Assay strategies for the discovery and validation of therapeutics targeting <i>Brugia pahangi</i> Hsp90

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target

Composite Films of Arabinoxylan and Fibrous Sepiolite: Morphological, Mechanical, and Barrier Properties

Hemicelluloses represent a largely unutilized resource for future bioderived films in packaging and other applications. However, improvement of film properties is needed in order to transfer this potential into reality. In this context, sepiolite, a fibrous clay, was investigated as an additive to enhance the properties of rye flour arabinoxylan. Composite films cast from arabinoxylan solutions and sepiolite suspensions in water were transparent or semitransparent at additive loadings in the 2.5-10 wt % range. Scanning electron microscopy showed that the sepiolite was well dispersed in the arabinoxylan films and sepiolite fiber aggregation was not found. FT-IR spectroscopy provided some evidence for hydrogen bonding between sepiolite and arabinoxylan. Consistent with these findings, mechanical testing showed increases in film stiffness and strength with sepiolite addition and the effect of poly(ethylene glycol) methyl ether (mPEG) plasticizer addition. Incorporation of sepiolite did not significantly influence the thermal degradation or the gas barrier properties of arabinoxylan films, which is likely a consequence of sepiolite fiber morphology. In summary, sepiolite was shown to have potential as an additive to obtain stronger hemicellulose films although other approaches, possibly in combination with the use of sepiolite, would be needed if enhanced film barrier properties are required for specific applications.</p

TLR15 Is Unique to Avian and Reptilian Lineages and Recognizes a Yeast-Derived Agonist

Abstract The TLRs represent a family of pattern recognition receptors critical in the induction of vertebrate immune responses. Between 10 and 13 different TLR genes can be identified in each vertebrate species, with many represented as orthologous genes in different species. The agonist specificity of orthologous TLR is also highly conserved. In contrast, TLR15 can only be identified in avian and reptilian genomes, suggesting that this receptor arose ∼320 million years ago after divergence of the bird/reptile and mammalian lineages. Transfection of a constitutively active form of chicken TLR15 led to NF-κB activation in HEK293 cells and induced cytokine mRNA upregulation in chicken cell lines. Full-length TLR15 mediated NF-κB induction in response to lysates from yeast, but not those derived from viral or bacterial pathogens, or a panel of well-characterized TLR agonists. TLR15 responses were induced by whole-cell lysates derived from Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, but not zymosan preparations from S. cerevisiae. The ability of yeast lysate to activate TLR15-dependent NF-κB pathways (in transfection assays) or stimulate IL-1β mRNA upregulation in chicken macrophages was abrogated by heat inactivation or pre-exposure of the lysate to PMSF. Identification of yeast as an agonist source for TLR15 provides a functional framework for consideration of this TLR within the context of pattern recognition receptor evolution and may impact on the development of novel adjuvants

Signalling pathways involved in TLR3 mediated responses.

<p>HD11 cells were pre-treated with inhibitors and then incubated with Poly(I:C) or medium (Untr). Expression of IFNα, IFNβ and TLR3 was assessed by qRT-PCR. Measured levels, shown by the tips of the triangles, are relative to those in cells treated with poly(I:C) but without inhibitor. The triangles are truncated on the left at 2 units. Blue triangles indicate significant differences from the induced but uninhibited level (p<0.01, two sided t-test with Holm correction for multiple testing).</p

A Critical Role for MAPK Signalling Pathways in the Transcriptional Regulation of Toll Like Receptors

