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Background: Prebiotics are non-digestible food ingredients that enhance the growth of certain microbes within the gut microbiota. Prebiotic consumption generates immune-modulatory effects that are traditionally thought to reflect microbial interactions within the gut. However, recent evidence suggests they may also impart direct microbe-independent effects on the host, though the mechanisms of which are currently unclear., Methods: Kinome arrays were used to profile the host intestinal signaling responses to prebiotic exposures in the absence of microbes. Identified pathways were functionally validated in Caco-2Bbe1 intestinal cell line and in vivo model of murine endotoxemia., Results: We found that prebiotics directly regulate host mucosal signaling to alter response to bacterial infection. Intestinal epithelial cells (IECs) exposed to prebiotics are hyporesponsive to pathogen-induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activations, and have a kinome profile distinct from non-treated cells pertaining to multiple innate immune signaling pathways. Consistent with this finding, mice orally gavaged with prebiotics showed dampened inflammatory response to lipopolysaccharide (LPS) without alterations in the gut microbiota., Conclusions: These findings provide molecular mechanisms of direct host-prebiotic interactions to support prebiotics as potent modulators of host inflammation.

The ongoing epidemic of chronic wasting disease (CWD) within cervid populations indicates the need for novel approaches for disease management. A vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. The development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. To address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (DSEs), whose exposure for antibody binding depends on disease-associated misfolding of PrP C into PrP Sc . Here, a DSE corresponding to the rigid loop (RL) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. This vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein G recombinant fusion with the RL epitope (hAd5:tgG-RL). Oral immunization of white-tailed deer with hAd5:tgG-RL induced PrP Sc -specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. By building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular PrP Sc -specific oral vaccine for CWD as well as providing a versatile platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based CWD vaccines.

Background: Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated., Methods: We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer., Results: The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated in common or unique ways., Conclusions: These two new kinase or "Kin-OMIC" analyses add another dimension of important data about these frequently used breast cancer cell lines. This will assist researchers in selecting the most appropriate cell lines to use for breast cancer studies and provide context for the interpretation of the emerging results.

There is increasing appreciation among researchers and clinicians of the value of investigating biology and pathobiology at the level of cellular kinase (kinome) activity. Kinome analysis provides valuable opportunity to gain insights into complex biology (including disease pathology), identify biomarkers of critical phenotypes (including disease prognosis and evaluation of therapeutic efficacy), and identify targets for therapeutic intervention through kinase inhibitors. The growing interest in kinome analysis has fueled efforts to develop and optimize technologies that enable characterization of phosphorylation-mediated signaling events in a cost-effective, high-throughput manner. In this review, we highlight recent advances to the central technologies currently available for kinome profiling and offer our perspectives on the key challenges remaining to be addressed., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Prebiotics are non-digestible oligosaccharides that promote the growth of beneficial gut microbes, but it is unclear whether they also have direct effects on the intestinal mucosal barrier. Here we demonstrate two commercial prebiotics, inulin and short-chain fructo-oligosaccharide (scFOS), when applied onto intestinal epithelia in the absence of microbes, directly promote barrier integrity to prevent pathogen-induced barrier disruptions. We further show that these effects involve the induction of select tight junction (TJ) proteins through a protein kinase C (PKC) δ-dependent mechanism. These results suggest that in the absence of microbiota, prebiotics can directly exert barrier protective effects by activating host cell signaling in the intestinal epithelium, which represents a novel alternative mechanism of action of prebiotics., Competing Interests: Research efforts of the authors are supported by Lallemand Health Solutions (Montreal, Quebec, Canada) and PMS has received honoraria from Abbott Nutrition, Mead Johnson Nutritionals and Nestlé Nutrition

As a part of their pathogenic mechanism, many pathogens causing persistent infections, including bovine viral diarrhea virus (BVDV), immunosuppress their hosts, often by limiting the ability to either produce, or respond to, interferon. The objective of this study was to quantify the extent to which an acute infection of cattle with a non-cytopathic strain of BVDV induces interferon responses and to establish the functionality of these responses. Functionality of responses was investigated using a bovine specific peptide array to monitor kinase-mediated signal transduction activity within peripheral blood mononuclear cells (PBMCs) at time points corresponding to the interferon gamma (IFN-γ) and alpha (IFN-α) responsive phases of acute BVDV infection. Further, with an appreciation of diverse mechanisms and levels at which pathogens modulate host cell defences, patterns of expression of IFN-γ and -α responsive genes were also quantified within PBMCs. Infection of cows with ncpBVDV2-1373 induced significant increases in levels of serum IFN-γ and IFN-α. Within the PBMCs of the infected animals, distinct patterns of kinase-mediated signal transduction activity, in particular with respect to activation of classic IFN-activated signalling pathways, such as Jak-Stat, as well as induced expression of IFN-γ and IFN-α regulated genes, support the functionality of the host interferon response., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The post-translational modification of proteins is critical for regulating their function. Although many post-translational modification sites have been experimentally determined, particularly in certain model organisms, experimental knowledge of these sites is severely lacking for many species. Thus, it is important to be able to predict sites of post-translational modification in such species. Previously, we described DAPPLE, a tool that facilitates the homology-based prediction of one particular post-translational modification, phosphorylation, in an organism of interest using known phosphorylation sites from other organisms. Here, we describe DAPPLE 2, which expands and improves upon DAPPLE in three major ways. First, it predicts sites for many post-translational modifications (20 different types) using data from several sources (15 online databases). Second, it has the ability to make predictions approximately 2-7 times faster than DAPPLE depending on the database size and the organism of interest. Third, it simplifies and accelerates the process of selecting predicted sites of interest by categorizing them based on gene ontology terms, keywords, and signaling pathways. We show that DAPPLE 2 can successfully predict known human post-translational modification sites using, as input, known sites from species that are either closely (e.g., mouse) or distantly (e.g., yeast) related to humans. DAPPLE 2 can be accessed at http://saphire.usask.ca/saphire/dapple2 .

