Inhibition of cytosolic Phospholipase A2 prevents prion peptide-induced neuronal damage and co-localisation with Beta III Tubulin.

BACKGROUND: Activation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites. RESULTS: Exposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3) reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death. CONCLUSIONS: Collectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Toll-like receptors in domestic animals

Toll-like receptors are pattern recognition receptors with which hosts recognize pathogen-associated molecular patterns (PAMP). This recognition process is translated rapidly into a meaningful defense reaction. This form of innate host defense is preserved in the animal kingdom: invertebrates heavily depend on it; higher vertebrates also have an adaptive immune system. Both adaptive and innate immune systems are intertwined in that the former also depends on an intact innate recognition and response system. Members of the TLR system cover recognition of parasitic, bacterial or viral germs. Due to the constraints imposed by the necessity to recognize PAMP and to interact with downstream signaling molecules, the TLR system is relatively conserved in evolution. Nevertheless, subtle species differences have been reported for several mammalian TLR members. Examples of this will be given. In all mammalian species investigated, part of the coding sequence is available for the most important TLR members, thus allowing study of expression of these TLR members in various tissues by reverse-transcription polymerase chain reaction in its classical (RT-PCR) and quantitative real time RT-PCR (qRT-PCR) form. In some species, the whole coding sequences of the most important or even all TLR members are known. This allows construction of cDNA and transfection of common host cells, thus permitting functional studies. Extensive investigations were devoted to the study of non-synonymous single nucleotide polymorphisms. In a few cases, expression of a given amino acid in the extracellular (ligand-binding) portion of TLR members could be associated with infectious diseases. This will be discussed belo

Genome-wide association studies of inflammatory bowel disease in German shepherd dogs

Canine Inflammatory Bowel Disease (IBD) is considered a multifactorial disease caused by complex interactions between the intestinal immune system, intestinal microbiota and environmental factors in genetically susceptible individuals. Although IBD can affect any breed, German shepherd dogs (GSD) in the UK are at increased risk of developing the disease. Based on previous evidence, the aim of the present study was to identify single nucleotide polymorphisms (SNPs), which may confer genetic susceptibility or resistance to IBD using a genome-wide association study (GWAS). Genomic DNA was extracted from EDTA blood or saliva samples of 96 cases and 98 controls. Genotyping of cases and controls was performed on the Canine Illumina HD SNP array and data generated was analyzed using PLINK. Several SNPs and regions on chromosomes 7,9,11 and 13 were detected to be associated with IBD using different SNP-by-SNP association methods and FST windows approach. Searching one Mb up-and down-stream of the most significant SNPs, as identified by single SNP analysis as well as 200Kb before and after the start and the end position of the associated regions identified by FST windows approach, we identified 63 genes. Using a combination of pathways analysis and a list of genes that have been reported to be involved in human IBD, we identified 16 candidate genes potentially associated with IBD in GSD

Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle

Transcriptomic analysis of circulating leukocytes obtained during the recovery from clinical mastitis caused by Escherichia coli in Holstein dairy cows

SIMPLE SUMMARY: Escherichia coli is a bacterium which infects cow udders causing clinical mastitis, a potentially severe disease with welfare and economic consequences. During an infection, white blood cells (leukocytes) enter the udder to provide immune defence and assist tissue repair. We sequenced RNA derived from circulating leukocytes to investigate which genes are up- or down-regulated in dairy cows with naturally occurring cases of clinical mastitis in comparison with healthy control cows from the same farm. We also looked for genetic variations between infected and healthy cows. Blood samples were taken either EARLY (around 10 days) or LATE (after 4 weeks) during the recovery phase after diagnosis. Many genes (1090) with immune and inflammatory functions were up-regulated during the EARLY phase. By the LATE phase only 29 genes were up-regulated including six haemoglobin subunits, possibly important for the production of new red blood corpuscles. Twelve genetic variations which were associated with an increased or decreased expression of some important immune genes were identified between the infected and control cows. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite the cows having received prompt veterinary treatment, but they had largely recovered within 4 weeks. Genetic differences between cows may predispose some animals to infection. ABSTRACT: The risk and severity of clinical infection with Escherichia coli as a causative pathogen for bovine mastitis is influenced by the hosts’ phenotypic and genotypic variables. We used RNA-Seq analysis of circulating leukocytes to investigate global transcriptomic profiles and genetic variants from Holstein cows with naturally occurring cases of clinical mastitis, diagnosed using clinical symptoms and milk microbiology. Healthy lactation-matched cows served as controls (CONT, n = 6). Blood samples were collected at two time periods during the recovery phase post diagnosis: EARLY (10.3 ± 1.8 days, n = 6) and LATE (46.7 ± 11 days, n = 3). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21 and subjected to enrichment analysis. Variant calling was performed following GATKv3.8 best practice. The comparison of E. coli(+) EARLY and CONT cows found the up-regulation of 1090 DEGs, mainly with immune and inflammatory functions. The key signalling pathways involved NOD-like and interleukin-1 receptors and chemokines. Many up-regulated DEGs encoded antimicrobial peptides including cathelicidins, beta-defensins, S100 calcium binding proteins, haptoglobin and lactoferrin. Inflammation had largely resolved in the E. coli(+) LATE group, with only 29 up-regulated DEGs. Both EARLY and LATE cows had up-regulated DEGs encoding ATP binding cassette (ABC) transporters and haemoglobin subunits were also up-regulated in LATE cows. Twelve candidate genetic variants were identified in DEGs between the infected and CONT cows. Three were in contiguous genes WIPI1, ARSG and SLC16A6 on BTA19. Two others (RAC2 and ARHGAP26) encode a Rho-family GTPase and Rho GTPase-activating protein 26. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite prompt treatment and provide preliminary evidence for genetic differences between cows that may predispose them to infection

Enterobactin Deficiency in a Coliform Mastitis Isolate Decreases Its Fitness in a Murine Model:A Preliminary Host–Pathogen Interaction Study

Iron is an essential nutrient for bacterial growth. Therefore, bacteria have evolved chelation mechanisms to acquire iron for their survival. Enterobactin, a chelator with high affinity for ferric iron, is secreted by Escherichia coli and contributes to its improved bacterial fitness. In this preliminary study, we evaluated enterobactin deficiency both in vitro and in vivo in the context of E. coli mastitis. Firstly, we showed that expression of lipocalin 2, a protein produced by the host that is able to both bind and deplete enterobactin, is increased upon E. coli infection in the cow’s mastitic mammary gland. Secondly, we demonstrated in vitro that enterobactin deficiency does not alter interleukin (IL)-8 expression in bovine mammary epithelial cells and its associated neutrophil recruitment. However, a significantly increased reactive oxygen species production of these neutrophils was observed. Thirdly, we showed there was no significant difference in bacterial in vitro growth between the enterobactin-deficient mutant and its wild-type counterpart. However, when further explored in a murine model for bovine mastitis, the enterobactin-deficient mutant vs. the wild-type strain revealed a significant reduction of the bacterial load and, consequently, a decrease in pro-inflammatory cytokines (IL-1a,−1b,−4,−6, and−8). A reduced neutrophilic influx was also observed immunohistochemically. These findings therefore identify interference of the enterobactin iron-scavenging mechanism as a potential measure to decrease the fitness of E. coli in the mastitic mammary gland

A Novel Whole Yeast-Based Subunit Oral Vaccine Against Eimeria tenella in Chickens

Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs)