Short communication: J-5 Escherichia coli vaccination does not influence severity of an Escherichia coli intramammary challenge in primiparous cows.

The objective of the present study was to evaluate the efficacy of a J-5 Escherichia coli vaccine in a mild to moderate inflammatory challenge model using primiparous dairy cows for inoculation only. We hypothesized a clinical difference between placebo and J-5 E. coli vaccinated animals with the mild to moderate inflammatory challenge model. In case the null hypothesis could not be confirmed, the alternate hypothesis was no clinical difference between both treatment groups. Therefore, 23 primiparous cows in mo 7 of pregnancy were randomly assigned to 1 of 2 treatment groups: J-5 E. coli vaccine (n = 12) or placebo (n = 11). Animals were vaccinated 3 times at 56 (±7) and 28 (±7) d before expected calving date and within 14 d postcalving (DIM 5 ± 3). All cows were challenged by infusion with E. coli P4:O32 into 2 left mammary quarters between 14 and 28 d postparturition, at least 10 d after the 3rd vaccination, immediately after the morning milking. Clinical observations and blood and milk samples were collected at several time points from 7 d pre-challenge until 13 d post-challenge. Primiparous cows responded mild to moderately to intramammary E. coli challenge with little clinical difference between treatment groups. Rectal temperature increased earlier in the vaccinated animals, which also eliminated bacteria faster during the early hours after intramammary E. coli challenge. At post-infusion hour 9, the bacterial population was significantly lower in the infected quarters of the vaccinated animals. Blood leukocyte number was only numerically higher in the vaccinated animals, in combination with a numerically higher percentage of late immature polymorphonuclear leukocytes (band cells) in circulation. Even in the nonvaccinated animals, the E. coli challenge in the primiparous cows elicited only a mild to moderate response. The absence of a pronounced clinical difference between vaccinated and nonvaccinated animals was therefore not surprising.

Methylation of selected CpG islands involved in the transcription of myeloperoxidase and superoxide dismutase 2 in neutrophils of periparturient and mid-lactation cows.

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 ß 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.

Mucosal and blood-brain barrier transport kinetics of the plant N-alkylamide spilanthol using in vitro and in vivo models.

BACKGROUND: N-alkylamides (NAAs) are a large group of secondary metabolites occurring in more than 25 plant families which are often used in traditional medicine. A prominent active NAA is spilanthol. The general goal was to quantitatively investigate the gut mucosa and blood-brain barrier (BBB) permeability pharmacokinetic properties of spilanthol. METHODS: Spilanthes acmella (L.) L. extracts, as well as purified spilanthol were used to investigate (1) the permeation of spilanthol through a Caco-2 cell monolayer in vitro, (2) the absorption from the intestinal lumen after oral administration to rats, and (3) the permeation through the BBB in mice after intravenous injection. Quantification of spilanthol was performed using a validated bio-analytical UPLC-MS(2) method. RESULTS: Spilanthol was able to cross the Caco-2 cell monolayer in vitro from the apical-to-basolateral side and from the basolateral-to-apical side with apparent permeability coefficients Papp between 5.2 · 10(-5) and 10.2 · 10(-5) cm/h. This in vitro permeability was confirmed by the in vivo intestinal absorption in rats after oral administration, where an elimination rate constant ke of 0.6 h(-1) was obtained. Furthermore, once present in the systemic circulation, spilanthol rapidly penetrated the blood-brain barrier: a highly significant influx of spilanthol into the brains was observed with a unidirectional influx rate constant K1 of 796 µl/(g · min). CONCLUSIONS: Spilanthol shows a high intestinal absorption from the gut into the systemic circulation, as well as a high BBB permeation rate from the blood into the brain.

The mycotoxin definition reconsidered towards fungal cyclic depsipeptides.

Currently, next to the major classes, cyclic depsipeptides beauvericin and enniatins are also positioned as mycotoxins. However, as there are hundreds more fungal cyclic depsipeptides already identified, should these not be considered as mycotoxins as well? The current status of the mycotoxin definition revealed a lack of consistency, leading to confusion about what compounds should be called mycotoxins. Because this is of pivotal importance in risk assessment prioritization, a clear and quantitatively expressed mycotoxin definition is proposed, based on data of widely accepted mycotoxins. Finally, this definition is applied to a set of fungal cyclic depsipeptides, revealing that some of these should indeed be considered as mycotoxins.

Human skin permeation of emerging mycotoxins (beauvericin and enniatins).

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.

Quorum Sensing Peptides Selectively Penetrate the Blood-Brain Barrier.

Bacteria communicate with each other by the use of signaling molecules, a process called 'quorum sensing'. One group of quorum sensing molecules includes the oligopeptides, which are mainly produced by Gram-positive bacteria. Recently, these quorum sensing peptides were found to biologically influence mammalian cells, promoting i.a. metastasis of cancer cells. Moreover, it was found that bacteria can influence different central nervous system related disorders as well, e.g. anxiety, depression and autism. Research currently focuses on the role of bacterial metabolites in this bacteria-brain interaction, with the role of the quorum sensing peptides not yet known. Here, three chemically diverse quorum sensing peptides were investigated for their brain influx (multiple time regression technique) and efflux properties in an in vivo mouse model (ICR-CD-1) to determine blood-brain transfer properties: PhrCACET1 demonstrated comparatively a very high initial influx into the mouse brain (Kin = 20.87 µl/(g×min)), while brain penetrabilities of BIP-2 and PhrANTH2 were found to be low (Kin = 2.68 µl/(g×min)) and very low (Kin = 0.18 µl/(g×min)), respectively. All three quorum sensing peptides were metabolically stable in plasma (in vitro) during the experimental time frame and no significant brain efflux was observed. Initial tissue distribution data showed remarkably high liver accumulation of BIP-2 as well. Our results thus support the potential role of some quorum sensing peptides in different neurological disorders, thereby enlarging our knowledge about the microbiome-brain axis.

Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo.

Cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47-57, transportan 10 (TP10) and TP10-2, were determined. The results of the multiple time regression (MTR) analysis revealed that CPPs show divergent BBB influx properties: Tat 47-57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 µl/(g × min), 5.63 µl/(g × min) and 6.02 µl/(g × min), respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA) did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.

Classification of Peptides According to their Blood-Brain Barrier Influx.

An increasing number of studies demonstrate the ability of peptides to cross the blood-brain barrier (BBB), opening perspectives for a new class of therapeutics for central nervous system diseases. However, information on the BBB transport of peptides suffer from a wide variety in used methods and experimental set-up. Therefore, it is currently difficult, if not impossible, to classify peptides according to their BBB influx characteristics. To allow direct comparison of BBB influx results of peptides, we introduce a classification method and unified response for BBB influx transport of peptides. First, the results of BBB influx response types (i.e. Kin (MTR), Kin (Perfusion), Pin vitro and Pin vivo), which quantitatively express brain influx, were classified into five classes of BBB influx magnitude based on the distribution of these results for the individual response types. Then, these classes were converted to a BBBin-response, representing a scaled value ranging from zero (no influx) to ten (high influx), independent from the BBB influx response type from which it was derived. This unified response can immediately be applied for new BBB influx results of peptides and represents a ballpark figure for BBB influx and allows direct comparison and ranking of peptides independent of the response type.

Reactive oxygen species generation by bovine blood neutrophils with different CXCR1 (IL8RA) genotype following Interleukin-8 incubation.