In cattle, Mycobacterium avium subsp. paratuberculosis infection is primarily mediated through M cells overlying Peyer's patches (PP) in the ileum. The capacity of M. avium subsp. paratuberculosis to invade ileal PP (IPP) versus discrete PP in the jejunum (JPP) and subsequent differences in mucosal immune responses were investigated. Intestinal segments were surgically prepared in both mid-jejunum, containing two JPPs, and in terminal small intestine containing continuous IPP. M. avium subsp. paratuberculosis (109 CFU) was injected into the lumen of half of each intestinal segment when calves were 10-14 days-old and infection confirmed 1-2 months later by PCR and immunohistochemistry. Thirteen recombinant M. avium subsp. paratuberculosis proteins, previously identified as immunogenic, were used to analyze pathogen-specific B- and T-cell responses in PP and mesenteric lymph nodes. IgA plasma cell responses to 9 of 13 recombinant proteins were detected in JPP but not in IPP. Secretory IgA reacting in ELISA with 9 of the 13 recombinant proteins was detected in luminal contents from both jejunal and ileal segments. These observations support the conclusion that pathogen-specific IgA B cells were induced in JPP but not IPP early after a primary infection. The presence of secretory IgA in intestinal contents is consistent with dissemination of IgA plasma cells from the identified mucosa-associated immune induction sites. This is the first direct evidence for M. avium subsp. paratuberculosis uptake by bovine JPP and for local induction of pathogen-specific IgA plasma cell responses after enteric infection. We also provide evidence that bacterial invasion of IPP, a primary B lymphoid tissue, provides a novel strategy to evade induction of mucosal immune responses. Over 60% of PPs in the newborn calf small intestine is primary lymphoid tissue, which has significant implications when designing oral vaccines or diagnostic tests to detect early M. avium subsp. paratuberculosis infections.

Protein kinase-mediated phosphorylation is among the most important post-translational modifications. However, few phosphorylation sites have been experimentally identified for most species, making it difficult to determine the degree to which phosphorylation sites are conserved. The goal of this study was to use computational methods to characterize the conservation of human phosphorylation sites in a wide variety of eukaryotes. Using experimentally-determined human sites as input, homologous phosphorylation sites were predicted in all 432 eukaryotes for which complete proteomes were available. For each pair of species, we calculated phosphorylation site conservation as the number of phosphorylation sites found in both species divided by the number found in at least one of the two species. A clustering of the species based on this conservation measure was concordant with phylogenies based on traditional genomic measures. For a subset of the 432 species, phosphorylation site conservation was compared to conservation of both protein kinases and proteins in general. Protein kinases exhibited the highest degree of conservation, while general proteins were less conserved and phosphorylation sites were least conserved. Although preliminary, these data tentatively suggest that variation in phosphorylation sites may play a larger role in explaining phenotypic differences among organisms than differences in the complements of protein kinases or general proteins.

Prion diseases denote a distinct form of infectivity that is based in the misfolding of a self-protein (PrP(C)) into a pathological, infectious conformation (PrP(Sc)). Efforts to develop vaccines for prion diseases have been complicated by the potential dangers that are associated with induction of immune responses against a self-protein. As a consequence, there is considerable appeal for vaccines that specifically target the misfolded prion conformation. Such conformation-specific immunotherapy is made possible through the identification of vaccine targets (epitopes) that are exclusively presented as a consequence of misfolding. An immune response directed against these targets, termed disease-specific epitopes (DSEs), has the potential to spare the function of the native form of the protein while clearing, or neutralizing, the infectious isomer. Although identification of DSEs represents a critical first step in the induction of conformation-specific immune responses, substantial efforts are required to translate these targets into functional vaccines. Due to the poor immunogenicity that is inherent to self-proteins, and that is often associated with short peptides, substantial efforts are required to overcome tolerance-to-self and maximize the resultant immune response following DSE-based immunization. This often includes optimization of target sequences in terms of immunogenicity and development of effective formulation and delivery strategies for the associated peptides. Further, these vaccines must satisfy additional criteria from perspectives of specificity (PrP(C) vs. PrP(Sc)) and safety (antibody-induced template-driven misfolding of PrP(C)). The emphasis of this report is on the steps required to translate DSEs into prion vaccines and subsequent evaluation of the resulting immune responses.

There is a growing appreciation within the livestock industry and throughout society that animal stress is an important issue that must be addressed. With implications for animal health, well-being, and productivity, minimizing animal stress through improved animal management procedures and/or selective breeding is becoming a priority. Effective management of stress, however, depends on the ability to identify and quantify the effects of various stressors and determine if individual or combined stressors have distinct biological effects. Furthermore, it is critical to determine the duration of stress-induced biological effects if we are to understand how stress alters animal production and disease susceptibility. Common stress models used to evaluate both psychological and physical stressors in cattle are reviewed. We identify some of the major gaps in our knowledge regarding responses to specific stressors and propose more integrated methodologies and approaches to measuring these responses. These approaches are based on an increased knowledge of both the metabolic and immune effects of stress. Finally, we speculate on how these findings may impact animal agriculture, as well as the potential application of large animal models to understanding human stress.

The majority of scientific resources are devoted to studying a relatively small number of model species, meaning that the ability to translate knowledge across species is of considerable importance. Obtaining species-specific knowledge enables targeted investigations of the biology and pathobiology of a particular species, and facilitates comparative analyses. Phosphorylation is the most widespread posttranslational modification in eukaryotes, and although many phosphorylation sites have been experimentally identified for some species, little or no data are available for others. Using the honeybee as a test organism, this case study illustrates the process of using protein sequence homology to identify putative phosphorylation sites in a species of interest using experimentally determined sites from other species. A number of issues associated with this process are examined and discussed. Several databases of experimentally determined phosphorylation sites exist; however, it can be difficult for the nonspecialist to ascertain how their contents compare. Thus, this case study assesses the content and comparability of several phosphorylation site databases. Additional issues examined include the efficacy of homology-based phosphorylation site prediction, the impact of the level of evolutionary relatedness between species in making these predictions, the ability to translate knowledge of phosphorylation sites across large evolutionary distances and the criteria that should be used in selecting probable phosphorylation sites in the species of interest. Although focusing on phosphorylation, the issues discussed here also apply to the homology-based cross-species prediction of other posttranslational modifications, as well as to sequence motifs in general., (© The Author 2014. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.)

Unlabelled: Efforts to develop peptide-based vaccines, in particular those requiring site-specific targeting of self-proteins, rely on the ability to optimize the immunogenicity of the peptide epitopes. Currently, screening of candidate vaccines is typically performed through low-throughput, high-cost animal trials. To improve on this we present the program EpIC, which enables high-throughput prediction of peptide immunogenicity based on the endogenous occurrence of B-cell epitopes within native protein sequences. This information informs rational selection of immunogenicity-optimized epitopes for peptide vaccines., Availability and Implementation: EpIC is available as a web server at http://saphire.usask.ca/saphire/epic., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Defining cellular responses at the level of global cellular kinase (kinome) activity is a powerful approach to deciphering complex biology and identifying biomarkers. Here we report on the development of a chicken-specific peptide array and its application to characterizing kinome responses within the breast (pectoralis major) and thigh (iliotibialis) muscles of poultry subject to temperature stress to mimic conditions experienced by birds during commercial transport. Breast and thigh muscles exhibited unique kinome profiles, highlighting the distinct nature of these tissues. Against these distinct backgrounds, tissue- and temperature-specific kinome responses were observed. In breast, both cold and hot stresses activated calcium-dependent metabolic adaptations. Also within breast, but specific to cold stress, was the activation of ErbB signaling as well as dynamic patterns of phosphorylation of AMPK, a key regulatory enzyme of metabolism. In thigh, cold stress induced responses suggestive of the occurrence of tissue damage, including activation of innate immune signaling pathways and tissue repair pathways (TGF-β). In contrast, heat stress in thigh activated pathways associated with protein and fat metabolism through adipocytokine and mammalian target of rapamycin (mTOR) signaling. Defining the responses of these tissues to these stresses through conventional markers of pH, glycolytic potential, and meat quality offered a similar conclusion of the tissue- and stressor-specific responses, validating the kinome results. Collectively, the results of this study highlight the unique cellular responses of breast and thigh tissues to heat and cold stresses and may offer insight into the unique susceptibilities, as well as functional consequences, of these tissues to thermal stress., (© 2015 Poultry Science Association Inc.)

The Syrian golden hamster has been increasingly used to study viral hemorrhagic fever (VHF) pathogenesis and countermeasure efficacy. As VHFs are a global health concern, well-characterized animal models are essential for both the development of therapeutics and vaccines as well as for increasing our understanding of the molecular events that underlie viral pathogenesis. However, the paucity of reagents or platforms that are available for studying hamsters at a molecular level limits the ability to extract biological information from this important animal model. As such, there is a need to develop platforms/technologies for characterizing host responses of hamsters at a molecular level. To this end, we developed hamster-specific kinome peptide arrays to characterize the molecular host response of the Syrian golden hamster. After validating the functionality of the arrays using immune agonists of defined signaling mechanisms (lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α), we characterized the host response in a hamster model of VHF based on Pichinde virus (PICV(1)) infection by performing temporal kinome analysis of lung tissue. Our analysis revealed key roles for vascular endothelial growth factor (VEGF), interleukin (IL) responses, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and Toll-like receptor (TLR) signaling in the response to PICV infection. These findings were validated through phosphorylation-specific Western blot analysis. Overall, we have demonstrated that hamster-specific kinome arrays are a robust tool for characterizing the species-specific molecular host response in a VHF model. Further, our results provide key insights into the hamster host response to PICV infection and will inform future studies with high-consequence VHF pathogens., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Prion diseases reflect the misfolding of a self-protein (PrP(C)) into an infectious, pathological isomer (PrP(Sc)). By targeting epitopes uniquely exposed by misfolding, our group developed PrP(Sc)-specific vaccines to 3 disease specific epitopes (DSEs). Here, antibodies induced by individual DSE vaccines are evaluated for their capacity to neutralize prions in vitro. For both purified antibodies and immunoreactive sera, the PrP(Sc)-specific antibodies were equally effective in neutralizing prions. Further, there was no significant increase in neutralizing activity when multiple DSEs were targeted within an assay. At a low antibody concentration, the PrP(Sc)-specific antibodies matched the neutralization achieved by an antibody that may act via both PrP(C) and PrP(Sc). At higher doses, however, this pan-specific antibody was more effective, potentially due to a combined deactivation of PrP(Sc) and depletion of PrP(C).

Reversible protein phosphorylation is a central mechanism for both the transfer of intracellular information and the initiation of cellular responses. Within human medicine, considerable emphasis is placed on understanding and controlling the enzymes (kinases) that are responsible for catalyzing these modifications. This is evident in the prominent use of kinase inhibitors as drugs as well as the trend to understand complex biology and identify biomarkers via characterizations of global kinase (kinome) activity. Despite the demonstrated value of focusing on kinome activity, the application of this perspective to livestock has been restricted by the absence of appropriate research tools. In this review, we discuss the development of software platforms that facilitate the development and application of species-specific peptide arrays for kinome analysis of livestock. Examples of the application of kinomic approaches to a number of priority species (cattle, pigs, and chickens) in a number of biological contexts (infections, biomarker discovery, and food quality) are presented as are emerging trends for kinome analysis of livestock.

Human cathelicidin LL-37 protects against infections and endotoxin-induced inflammation. In a recent study we have shown that IG-19, an LL-37-derived peptide, protects in a murine model of arthritis. Cytokine interleukin-32 (IL-32) is elevated and directly associated with the disease severity of inflammatory arthritis. Therefore, in this study we examined the effects of LL-37 and IG-19 on IL-32-induced responses in human peripheral blood-derived mononuclear cells (PBMC) and macrophages. We showed that CD14(+) monocytes are the primary cells that produce pro-inflammatory tumour necrosis factor-α (TNF-α) following stimulation of PBMC with IL-32. We demonstrated that LL-37 and IG-19 significantly suppress IL-32-induced production of pro-inflammatory cytokines, e.g. TNF-α and IL-1β, without altering chemokine production. In contrast, LL-37 and IG-19 enhance the production of the anti-inflammatory cytokine IL-1RA. Further mechanistic studies revealed that LL-37 and IG-19 suppress IL-32-mediated phosphorylation of Fyn (Y420) Src kinase. In contrast, IL-32-mediated phosphorylation of AKT-1 (T308) and MKP-1 (S359) is not suppressed by the peptides. LL-37 and IG-19 alone induce the phosphorylation of MKP-1 (S359), which is a known negative regulator of inflammation. Furthermore, the peptides induce the activity of p44/42 mitogen-activated protein kinase, which is known to phosphorylate MKP-1 (S359). This is the first study to demonstrate the regulation of IL-32-induced inflammation by LL-37 and its derivative peptide IG-19. The mechanistic results from this study suggest that regulation of immune-mediated inflammation by these peptides may be controlled by the dual phosphatase MKP-1. We speculate that LL-37 and its derivatives may contribute to the control of immune-mediated inflammatory diseases., (© 2014 John Wiley & Sons Ltd.)

Unlabelled: Ebola virus (EBOV) causes a severe hemorrhagic disease in humans and nonhuman primates, with a median case fatality rate of 78.4%. Although EBOV is considered a public health concern, there is a relative paucity of information regarding the modulation of the functional host response during infection. We employed temporal kinome analysis to investigate the relative early, intermediate, and late host kinome responses to EBOV infection in human hepatocytes. Pathway overrepresentation analysis and functional network analysis of kinome data revealed that transforming growth factor (TGF-β)-mediated signaling responses were temporally modulated in response to EBOV infection. Upregulation of TGF-β signaling in the kinome data sets correlated with the upregulation of TGF-β secretion from EBOV-infected cells. Kinase inhibitors targeting TGF-β signaling, or additional cell receptors and downstream signaling pathway intermediates identified from our kinome analysis, also inhibited EBOV replication. Further, the inhibition of select cell signaling intermediates identified from our kinome analysis provided partial protection in a lethal model of EBOV infection. To gain perspective on the cellular consequence of TGF-β signaling modulation during EBOV infection, we assessed cellular markers associated with upregulation of TGF-β signaling. We observed upregulation of matrix metalloproteinase 9, N-cadherin, and fibronectin expression with concomitant reductions in the expression of E-cadherin and claudin-1, responses that are standard characteristics of an epithelium-to-mesenchyme-like transition. Additionally, we identified phosphorylation events downstream of TGF-β that may contribute to this process. From these observations, we propose a model for a broader role of TGF-β-mediated signaling responses in the pathogenesis of Ebola virus disease., Importance: Ebola virus (EBOV), formerly Zaire ebolavirus, causes a severe hemorrhagic disease in humans and nonhuman primates and is the most lethal Ebola virus species, with case fatality rates of up to 90%. Although EBOV is considered a worldwide concern, many questions remain regarding EBOV molecular pathogenesis. As it is appreciated that many cellular processes are regulated through kinase-mediated phosphorylation events, we employed temporal kinome analysis to investigate the functional responses of human hepatocytes to EBOV infection. Administration of kinase inhibitors targeting signaling pathway intermediates identified in our kinome analysis inhibited viral replication in vitro and reduced EBOV pathogenesis in vivo. Further analysis of our data also demonstrated that EBOV infection modulated TGF-β-mediated signaling responses and promoted "mesenchyme-like" phenotypic changes. Taken together, these results demonstrated that EBOV infection specifically modulates TGF-β-mediated signaling responses in epithelial cells and may have broader implications in EBOV pathogenesis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Recent investigations associate Varroa destructor (Mesostigmata: Varroidae) parasitism and its associated pathogens and agricultural pesticides with negative effects on colony health, resulting in sporadic global declines in domestic honeybee (Apis mellifera) populations. These events have motivated efforts to develop research tools that can offer insight into the causes of declining bee health as well as identify biomarkers to guide breeding programs. Here we report the development of a bee-specific peptide array for characterizing global cellular kinase activity in whole bee extracts. The arrays reveal distinct, developmentally-specific signaling profiles between bees with differential susceptibility to infestation by Varroa mites. Gene ontology analysis of the differentially phosphorylated peptides indicates that the differential susceptibility to Varroa mite infestation does not reflect compromised immunity; rather, there is evidence for mite-mediated immune suppression within the susceptible phenotype that may reduce the ability of these bees to counter secondary viral infections. This hypothesis is supported by the demonstration of more diverse viral infections in mite-infested, susceptible adult bees. The bee-specific peptide arrays are an effective tool for understanding the molecular basis of this complex phenotype as well as for the discovery and utilization of phosphorylation biomarkers for breeding programs.

Johne's disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts to control JD through traditional animal management practices are complicated by MAP's ability to cause long-term environmental contamination as well as difficulties associated with diagnosis of JD in the pre-clinical stages. As such, there is particular emphasis on the development of an effective vaccine. This is a daunting challenge, in large part due to MAP's ability to subvert protective host immune responses. Accordingly, there is a priority to understand MAP's interaction with the bovine host: this may inform rational targets and approaches for therapeutic intervention. Here we review the early host defenses encountered by MAP and the strategies employed by the pathogen to avert or subvert these responses, during the critical period between ingestion and the establishment of persistent infection in macrophages.

Prion diseases represent a novel form of infectivity caused by the propagated misfolding of a self-protein (PrP(C)) into a pathological, infectious conformation (PrP(Sc)). Efforts to develop a prion vaccine have been complicated by challenges and potential dangers associated with induction of strong immune responses to a self protein. There is considerable value in the development of vaccines that are specifically targeted to the misfolded conformation. Conformation specific immunotherapy depends on identification and optimization of disease-specific epitopes (DSEs)(1) that are uniquely exposed upon misfolding. Previously, we reported development of a PrP(Sc)-specific vaccine through empirical expansions of a YYR DSE. Here we describe optimization of two additional prion DSEs, YML of β-sheet 1 and a rigid loop (RL) linking β-sheet 2 to α-helix 2, through in silico predictions of B cell epitopes and further translation of these epitopes into PrP(Sc)-specific vaccines. The optimized YML and RL vaccines retain their properties of immunogenicity, specificity and safety when delivered individually or in a multivalent format. This investigation supports the utility of combining DSE prediction models with algorithms to infer logical peptide expansions to optimize immunogenicity. Incorporation of optimized DSEs into established vaccine formulation and delivery strategies enables rapid development of peptide-based vaccines for protein misfolding diseases., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mycoplasma bovis is one of the major causative pathogens of bovine respiratory complex disease (BRD), which is characterized by enzootic pneumonia, mastitis, pleuritis, and polyarthritis. M. bovis enters and colonizes bovine respiratory epithelial cells through inhalation of aerosol from contaminated air. The nature of the interaction between M. bovis and the bovine innate immune system is not well understood. We hypothesized that M. bovis invades blood monocytes and regulates cellular function to support its persistence and systemic dissemination. We used bovine-specific peptide kinome arrays to identify cellular signaling pathways that could be relevant to M. bovis-monocyte interactions in vitro. We validated these pathways using functional, protein, and gene expression assays. Here, we show that infection of bovine blood monocytes with M. bovis delays spontaneous or tumor necrosis factor alpha (TNF-α)/staurosporine-driven apoptosis, activates the NF-κB p65 subunit, and inhibits caspase-9 activity. We also report that M. bovis-infected bovine monocytes do not produce gamma interferon (IFN-γ) and TNF-α, although the level of production of interleukin-10 (IL-10) is elevated. Our findings suggest that M. bovis takes over the cellular machinery of bovine monocytes to prolong bacterial survival and to possibly facilitate subsequent systemic distribution.

Prions are a novel form of infectivity based on the misfolding of a self-protein (PrP(C)) into a pathological, infectious isomer (PrP(Sc)). The current uncontrolled spread of chronic wasting disease in cervids, coupled with the demonstrated zoonotic nature of select livestock prion diseases, highlights the urgent need for disease management tools. While there is proof-of-principle evidence for a prion vaccine, these efforts are complicated by the challenges and risks associated with induction of immune responses to a self-protein. Our priority is to develop a PrP(Sc)-specific prion vaccine based on epitopes that are uniquely exposed upon misfolding. These disease specific epitopes (DSEs) have the potential to enable specific targeting of the pathological species through immunotherapy. Here we review outcomes of the translation of a prion DSE into a PrP(Sc)-specific vaccine based on the criteria of immunogenicity, safety and specificity.

Background: Recently, questions have been raised regarding the ability of animal models to recapitulate human disease at the molecular level. It has also been demonstrated that cellular kinases, individually or as a collective unit (the kinome), play critical roles in regulating complex biology. Despite the intimate relationship between kinases and health, little is known about the variability, consistency and stability of kinome profiles across species and individuals., Results: As a preliminary investigation of the existence of species- and individual-specific kinotypes (kinome signatures), peptide arrays were employed for the analysis of peripheral blood mononuclear cells collected weekly from human and porcine subjects (n = 6) over a one month period. The data revealed strong evidence for species-specific signalling profiles. Both humans and pigs also exhibited evidence for individual-specific kinome profiles that were independent of natural changes in blood cell populations., Conclusions: Species-specific kinotypes could have applications in disease research by facilitating the selection of appropriate animal models or by revealing a baseline kinomic signature to which treatment-induced profiles could be compared. Similarly, individual-specific kinotypes could have implications in personalized medicine, where the identification of molecular patterns or signatures within the kinome may depend on both the levels of kinome diversity and temporal stability across individuals.

Kinome microarrays are comprised of peptides that act as phosphorylation targets for protein kinases. This platform is growing in popularity due to its ability to measure phosphorylation-mediated cellular signaling in a high-throughput manner. While software for analyzing data from DNA microarrays has also been used for kinome arrays, differences between the two technologies and associated biologies previously led us to develop Platform for Intelligent, Integrated Kinome Analysis (PIIKA), a software tool customized for the analysis of data from kinome arrays. Here, we report the development of PIIKA 2, a significantly improved version with new features and improvements in the areas of clustering, statistical analysis, and data visualization. Among other additions to the original PIIKA, PIIKA 2 now allows the user to: evaluate statistically how well groups of samples cluster together; identify sets of peptides that have consistent phosphorylation patterns among groups of samples; perform hierarchical clustering analysis with bootstrapping; view false negative probabilities and positive and negative predictive values for t-tests between pairs of samples; easily assess experimental reproducibility; and visualize the data using volcano plots, scatterplots, and interactive three-dimensional principal component analyses. Also new in PIIKA 2 is a web-based interface, which allows users unfamiliar with command-line tools to easily provide input and download the results. Collectively, the additions and improvements described here enhance both the breadth and depth of analyses available, simplify the user interface, and make the software an even more valuable tool for the analysis of kinome microarray data. Both the web-based and stand-alone versions of PIIKA 2 can be accessed via http://saphire.usask.ca.

Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP(C)). Species barriers to transmission also reflect PrP(C) sequence differences. Defining the structure-activity relationship of PrP(C)/PrP(Sc) with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP(C) corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP(Sc)-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP(Sc)-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP(Sc)-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders caused by misfolding of a cellular protein PrP(C) into an infectious conformation PrP(Sc). Previously our group demonstrated induction of PrP(Sc)-specific antibodies with a SN6b vaccine that targets regions of the protein that are exposed upon misfolding. There are concerns that these antibodies could function as templates to promote misfolding and cause disease. To evaluate the consequences of prolonged exposure to PrP(Sc)-specific antibodies in a prion sensitized animal, tga20 mice were vaccinated with the SN6b vaccine. No clinical signs of disease were detected up to 255 d post-vaccination, and postmortem assay of brains and spleens revealed no proteinase-K resistant PrP. These results suggest that vaccinating against TSEs with the SN6b antigen is safe from the standpoint of prion disease induction.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle. M. avium subsp. paratuberculosis infects the gastrointestinal tract of calves, localizing and persisting primarily in the distal ileum. A high percentage of cattle exposed to M. avium subsp. paratuberculosis do not develop JD, but the mechanisms by which they resist infection are not understood. Here, we merge an established in vivo bovine intestinal segment model for M. avium subsp. paratuberculosis infection with bovine-specific peptide kinome arrays as a first step to understanding how infection influences host kinomic responses at the site of infection. Application of peptide arrays to in vivo tissue samples represents a critical and ambitious step in using this technology to understand host-pathogen interactions. Kinome analysis was performed on intestinal samples from 4 ileal segments subdivided into 10 separate compartments (6 M. avium subsp. paratuberculosis-infected compartments and 4 intra-animal controls) using bovine-specific peptide arrays. Kinome data sets clustered into two groups, suggesting unique binary responses to M. avium subsp. paratuberculosis. Similarly, two M. avium subsp. paratuberculosis-specific immune responses, characterized by different antibody, T cell proliferation, and gamma interferon (IFN-γ) responses, were also observed. Interestingly, the kinomic groupings segregated with the immune response groupings. Pathway and gene ontology analyses revealed that differences in innate immune and interleukin signaling and particular differences in the Wnt/β-catenin pathway distinguished the kinomic groupings. Collectively, kinome analysis of tissue samples offers insight into the complex cellular responses induced by M. avium subsp. paratuberculosis in the ileum and provides a novel method to understand mechanisms that alter the balance between cell-mediated and antibody responses to M. avium subsp. paratuberculosis infection.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrP(C)) into an infectious, pathological conformation (PrP(Sc)). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrP(Sc)-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrP(C) associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrP(C) that is associated with early onset of familial dementia. With nanopore analysis, under non-denaturing conditions, we observed binding of the SN6b antibody to bPrP(T194A). This interaction was confirmed through ELISAs as well as immunoprecipitation of the recombinant and cellularly expressed forms of bPrP(T194A). This interaction did not promote formation of a protease resistant conformation of PrP in vitro. Collectively, these findings support the disease-specific approach for immunotherapy of prion diseases but also suggest that the concept of conformation-specific immunotherapy may be complicated in individuals who are genetically predisposed to PrP(C) misfolding.

Summary: While many experimentally characterized phosphorylation sites exist for certain organisms, such as human, rat and mouse, few sites are known for other organisms, hampering related research efforts. We have developed a software pipeline called DAPPLE that automates the process of using known phosphorylation sites from other organisms to identify putative sites in an organism of interest., Availability: DAPPLE is available as a web server at http://saphire.usask.ca., Supplementary Information: Supplementary data are available at Bioinformatics online.

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection of chickens that are more than a few days old results in asymptomatic cecal colonization with persistent shedding of bacteria. We hypothesized that while the bacterium colonizes and persists locally in the cecum it has systemic effects, including changes to metabolic pathways of skeletal muscle, influencing the physiology of the avian host. Using species-specific peptide arrays to perform kinome analysis on metabolic signaling pathways in skeletal muscle of Salmonella Typhimurium infected chickens, we have observed key metabolic changes that affected fatty acid and glucose metabolism through the 5'-adenosine monophosphate-activated protein kinase (AMPK) and the insulin/mammalian target of rapamycin (mTOR) signaling pathway. Over a three week time course of infection, we observed changes in the phosphorylation state of the AMPK protein, and proteins up and down the pathway. In addition, changes to a large subset of the protein intermediates of the insulin/mTOR pathway in the skeletal muscle were altered by infection. These changes occur in pathways with direct effects on fatty acid and glucose metabolism. This is the first report of significant cellular metabolic changes occurring systemically in chicken due to a Salmonella infection. These results have implications not only for animal production and health but also for the understanding of how Salmonella infection in the intestine can have widespread, systemic effects on the metabolism of chickens without disease-like symptoms.

A lack of appropriate disease models has limited our understanding of the pathogenesis of persistent enteric infections with Mycobacterium avium subsp. paratuberculosis. A model was developed for the controlled delivery of a defined dose of M. avium subsp. paratuberculosis to surgically isolated ileal segments in newborn calves. The stable intestinal segments enabled the characterization of host responses to persistent M. avium subsp. paratuberculosis infections after a 9-month period, including an analysis of local mucosal immune responses relative to an adjacent uninfected intestinal compartment. M. avium subsp. paratuberculosis remained localized at the initial site of intestinal infection and was not detected by PCR in the mesenteric lymph node. M. avium subsp. paratuberculosis-specific T cell proliferative responses included both CD4 and γδ T cell receptor (γδTcR) T cell responses in the draining mesenteric lymph node. The levels of CD8(+) and γδTcR(+) T cells increased significantly (P < 0.05) in the lamina propria, and M. avium subsp. paratuberculosis-specific tumor necrosis factor alpha (TNF-α) and gamma interferon secretion by lamina propria leukocytes was also significantly (P < 0.05) increased. There was a significant (P < 0.05) accumulation of macrophages and dendritic cells (DCs) in the lamina propria, but the expression of mucosal toll-like receptors 1 through 10 was not significantly changed by M. avium subsp. paratuberculosis infection. In conclusion, surgically isolated ileal segments provided a model system for the establishment of a persistent and localized enteric M. avium subsp. paratuberculosis infection in cattle and facilitated the analysis of M. avium subsp. paratuberculosis-specific changes in mucosal leukocyte phenotype and function. The accumulation of DC subpopulations in the lamina propria suggests that further investigation of mucosal DCs may provide insight into host responses to M. avium subsp. paratuberculosis infection and improve vaccine strategies to prevent M. avium subsp. paratuberculosis infection.

Transmissible spongiform encephalopathies (TSEs) are fatal, untreatable neurodegenerative diseases. While the impact of TSEs on human health is relatively minor, these diseases are having a major influence on how we view, and potentially treat, other more common neurodegenerative disorders. Until recently, TSEs encapsulated a distinct category of neurodegenerative disorder, exclusive in their defining characteristic of infectivity. It now appears that similar mechanisms of self-propagation may underlie other proteinopathies such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, and Huntington's disease. This link is of scientific interest and potential therapeutic importance as this route of self-propagation offers conceptual support and guidance for vaccine development efforts. Specifically, the existence of a pathological, self-promoting isoform offers a rational vaccine target. Here, we review the evidence of prion-like mechanisms within a number of common neurodegenerative disorders and speculate on potential implications and opportunities for vaccine development.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle. The complex, multifaceted interaction of M. avium subsp. paratuberculosis with its host includes dampening the ability of infected cells to respond to stimuli that promote M. avium subsp. paratuberculosis clearance. By disrupting host defenses, M. avium subsp. paratuberculosis creates an intracellular environment that favors the establishment and maintenance of infection. Toll-like receptors (TLRs) are important sensors that initiate innate immune responses to microbial challenge and are also immunotherapeutic targets. For example, TLR9 contributes to host defense against M. avium subsp. paratuberculosis, and its agonists (CpG oligodeoxynucleotides [ODNs]) are under investigation for treatment of Johne's disease and other infections. Here we demonstrate that M. avium subsp. paratuberculosis infection changes the responsiveness of bovine monocytes to TLR9 stimulation. M. avium subsp. paratuberculosis inhibits classical TLR9-mediated responses despite a 10-fold increase in TLR9 expression and maintained uptake of CpG ODNs. Other TLR9-mediated responses, such as oxidative burst, which occur through noncanonical signaling, remain functional. Kinome analysis verifies that classic TLR9 signaling is blocked by M. avium subsp. paratuberculosis infection and that signaling instead proceeds through a Pyk2-mediated mechanism. Pyk2-mediated signaling does not hinder infection, as CpG ODNs fail to promote M. avium subsp. paratuberculosis clearance. Indeed, Pyk2 signaling appears to be an important aspect of M. avium subsp. paratuberculosis infection, as Pyk2 inhibitors significantly reduce the number of intracellular M. avium subsp. paratuberculosis bacteria. The actions of M. avium subsp. paratuberculosis on TLR9 signaling may represent a strategy to generate a host environment which is better suited for infection, revealing potential new targets for therapeutic intervention.

