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To derive porcine embryonic stem (ES) cell lines, the time window during which porcine embryos contain pluripotent cells that are predisposed to undifferentiated self-renewal in vitro must be identified. Therefore we first studied the spatial and temporal expression pattern of key factors in pluripotency and lineage segregation of blastocyst-stage porcine embryos between embryonic days (E) 6.5 and E10.5 using whole mount in situ hybridization, quantitative reverse transcription (RT)-PCR and whole mount immunofluorescence. Expression of NANOG and SOX2 was detected in both the ICM and epiblast, while OCT4 expression became restricted to the epiblast at E9.5. Surprisingly ICM and epiblast cells also expressed CK18. Consequently, growth factors which sustain the undifferentiated growth of human ES cells and mouse epiblast stem cells (EpiSCs) were tested for their ability to sustain undifferentiated self-renewal of porcine ICM and epiblast cells in vitro. Cultures of ICM cells resulted in a higher percentage of primary colonies with an ES-like morphology compared to primary cultures derived from epiblast cells. These undifferentiated colonies sustained expression of OCT4, NANOG, SOX2 and CK18. The expression of CK18 suggests that these cells are more similar to human ES cells and mouse EpiSCs than to mouse ES cells. Although undifferentiated cultures were maintained for limited passages, ICM and epiblast cultures rapidly differentiated into cell types of mesodermal, ectodermal, and endodermal origin, as characterized by RT-PCR. These results demonstrate that porcine ICM and epiblast cells can not be cultured in vitro with currently used human ES cell culture conditions. Importantly however, the trio of OCT4, NANOG and SOX2, which are known to form an autoregulatory network for pluripotency in other systems, are co-expressed also by porcine epiblasts, and by undifferentiated primary colonies in culture.

Carvacrol is known to inhibit a number of food borne pathogens. The activity of carvacrol vapour was evaluated against S. enterica serotype Enteritidis on tryptone soya agar and on pieces of raw chicken. On agar, the size of the inhibition zone increased with decreasing volume of agar, increasing vapour exposure period, increasing temperature, increasing volume of carvacrol used and with decreasing bacterial density. Inhibition was equally effective under aerobic and anaerobic conditions. On chicken pieces (10 x 10 x 5 mm, UV-sterilized and inoculated with approx. 5 x 10(3) cfu) carvacrol vapour significantly reduced viable numbers of salmonellae at 4, 20 and 37 degrees C and all viable cells were eliminated by a minimum of 3 h at 37 degrees C (p

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Notably, citrullinated LL-37 has not yet been detected in human samples. In addition, functional and biophysical properties of citrullinated LL-37 are not fully explored. The aim of this study was to detect citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid and to determine antibacterial and biophysical properties of citrullinated LL-37. BAL fluid was obtained from healthy human volunteers after intra-bronchial exposure to lipopolysaccharide. Synthetic peptides were used for bacterial killing assays, transmission electron microscopy, isothermal titration calorimetry, mass-spectrometry and circular dichroism. Using targeted proteomics, we were able to detect both native and citrullinated LL-37 in BAL fluid. The citrullinated peptide did not kill Escherichia coli nor lysed human red blood cells. Both peptides had similar α-helical secondary structures but citrullinated LL-37 was more stable at higher temperatures, as shown by circular dichroism. In conclusion, citrullinated LL-37 is present in the human airways and citrullination impaired bacterial killing, indicating that a net positive charge is important for antibacterial and membrane lysing effects. It is possible that citrullination serves as a homeostatic regulator of AMP-function by alteration of key functions.

Fungal infections in humans are increasing worldwide and are currently mostly treated with a relative limited set of antifungals. Resistance to antifungals is increasing, for example, in Aspergillus fumigatus and Candida auris, and expected to increase for many medically relevant fungal species in the near future. We have developed and patented a set of cathelicidin-inspired antimicrobial peptides termed 'PepBiotics'. These peptides were initially selected for their bactericidal activity against clinically relevant Pseudomonas aeruginosa and Staphylococcus aureus isolates derived from patients with cystic fibrosis and are active against a wide range of bacteria (ESKAPE pathogens). We now report results from studies that were designed to investigate the antifungal activity of PepBiotics against a set of medically relevant species encompassing species of Aspergillus, Candida, Cryptococcus, Fusarium, Malassezia, and Talaromyces. We characterized a subset of PepBiotics and show that these peptides strongly affected metabolic activity and/or growth of a set of medically relevant fungal species, including azole-resistant A. fumigatus isolates. PepBiotics showed a strong inhibitory activity against a large variety of filamentous fungi and yeasts species at low concentrations (≤1 μM) and were fungicidal for at least a subset of these fungal species. Interestingly, the concentration of PepBiotics required to interfere with growth or metabolic activity varied between different fungal species or even between isolates of the same fungal species. This study shows that PepBiotics display strong potential for use as novel antifungal compounds to fight a large variety of clinically relevant fungal species.

To develop a Bordetella bronchiseptica vaccine with reduced endotoxicity, we previously inactivated lpxL1, the gene encoding the enzyme that incorporates a secondary 2-hydroxy-laurate in lipid A. The mutant showed a myriad of phenotypes. Structural analysis showed the expected loss of the acyl chain but also of glucosamine (GlcN) substituents, which decorate the phosphates in lipid A. To determine which structural change causes the various phenotypes, we inactivated here lgmB, which encodes the GlcN transferase, and lpxL1 in an isogenic background and compared the phenotypes. Like the lpxL1 mutation, the lgmB mutation resulted in reduced potency to activate human TLR4 and to infect macrophages and in increased susceptibility to polymyxin B. These phenotypes are therefore related to the loss of GlcN decorations. The lpxL1 mutation had a stronger effect on hTLR4 activation and additionally resulted in reduced murine TLR4 activation, surface hydrophobicity, and biofilm formation, and in a fortified outer membrane as evidenced by increased resistance to several antimicrobials. These phenotypes, therefore, appear to be related to the loss of the acyl chain. Moreover, we determined the virulence of the mutants in the Galleria mellonella infection model and observed reduced virulence of the lpxL1 mutant but not of the lgmB mutant.

Salmonella Infantis is a poultry-adapted Salmonella enterica serovar that is increasingly reported in broilers and is also regularly identified among human salmonellosis cases. An emerging S. Infantis mega-plasmid (pESI), carrying fitness, virulence and antimicrobial resistance genes, is also increasingly found. We investigated the prevalence, genetic characteristics and risk factors for (pESI-carrying) S. Infantis in broilers. Faecal samples from 379 broiler flocks (in 198 farms with ≥3000 birds) in the Netherlands were tested. A questionnaire about farm characteristics was also administered. Sampling was performed in July 2018-May 2019, three weeks before slaughter. Fourteen flocks (in 10 farms) were S. Infantis-positive, resulting in a 3.7 % flock-level and 5.1 % farm-level prevalence. Based on multi-locus sequence typing (MLST), all isolates belonged to sequence type 32. All but one isolate carried a pESI-like mega-plasmid. Core-genome MLST showed considerable heterogeneity among the isolates, even within the same farm, with a few small clusters detected. The typical pESI-borne multi-resistance pattern to aminoglycosides, sulphonamide and tetracycline (93 %), as well as trimethoprim (71 %), was found. Additionally, resistance to (fluoro)quinolones based on gyrA gene mutations was detected. S. Infantis was found more often in flocks using salinomycin as coccidiostat, where flock thinning was applied or litter quality was poor, whereas employing external cleaning companies, wheat in feed, and vaccination against infectious bronchitis, were protective. Suggestive evidence for vertical transmission from hatcheries was found. A heterogeneous (pESI-carrying) S. Infantis population has established itself in Dutch broiler flocks, calling for further monitoring of its spread and a comprehensive appraisal of control options.

Equine recurrent uveitis (ERU) is considered one of the most important eye diseases in horses and typically appears with relapsing inflammatory episodes without systemic effects. Various disorders have been described as an initial trigger, including infections. Independent of the initiating cause, there are numerous indications that ERU is an immune-mediated disease. We investigated whether neutrophil extracellular traps (NETs) are part of the ERU pathogenesis. Therefore, vitreous body fluids (VBF), sera, and histological sections of the eye from ERU-diseased horses were analyzed for the presence of NET markers and compared with horses with healthy eyes. In addition, NET formation by blood derived neutrophils was investigated in the presence of VBF derived from horses with healthy eyes versus ERU-diseased horses using immunofluorescence microscopy. Interestingly, NET markers like free DNA, histone-complexes, and myeloperoxidase were detected in higher amounts in samples from ERU-diseased horses. Furthermore, in vitro NET formation was higher in neutrophils incubated with VBF from diseased horses compared with those animals with healthy eyes. Finally, we characterized the ability of equine cathelicidins to induce NETs, as potential NET inducing factors in ERU-diseased horses. In summary, our findings lead to the hypothesis that ERU-diseased horses develop more NETs and that these may contribute to the pathogenesis of ERU.

Keratinocytes (KC) are the main cellular components of the stratum corneum that constitutes a solid physical skin barrier representing the first line of defense against pathogens. Moreover, KC are potent producers of inflammatory mediators and antimicrobial peptides (AMP) when activated through their pattern recognition receptors. In atopic dermatitis (AD) the protective skin barrier may be compromised due to barrier disruption, secondary infection and accelerated secretion of inflammatory cytokines which may also affect AMP expression in the skin. In the present study, we addressed the responses of a canine KC cell line upon exposure to Staphylococcus pseudintermedius, typically found on canine atopic skin during secondary infections, and stimulation by individual AD-associated ligands and cytokines. All stimuli induced a significant increase in expression of the pro-inflammatory cytokine genes tumor necrosis factor (TNF)-α and interleukin (IL)-8, but with different kinetics. Limited effects were observed on AMP gene expression except for K9CATH which was significantly upregulated upon bacterial infection but with none of the individual AD-associated ligands. Interestingly, K9CATH possessed antimicrobial activity towards Staphylococcus pseudintermedius, indicating that K9CATH expression is a specific defense reaction towards bacterial infection and not part of a general pro-inflammatory profile of KC.

Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells (PBEC) to pro-inflammatory cytokines alters their vitamin D metabolism, antibacterial activity and expression of hCAP18/LL-37. To investigate this, PBEC were differentiated at the air-liquid interphase for 14 days in presence of the pro-inflammatory cytokines TNF-α and IL-1β (TNF-α/IL-1β), and subsequently exposed to vitamin D (inactive 25(OH)D3 and active 1,25(OH)2D3). Expression of hCAP18/LL-37, vitamin D receptor (VDR) and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using qPCR, Western blot and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using non-typeable Haemophilus influenzae (NTHi). We found that TNF-α/IL-1β treatment reduced vitamin D-induced expression of hCAP18/LL-37 and killing of NTHi. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-α/IL-1β, whereas CYP27B1 (that converts 25(OH)D3 to its active form) and VDR expression remained unaffected. Furthermore, we demonstrated that the TNF-α/IL-1β-mediated induction of CYP24A1 was at least in part mediated by the transcription factor specific protein 1 (Sp1) and the EGFR-MAPK-pathway. These findings indicate that TNF-α/IL-1β decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1, and suggests that chronic inflammation impairs protective responses induced by vitamin D.

Histones are known for their ability to bind to and regulate expression of DNA. However, histones are also present in cytoplasm and extracellular fluids where they serve host defense functions and promote inflammatory responses. Histones are a major component of neutrophil extracellular traps that contribute to bacterial killing but also to inflammatory injury. Histones can act as antimicrobial peptides and directly kill bacteria, fungi, parasites and viruses, in vitro and in a variety of animal hosts. In addition, histones can trigger inflammatory responses in some cases acting through Toll-like receptors or inflammasome pathways. Extracellular histones mediate organ injury (lung, liver), sepsis physiology, thrombocytopenia and thrombin generation and some proteins can bind histones and reduce these potentially harmful effects.

A truncated version of host defense peptide chicken cathelicidin-2, C1-15, possesses potent, broad spectrum antibacterial activity. A variant of this peptide, F2,5,12W, which contains 3 phenylalanine to tryptophan substitutions, possesses improved antibacterial activity and lipopolysaccharide (LPS) neutralizing activity compared to C1-15. In order to improve the proteolytic resistance of both peptides we engineered novel chicken cathelicidin-2 analogs by substitution of l- with d-amino acids and head-to-tail cyclization. Both cyclic and d-amino acid variants showed enhanced stability in human serum compared to C1-15 and F2,5,12W. The d-amino acid variants were fully resistant to proteolysis by trypsin and bacterial proteases. Head-to-tail cyclization of peptide F2,5,12W resulted in a 3.5-fold lower cytotoxicity toward peripheral blood mononuclear cells. In general, these modifications did not influence antibacterial and LPS neutralization activities. It is concluded that for the development of novel therapeutic compounds based on chicken cathelicidin-2 d-amino acid substitutions and cyclization must be considered. These modifications increase the stability and lower cytotoxicity of the peptides without altering their antimicrobial potency. © 2011 Elsevier Inc. All rights reserved.

Host defence peptides (HDPs) are considered to be excellent candidates for the development of novel therapeutic agents. Recently, it was demonstrated that the peptide C1-15, an N-terminal segment of chicken HDP cathelicidin-2, exhibits potent antibacterial activity while lacking cytotoxicity towards eukaryotic cells. In the present study, we report that C1-15 is active against bacteria such as Bacillus anthracis and Yersinia pestis that may potentially be used by bioterrorists. Substitution of single and multiple phenylalanine (Phe) residues to tryptophan (Trp) in C1-15 resulted in variants with improved antibacterial activity against B. anthracis and Y. pestis as well as decreased salt sensitivity. In addition, these peptides exhibited enhanced neutralisation of lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). The antibacterial and LPS-neutralising activities of these C1-15-derived peptides are exerted at concentrations far below the concentrations that are toxic to human PBMCs. Taken together, we show that Phe → Trp substitutions in C1-15 variants enhances the antibacterial and LPS-neutralising activities against pathogenic bacteria, including those that may potentially be used as biological warfare agents.Keywords: Host defence peptide; Cathelicidin; Antibacterial activity; Cytotoxicity; Immunomodulatory activity; Biological warfare agents

This review is focused on the evolution and function of alveolar proteins The lung faces physical and environmental challenges, due to changing pressures/volumes and foreign pathogens, respectively The pulmonary surfactant system is integral in protecting the lung from these challenges via two groups of surfactant proteins - the small molecular weight hydrophobic SPs. SP-B and -C, that regulate interfacial adsorption of the lipids, and the large hydrophilic SPs. SP-A and -D, which are surfactant collectins capable of inhibiting foreign pathogens Further aiding pulmonary host defence are non-surfactant collectins and antimicrobial peptides that are expressed across the biological kingdoms Linking to the first symposium session, which emphasised molecular structure and biophysical function of surfactant lipids and proteins, this review begins with a discussion of the role of temperature and hydrostatic pressure in shaping the evolution of SP-C in mammals Transitioning to the role of the alveolus in innate host defence we discuss the structure, function and regulation of antimicrobial peptides, the defensins and cathelicidins. We describe the recent discovery of novel avian collectins and provide evidence for their role in preventing influenza infection This is followed by discussions of the roles of SP-A and SP-D in mediating host defence at the alveolar surface and in mediating inflammation and the allergic response of the airways Finally we discuss the use of animal models of lung disease including knockouts to develop an understanding of the role of these proteins in initiating and/or perpetuating disease with the aim of developing new therapeutic strategies (C) 2010 Elsevier B V All rights reserved

Chicken host defense peptide cathelicidin-2 (CATH-2) is known to exert antimicrobial and immunomodulatory activities and consists of two α-helices connected by a hinge region. Here we report the biological properties of the separate α-helical segments and the importance of the proline residue in the hinge region. Substitution of proline-14 in the CATH-2 hinge region by leucine, but not by glycine, strongly reduced antibacterial and hemolytic activity. Furthermore, substitution by leucine strongly reduced the neutralization of LPS-induced cytokine production and peptide-induced monocyte chemotactic protein-1 (MCP-1) production by human peripheral blood mononuclear cells (PBMCs). This indicates that the hinge region is important for rapid penetration of the bacterial membrane as well as indirect and direct immunomodulatory activities. The highly cationic and amphipathic N-terminal segment (C1-15) exhibited very potent antibacterial activity and fast killing kinetics, while displaying low cytotoxicity towards chicken erythrocytes and PBMCs. The N-terminal and, to a lesser extent, the C-terminal helical regions potently neutralized LPS-induced release of TNFα, IL-6 and IL-10 by PBMCs, while IL-8 production was only moderately affected. These results indicate that core elements within mature CATH-2 can be identified that are linked to antibacterial and/or immunomodulatory activities. Further studies may lead to the development of peptide antibiotics with specific properties that can be used for prophylactic and/or therapeutic applications.

The biological functions of avian cathelicidins are poorly defined. In mammals, cathelicidins have shown to possess potent broad-range antimicrobial activity as well as immunomodulatory activities. Therefore, we investigated the microbicidal activities and localization of Cathelicidin-2 in non-infected and Salmonella-challenged broiler chickens. Using immunohistochemistry, Cathelicidin-2 was shown to be abundantly present in heterophils, localized in the large rod-shaped granules, but absent in other peripheral blood cells and intestinal epithelial cells. Cathelicidin-2 synthesis was observed to be initiated at the early promyelocyte stage. Considerable infiltration of Cathelicidin-2 containing heterophils was observed in the jejunum of Salmonella enteritidis-challenged broilers within 8 h post-infection. Heterophils were shown to release mature Cathelicidin-2 peptide upon stimulation with Salmonella-derived LPS in a time-dependent way. Processing of the Cathelicidin-2 precursor was mediated by serine proteases with a divalent cation dependency. Cathelicidin-2 peptide showed potent bactericidal and fungicidal activity against all tested microorganisms, including chicken-specific Salmonella isolates. These results underscore the importance of avian heterophils as a first line of defence against invading pathogens and implicate that via heterophil-mediated release, cathelicidins may greatly contribute to avian innate immunity.

Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an important role in innate immunity. They can bind to a broad range of carbohydrates, which enables them to interact with ligands present on the surface of micro-organisms. We previously reported the finding of a new putative chicken lectin, which was predominantly localized to the respiratory tract, and thus termed chicken lung lectin (cLL). In order to investigate the biochemical and biophysical properties of cLL, the recombinant protein was expressed, affinity purified and characterized. Recombinant cLL was expressed as four differently sized peptides, which is most likely due to post-translational modification. Crosslinking of the protein led to the formation of two high-molecular weight products, indicating that cLL forms trimeric and possibly even multimeric subunits. cLL was shown to have lectin activity, preferentially binding to a-mannose in a calcium-dependent manner. Furthermore, cLL was shown to inhibit the haemagglutination-activity of human isolates of influenza A virus, subtype H3N2 and H1N1. These result show that cLL is a true C-type lectin with a very distinct sugar specificity, and that this chicken lectin could play an important role in innate immunity. (c) 2008 Published by Elsevier B.V.

The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mMor 10 mMdid not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7.

Salmonella enterica serovar typhimurium (S. typhimurium) species are a leading cause of human invasive gastroenteritis. There is increasing in vitro evidence about Salmonella interaction with isolated cells or cell lines (macrophages, and enterocytes) on the molecular level, however, very little is known about in vivo interactions during actual invasion. We investigated the early interaction of S. typhimurium with intact small intestinal mucosa, in a pig model. Intestinal segments were infected with or without S. typhimurium DT104, and perfused. Whole mucosal gene expression was analyzed by cDNA array on 0, 2, 4, and 8 h post-infection. Invasion resulted in the upregulation of only eight transcripts in jejunal mucosa, among those the proinflammatory IL-8 (at 4 h only), and the antiinflammatory STAT3 (at 4 and 8 h). The limited number of differentially expressed genes found here in vivo compared to in vitro is most likely due to the presence of multiple, heterogenous cell interactions in intact mucosa. Furthermore, it is concluded that S. typhimurium evades strong host responses by downregulating the local inflammatory response.