<div><p>Toll-like Receptors (TLR) are phylogenetically conserved transmembrane proteins responsible for detection of pathogens and activation of immune responses in diverse animal species. The stimulation of TLR by pathogen-derived molecules leads to the production of pro-inflammatory mediators including cytokines and nitric oxide. Although TLR-induced events are critical for immune induction, uncontrolled inflammation can be life threatening and regulation is a critical feature of TLR biology. We used an avian macrophage cell line (HD11) to determine the relationship between TLR agonist-induced activation of inflammatory responses and the transcriptional regulation of TLR. Exposure of macrophages to specific TLR agonists induced upregulation of cytokine and nitric oxide pathways that were inhibited by blocking various components of the TLR signalling pathways. TLR activation also led to changes in the levels of mRNA encoding the TLR responsible for recognising the inducing agonist (cognate regulation) and cross-regulation of other TLR (non-cognate regulation). Interestingly, in most cases, regulation of TLR mRNA was independent of NFκB activity but dependent on one or more of the MAPK pathway components. Moreover, the relative importance of ERK, JNK and p38 was dependent upon both the stimulating agonist and the target TLR. These results provide a framework for understanding the complex pathways involved in transcriptional regulation of TLR, immune induction and inflammation. Manipulation of these pathways during vaccination or management of acute inflammatory disease may lead to improved clinical outcome or enhanced vaccine efficacy.</p> </div

MAPK signalling pathways are responsible for LPS mediated cross-regulation.

<p>Pre-treatment of HD11 cells with MAPK inhibitors was followed by LPS stimulation. TLR mRNA levels were assessed by qRT-PCR. The data for each inflammatory mediator are represented in relation to the agonist employed (top) and the inhibitor (left); with the target indicated on the right. Tips of triangles represent the ratio of TLR expression in the presence of inhibitor to that in its absence, on a logarithmic scale with triangles truncated at 2 units either side of the axis. Blue and orange triangles indicate significant inhibition or enhancement, respectively (p<0.006, two sided t-test with Holm correction for multiple testing).</p

Differential involvement of signalling pathways in cognate regulation of TLR expression.

<p>HD11 cells were pre-treated with inhibitors and then incubated with TLR agonists or media (Untr). Expression of cognate TLR mRNA was assessed by qRT-PCR. The data for each inflammatory mediator are represented in relation to the agonist employed (top) and the inhibitor (left); with the target indicated on the right. Tips of triangles represent the ratio of TLR expression in the presence of inhibitor to that in its absence, on a logarithmic scale. The triangles are truncated at 2 units either side of the axis (99% inhibition on the left and 100 fold enhancement on the right). Blue and orange triangles indicate significant inhibition or enhancement, respectively (p<0.006, two sided t-test with Holm correction for multiple testing).</p

Agonist induced cross-regulation of TLR mRNA levels.

<p>HD11 cells were stimulated with agonists for 10, 24 and 48 h and the levels of all TLR mRNA were measured in all samples. Data are represented in relation to the agonist employed (top) and the analysed TLR (left) at different times. The tips of triangles represent the change in adjusted Ct for the indicated TLR mRNA (inner rows) after exposure of HD11 cells to the indicated agonists (columns) for the indicated times (outer rows), reduced expression to the left and increased expression to the right using a logarithmic (Ct) scale. Triangles are truncated at +/−4 Ct units (about 16-fold difference). Triangles coloured orange or blue indicate significant increase or decrease in expression (Welsh's two-sided t test; p<0.05 after correction for multiple testing using “fdr” method of Benjamini and Hochberg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051243#pone.0051243-Benjamini1" target="_blank">[99]</a>).</p

Differential involvement of signalling pathways in TLR mediated responses.

<p>HD11 cells were pre-treated with inhibitors and then incubated with TLR agonists. Expression of IL1β, IL6 and iNOS was assessed by qRT-PCR. NO concentrations in cell supernatants were determined using the Griess reagent system. The data for each inflammatory mediator are represented in relation to the agonist employed (top) and the inhibitor (left); with the target indicated on the right. Tips of triangles represent the ratio of agonist response in the presence of inhibitor to that in its absence, on a logarithmic scale. The triangles are truncated on the left at −1.3 units (95% inhibition) and on the right at 1.3 (20 fold enhancement). Blue and orange triangles indicate significant inhibition or enhancement, respectively (p<0. 002, two sided t-test with Holm correction for multiple testing).</p

Towards a universal vaccine for avian influenza: Protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus

AbstractCurrent vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP+M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP+M1 and a secondary vaccination with MVA-NP+M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry