an increase in somatic cell count. Management strategies, including greater awareness for efficient milking and hygienic measures, have limited the spread of Gram-positive bacteria and resulted in a significant decrease of proportion of S. aureus isolates and subclinical mastitis worldwide. Other organisms such as coliform subspecies and Streptococcus uberis, both environmental bacteria that cause clinical mastitis, have received less attention. Escherichia coli causes inflammation of the mammary gland in dairy cows around parturition and during early lactation with striking local and sometimes severe systemic clinical symptoms. This disease affects many high producing cows in dairy herds and may cause several cases of death per year in the most severe cases. It is well known that bacterial, cow and environmental factors are interdependent and influence mastitis susceptibility. Many studies, executed during the last decade, indicate that the severity of E. coli mastitis is mainly determined by cow factors rather than by E. coli pathogenicity. During E. coli mastitis, the host defense status is a cardinal factor determining the outcome of the disease. Today, we know that the neutrophil is a key factor in the cows' defense against intramammary infection with E. coli. Effective elimination of the pathogen by neutrophils is important for the resolution of infection and the outcome of E. coli mastitis. This review is a compilation of some major findings over the last 15 years concerning mainly host factors that modulate and influence neutrophil function and the mammary inflammatory reaction. The individual chapters address: virulence factors of E. coli strains, how neutrophils kill E. coli, connection between endotoxins, tumor necrosis factor-alpha and nitric oxide, severity classification of E. coli mastitis, lifespan of neutrophils, host factors that influence severity, tissue damage and production loss.
Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that ...need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples—even single cell samples—can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10cells for reverse transcription quantitative PCR (RT–qPCR) analysis, the low RNA quality should be taken into account when designing the RT–qPCR assays.
The dynamics of blood neutrophil acyloxyacyl hydrolase (AOAH) activity, the appearance of endotoxin (lipopolysaccharide, LPS) in blood and the role of blood neutrophil AOAH in the severity of ...Escherichia coli and endotoxin mastitis were investigated in early postpartum dairy cows experimentally challenged with either endotoxin (n = 6) or E. coli (n = 6). The AOAH activity of blood neutrophils started to decrease significantly at post challenge hours (PCH) 6-24 and 12-24 in the endotoxin and E. coli-challenged groups, respectively; it returned to pre-challenged values at PCH 48 in both endotoxin- and E. coli-challenged groups. The cows were classified as moderate and severe responders according to milk production loss in the non-challenged quarters at PCH 48. There were no severe responders in the endotoxin-challenged group. In the E. coli-challenged group, only 1 severe responder was identified. The pre-challenge neutrophil AOAH activity of the severe responder was approximately 30% lower than that of moderate responders. No LPS was detected in the plasma of endotoxin-challenged cows; neither was it found in the plasma of moderate responders in the E. coli-challenged group at any PCH. However, at PCH 6, a remarkable amount of LPS was detected in the plasma of the severe responder from the E. coli-challenged group. Furthermore, neutrophil AOAH activity was increased by approximately 70% in the severe responder at PCH 6, but it increased by only approximately 15% in moderate responders. This was followed by a decreased neutrophil AOAH activity at PCH 12-24 and 24-72 in moderate and severe responders, respectively; the decreased AOAH activity at those PCH was more pronounced in the severe responder. The pronounced decreased neutrophil AOAH activity during mastitis often coincided with extreme leukopenia, neutropenia and a maximal number of immature neutrophils in the blood. Our results demonstrate that a decrease in neutrophil AOAH activity results in the appearance of LPS in the blood, and low blood neutrophil deacylation activity could be considered as a risk factor for severe clinical coliform mastitis.
To evaluate L-selectin (CD62L) and Mac-1 (CD11b) expression at the surface of blood and milk neutrophils during the early inflammatory response to Escherichia coli-induced mastitis in cows.
6 healthy ...Holstein heifers in early lactation.
Blood and milk samples were collected before and after intramammary administration of 10(4) CFU's of E coli in the left mammary gland quarters. Bacterial counts and electrolyte concentrations in milk, rectal temperature, differential blood leukocyte counts, milk somatic cell counts, neutrophil viability, and the expression of CD62L and CD11b on blood and milk neutrophils were determined longitudinally.
Bacteria grew during the first 6 hours after inoculation with a pronounced leukocytic influx. Coincident with neutrophil influx was an increase in CD62L+ and CD11b+ milk neutrophils, as well as an improved viability of milk neutrophils. The peak of the inflammatory reaction was reached approximately 12 hours after E coli inoculation. From that time forward, changes in CD62L and CD11b expression were opposed to each other, with a decrease in CD62L expression and an increase in CD11b expression on blood and milk neutrophils; the magnitude of the differences in CD62L and CD11b expression between blood and milk neutrophils decreased. Percentages of CD62L+ and CD11b+ milk neutrophils increased to percentages that were similar to blood neutrophils (ie, approx 92%).
The presence of adhesion molecules on a large percentage of milk neutrophils during the acute inflammatory response, together with the changes in receptor density, suggest a major role for CD62L and CD11b in neutrophil function during coliform mastitis.