BACKGROUND: Associations between polymorphisms in the bovine CXCR1 gene, encoding the chemokine (C-X-C motif) receptor 1 (IL8RA), and neutrophil traits and mastitis have been described. In the present study, blood neutrophils were isolated from 20 early lactating heifers with different CXCR1 genotype at position 735 or 980. The cells were incubated with different concentrations of recombinant bovine IL-8 (rbIL-8) for 2 or 6 h and stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan particles (OZP). Potential association between CXCR1 genotype and production of reactive oxygen species (ROS) was studied. RESULTS: Although on single nucleotide polymorphisms (SNPs) may potentially affect CXCR1 function, SNPs c.735C > G and c.980A > G showed no association with ROS production with or without incubation of rbIL-8. Neutrophils incubated with rbIL-8 for 2 or 6 h showed higher PMA- and lower OZP-induced ROS production compared to control without rbIL-8. CONCLUSIONS: In the present study no association could be detected between superoxide production by isolated bovine neutrophils during early lactation and CXCR1 gene polymorphism. IL-8 showed to possess inhibitory effects on ROS generation in bovine neutrophils.

The quorum sensing peptides PhrG, CSP and EDF promote angiogenesis and invasion of breast cancer cells in vitro.

The role of the human microbiome on cancer progression remains unclear. Therefore, in this study, we investigated the influence of some quorum sensing peptides, produced by diverse commensal or pathogenic bacteria, on breast cancer cell invasion and thus cancer outcome. Based on microscopy, transcriptome and Chick Chorioallantoic Membrane (CAM) analyses, four peptides (PhrG from B. subtilis, CSP from S. mitis and EDF from E. coli, together with its tripeptide analogue) were found to promote tumour cell invasion and angiogenesis, thereby potentially influencing tumour metastasis. Our results offer not only new insights on the possible role of the microbiome, but also further opportunities in cancer prevention and therapy by competing with these endogenous molecules and/or by modifying people's life style.

Crosstalk between the microbiome and cancer cells by quorum sensing peptides.

To date, the precise role of the human microbiome in health and disease states remains largely undefined. Complex and selective crosstalk systems between the microbiome and mammalian cells are also not yet reported. Research up till now mainly focused on bacterial synthesis of virulence factors, reactive oxygen/nitrogen species (ROS/RNS) and hydrogen sulphide, as well as on the activation of exogenous mutagen precursors by intestinal bacteria. We discovered that certain quorum sensing peptides, produced by bacteria, interact with mammalian cells, in casu cancer cells: Phr0662 (Bacillus sp.), EntF-metabolite (Enterococcus faecium) and EDF-derived (Escherichia coli) peptides initiate HCT-8/E11 colon cancer cell invasion, with Phr0662 also promoting angiogenesis. Our findings thus indicate that the human microbiome, through their quorum sensing peptides, may be one of the factors responsible for cancer metastasis.

Blood-brain transfer and antinociception of linear and cyclic N-methyl-guanidine and thiourea-enkephalins.

Enkephalins are active in regulation of nociception in the body and are key in development of new synthetic peptide analogs that target centrally located opioid receptors. In this study, we investigated the in vivo blood-brain barrier (BBB) penetration behavior and antinociceptive activity of two cyclic enkephalin analogs with a thiourea (CycS) or a N-methyl-guanidine bridge (CycNMe), and their linear counterparts (LinS and LinNMe) in mice, as well as their in vitro metabolic stability. (125)I-LinS had the highest blood-brain clearance (K1=3.46µL/gmin), followed by (125)I-LinNMe, (125)I-CycNMe, and (125)I-CycS (K1=1.64, 0.31, and 0.11µL/gmin, respectively). Also, these peptides had a high metabolic stability (t1/2>1h) in mouse serum and brain homogenate, and half-inhibition constant (Ki) values in the nanomolar range with predominantly µ-opioid receptor selectivity. The positively charged NMe-enkephalins showed a higher antinociceptive activity (LinNMe: 298% and CycNMe: 205%), expressed as molar-dose normalized area under the curve (AUC) relative to morphine, than the neutral S-enkephalins (CycS: 122% and LinS: 130%).

Quantitative transdermal behavior of pellitorine from Anacyclus pyrethrum extract.

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(-4)cm/h) was administered, compared to purified pellitorine (Kp=1.1 × 10(-4)cm/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.

Identification of a microscopically selected microorganism in milk samples.

Identification of unwanted microbial contaminants microscopically observed in food products is challenging due to their low abundance in a complex matrix, quite often containing other microorganisms. Therefore, a selective identification method was developed using laser capture microdissection in combination with direct-captured cell PCR. This procedure was validated with Geobacillus stearothermophilus and further used to identify microbial contaminants present in some industrial milk samples. The microscopically observed contaminants were identified as mainly Methylobacterium species.

LC-UV/MS quality analytics of paediatric artemether formulations.

A highly selective and stability-indicating HPLC-method, combined with appropriate sample preparation steps, is developed for ß-artemether assay and profiling of related impurities, including possible degradants, in a complex powder for oral suspension. Following HPLC conditions allowed the required selectivity: a Prevail organic acid (OA) column (250 mm×4.6 mm, 5 µm), flow rate set at 1.5 mL/min combined with a linear gradient (where A=25 mM phosphate buffer (pH 2.5), and B=acetonitrile) from 30% to 75% B in a runtime of 60 min. Quantitative UV-detection was performed at 210 nm. Acetonitrile was applied as extraction solvent for sample preparation. Using acetonitrile-water mixtures as extraction solvent, a compartmental behaviour by a non-solving excipient-bound fraction and an artemether-solubilising free fraction of solvent was demonstrated, making a mobile phase based extraction not a good choice. Method validation showed that the developed HPLC-method is considered to be suitable for its intended regulatory stability-quality characterisation of ß-artemether paediatric formulations. Furthermore, LC-MS on references as well as on stability samples was performed allowing identity confirmation of the ß-artemether related impurities. MS-fragmentation scheme of ß-artemether and its related substances is proposed, explaining the m/z values of the in-source fragments obtained.

Risk evaluation of impurities in topical excipients: The acetol case.

Pharmaceutical excipients for topical use may contain impurities, which are often neglected from a toxicity qualification viewpoint. The possible impurities in the most frequently used topical excipients were evaluated in-silico for their toxicity hazard. Acetol, an impurity likely present in different topical pharmaceutical excipients such as propylene glycol and glycerol, was withheld for the evaluation of its health risk after dermal exposure. An ex-vivo in-vitro permeation study using human skin in a Franz Diffusion Cell set-up and GC as quantification methodology showed a significant skin penetration with an overall Kp value of 1.82×10-3 cm/h. Using these data, limit specifications after application of a dermal pharmaceutical product were estimated. Based on the TTC approach of Cramer class I substances, i.e. 1800 µg/(day∙person), the toxicity-qualified specification limits of acetol in topical excipients were calculated to be 90 µg/mL and 180 µg/mL for propylene glycol and glycerol, respectively. It is concluded that setting specification limits for impurities within a quality-by-design approach requires a case-by-case evaluation as demonstrated here with acetol.

Blood-brain barrier transport of short proline-rich antimicrobial peptides.

Infections by antibiotic-resistant bacteria are becoming a great risk for human health, leading to an urgent need for new efficient antibacterial therapies. The short, proline-rich antimicrobial peptides from insects gained a lot of interest as a potential antibacterial treatment, having a low toxicity profile and being mainly active against Gram-negative bacteria. To know whether these antimicrobial peptides can be used for the treatment of cerebral infections, the blood-brain barrier transport characteristics of these peptides were investigated. This study describes the results of the in vivo blood-brain barrier experiments in mice, as well as the in vitro metabolic stability in mouse plasma and brain of apidaecin Api137, oncocin, drosocin and drosocin Pro5Hyp. The four investigated peptides showed a significant influx into the brain with a K(in) ranging between 0.37 and 0.86 µL/g x min and brain distribution volumes of 19.6 to 25.8 µL/g. Only for drosocin, a significant efflux was determined, with a k(out) of 0.22 min(-1). After entering the brain, oncocin was for approximately 80% trapped in the endothelial cells, while the other peptides reached the brain parenchyma for about 70%. All peptides were stable in plasma and brain during the experiments, with estimated metabolic half-lives ranging between 47 min and 637 min. We conclude that the investigated short, proline-rich antimicrobial peptides show an influx into the brain, which make them a promising antibacterial treatment of cerebral infections.

LC-UV/MS quality analytics of paediatric artemether formulations / 药物分析学报

A highly selective and stability-indicating HPLC-method, combined with appropriate sample preparation steps, is developed for β-artemether assay and profiling of related impurities, including possible degradants, in a complex powder for oral suspension. Following HPLC conditions allowed the required selectivity: a Prevail organic acid (OA) column (250 mm×4.6 mm, 5μm), flow rate set at 1.5 mL/min combined with a linear gradient (where A ? 25 mM phosphate buffer (pH 2.5), and B ? acetonitrile) from 30% to 75% B in a runtime of 60 min. Quantitative UV-detection was performed at 210 nm. Acetonitrile was applied as extraction solvent for sample preparation. Using acetonitrile-water mixtures as extraction solvent, a compartmental behaviour by a non-solving excipient-bound fraction and an artemether-solubilising free fraction of solvent was demonstrated, making a mobile phase based extraction not a good choice. Method validation showed that the developed HPLC-method is considered to be suitable for its intended regulatory stability-quality characterisation of β-artemether paediatric formulations. Furthermore, LC-MS on references as well as on stability samples was performed allowing identity confirmation of the β-artemether related impurities. MS-fragmentation scheme of β-artemether and its related substances is proposed, explaining the m/z values of the in-source fragments obtained.

Risk evaluation of impurities in topical excipients:The acetol case / 药物分析学报

Pharmaceutical excipients for topical use may contain impurities, which are often neglected from a toxicity qualification viewpoint. The possible impurities in the most frequently used topical excipients were evaluated in-silico for their toxicity hazard. Acetol, an impurity likely present in different topical pharmaceutical excipients such as propylene glycol and glycerol, was withheld for the evaluation of its health risk after dermal exposure. An ex-vivo in-vitro permeation study using human skin in a Franz Diffusion Cell set-up and GC as quantification methodology showed a significant skin penetration with an overall Kp value of 1.82 ? 10 ? 3 cm/h. Using these data, limit specifications after application of a dermal pharmaceutical product were estimated. Based on the TTC approach of Cramer class I substances, i.e. 1800 mg/(day?person), the toxicity-qualified specification limits of acetol in topical excipients were calculated to be 90 mg/mL and 180 mg/mL for propylene glycol and glycerol, respectively.

Chemical-functional diversity in cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are a promising tool to overcome cell membrane barriers. They have already been successfully applied as carriers for several problematic cargoes, like e.g. plasmid DNA and (si)RNA, opening doors for new therapeutics. Although several hundreds of CPPs are already described in the literature, only a few commercial applications of CPPs are currently available. Cellular uptake studies of these peptides suffer from inconsistencies in used techniques and other experimental conditions, leading to uncertainties about their uptake mechanisms and structural properties. To clarify the structural characteristics influencing the cell-penetrating properties of peptides, the chemical-functional space of peptides, already investigated for cellular uptake, was explored. For 186 peptides, a new cell-penetrating (CP)-response was proposed, based upon the scattered quantitative results for cellular influx available in the literature. Principal component analysis (PCA) and a quantitative structure-property relationship study (QSPR), using chemo-molecular descriptors and our newly defined CP-response, learned that besides typical well-known properties of CPPs, i.e. positive charge and amphipathicity, the shape, structure complexity and the 3D-pattern of constituting atoms influence the cellular uptake capacity of peptides.