The plant Anacyclus pyrethrum (AP) consists of several N-alkylamides with pellitorine as main constituent. AP extracts are known to be biologically active and some products for topical administration ...containing AP plant extracts are already commercially available with functional cosmeceutical claims. However, no transdermal data for pellitorine are currently available. Therefore, our general goal was to investigate the local skin pharmacokinetics of the plant N-alkylamide pellitorine using a Franz diffusion cell set-up. Two different forms were applied on human skin: purified pellitorine and the AP extract. Our study demonstrated that pellitorine is able to cross the stratum corneum and the subsequent skin layers. A significantly higher permeability coefficient was observed when the AP extract (Kp=2.3×10−4cm/h) was administered, compared to purified pellitorine (Kp=1.1×10−4cm/h). With the obtained pellitorine concentrations in the skin layers and the receptor fluid, it is concluded that local and systemic effects can be expected after topical application. Due to these findings and as a regulatory consequence, products containing reasonable concentrations of pellitorine are recommended to be classified as a medicinal product.
Peptides are able to cross the blood–brain barrier (BBB) through various mechanisms, opening new diagnostic and therapeutic avenues. However, their BBB transport data are scattered in the literature ...over different disciplines, using different methodologies reporting different influx or efflux aspects. Therefore, a comprehensive BBB peptide database (Brainpeps) was constructed to collect the BBB data available in the literature. Brainpeps currently contains BBB transport information with positive as well as negative results. The database is a useful tool to prioritize peptide choices for evaluating different BBB responses or studying quantitative structure–property (BBB behaviour) relationships of peptides. Because a multitude of methods have been used to assess the BBB behaviour of compounds, we classified these methods and their responses. Moreover, the relationships between the different BBB transport methods have been clarified and visualized.
An N-alkylamide profiling from an ethanolic Anacyclus pyrethrum DC. root extract was performed using a gradient reversed phase high performance liquid chromatography/UV/electrospray-ionization ...ion-trap mass spectrometry (HPLC/UV/ESI-MS) method on an embedded polar column. MS1 and MS2 fragmentation data were used for identification purposes while UV was used for quantification. Thirteen N-alkylamides (five N-isobutylamides, three N-methyl isobutylamides, four tyramides and one 2-phenylethylamide) were detected. Six of them, identified as undeca-2E,4E-diene-8,10-diynoic acid isobutylamide, undeca-2E,4E-diene-8,10-diynoic acid N-methyl isobutylamide, tetradeca-2E,4E-diene-8,10-diynoic acid tyramide, deca-2E,4E-dienoic acid N-methyl isobutylamide, tetradeca-2E,4E,XE/Z-trienoic acid tyramide and tetradeca-2E,4E,XE/Z,YE/Z-tetraenoic isobutylamide, are novel compounds which have never been reported before from Anacyclus pyrethrum.
Currently, dermal exposure data of cyclic depsipeptide mycotoxins are completely absent. There is a lack of understanding about the local skin and systemic kinetics and effects, despite their ...widespread skin contact and intrinsic hazard. Therefore, we provide a quantitative characterisation of their dermal kinetics. The emerging mycotoxins enniatins (ENNs) and beauvericin (BEA) were used as model compounds and their transdermal kinetics were quantitatively evaluated, using intact and damaged human skin in an in vitro Franz diffusion cell set-up and ultra high-performance liquid chromatography (UHPLC)-MS analytics. We demonstrated that all investigated mycotoxins are able to penetrate through the skin. ENN B showed the highest permeation (kp,v=9.44 × 10(-6) cm/h), whereas BEA showed the lowest (kp,v=2.35 × 10(-6) cm/h) and the other ENNs ranging in between. Combining these values with experimentally determined solubility data, Jmax values ranging from 0.02 to 0.35 μg/(cm(2) h) for intact skin and from 0.07 to 1.11 μg/(cm(2) h) for damaged skin were obtained. These were used to determine the daily dermal exposure (DDE) in a worst-case scenario. On the other hand, DDE's for a typical occupational scenario were calculated based on real-life mycotoxin concentrations for the industrial exposure of food-related workers. In the latter case, for contact with intact human skin, DDE's up to 0.0870 ng/(kg BW × day) for ENN A were calculated, whereas for impaired skin barrier this can even rise up to 0.3209 ng/(kg BW × day) for ENN B1. This knowledge is needed for the risk assessment after skin exposure of contaminated food, feed, indoor surfaces and airborne particles with mycotoxins.
Following several conflicting publications, the inability to reproduce the original findings on
in vitro obestatin binding and activation of GPR39 receptors was recently reported by its discoverers, ...and several hypotheses to rationalize these findings were proposed. Based on one of these postulations (
i.e., presence of impurities), peptide identity and impurity profiles were thoroughly evaluated on obestatin peptides obtained from five different manufacturers, as used by the different research groups. We found that one of the products examined was in reality a totally different peptide and that the quality of two-thirds of the other peptides was insufficient for
in vitro and
in vivo experiments (
i.e., peptide purity less than 95% and/or individual impurities exceeding 1%). These observations question the divergent conclusions reported in the literature about the activity of obestatin. Therefore, we strongly recommend appropriate quality control testing before using any peptides for biomedical research purposes.
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