Cellular prion protein (PrP (C) ) has attracted considerable attention for its role in transmissible spongiform encephalopathies (TSEs). In spite of being a point of intense research effort critical questions still remain regarding the physiological function of PrP (C) and how these functions may change with the conversion of the protein into the infectious and pathological conformation (PrP (Sc) ). While emerging evidence suggests PrP (C/Sc) are involved in signal transduction there is little consensus on the signaling pathways associated with the normal and diseased states. The purported involvement of PrP (C) in signal transduction, and the association of TSEs with neural pathology, makes kinome analysis of human neurons an interesting and appropriate model to characterize patterns of signal transduction following activation of PrP (C) by two commonly employed experimental ligands; antibody-induced dimerization by 6H4 and the amino acids 106-126 PrP peptide fragment (PrP 106-126). Analysis of the induced kinome responses reveals distinct patterns of signaling activity following each treatment. Specifically, stimulation of human neurons with the 6H4 antibody results in alterations in mitogen activated protein kinase (MAPK) signaling pathways while the 106-126 peptide activates growth factor related signaling pathways including vascular endothelial growth factor (VEGF) signaling and the phosphoinositide-3 kinase (PI3K) pathway. These pathways were validated through independent functional assays. Collectively these results indicate that stimulation of PrP (C) with distinct ligands, even within the same cell type, results in unique patterns of signaling. While this investigation highlights the apparent functional versatility of PrP (C) as a signaling molecule and may offer insight into cellular mechanisms of TSE pathology it also emphasizes the potential dangers associated with attributing activation of specific intracellular events to particular receptors through artificial models of receptor activation.

Cells from medulloblastoma lines do not contain detectable amounts of the basic leucine-zipper protein Zhangfei. However, we have previously shown that expression of this protein in cells of the ONS-76 and UW228 medulloblastoma lines causes the cells to stop growing and develop processes that resemble neurites. Our objective was to determine the molecular mechanisms by which Zhangfei influences ONS-76 cells. We infected ONS-76 cells with adenovirus vectors expressing either Zhangfei or the control protein LacZ and then compared the following parameters in Zhangfei and LacZ-expressing cells: (a) markers of apoptosis, autophagy and macropinocytosis, (b) transcripts for genes involved in neurogenesis and apoptosis, (c) phosphorylation of peptide targets of selected cellular protein kinases, and (d) activation of transcription factors. Zhangfei-expressing cells appeared to succumb to apoptosis. Increased staining for autophagic vesicles and upregulated expression of autophagy response genes in these cells indicated that they were undergoing autophagy, possibly associated with apoptosis. Within our analysis, patterns of gene expression and phosphorylation-mediated signal transduction activity in Zhangfei-expressing cells indicated that the mitogen-activated protein kinase (MAPK) pathway was active. In addition, we found that the transcription factor Brn3a as well as factors implicated in differentiation were also active in Zhangfei-expressing cells. We tested the hypothesis that Zhangfei enhances the expression of Brn3a, a known inducer of TrkA, the high-affinity receptor for nerve growth factor (NGF). TrkA then engages NGF in an autocrine manner triggering the MAPK pathway and leading to differentiation of ONS-76 cells into neuron and glia-like cells-a process that eventually brings about cell death. We showed that: (a) Zhangfei could enhance transcription from the isolated Brn3a promoter, (b) ONS-76 cells produced NGF and (c) antibodies against NGF and inhibitors of TrkA and selected components of the MAPK pathway could partially restore the growth of Zhangfei-expressing ONS-76 cells.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection by M. avium subsp. paratuberculosis depends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-γ) for clearance of this intracellular pathogen. To define the mechanism by which M. avium subsp. paratuberculosis subverts this critical host cell function, patterns of signal transduction to IFN-γ stimulation of uninfected and M. avium subsp. paratuberculosis-infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN-γ signaling, in uninfected monocytes. In contrast, IFN-γ stimulation of M. avium subsp. paratuberculosis-infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN-γ to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates that M. avium subsp. paratuberculosis blocks responsiveness at, or near, the IFN-γ receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN-γ receptors SOCS1 and SOCS3 as well as decreased expression of IFN-γ receptor chains 1 and 2 is observed in M. avium subsp. paratuberculosis-infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this critical M. avium subsp. paratuberculosis behavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.

Peptide modifications that improve pharmacological properties are of considerable therapeutic importance. Here we consider the retro (R), inversed (D) and retro-inversed (RI) isomers of glucagon with respect to structure, stability, toxicity and biological activity. Biologically, RI-glucagon demonstrated comparable in vivo activity as L-glucagon with respect to magnitude and duration of blood sugar elevation following i.p. administration to mice. Structurally, the isomers were investigated through circular dichroism (CD) and nanopore analysis. CD demonstrated a conserved potential for formation of secondary structure, which was independent of the direction of the peptide (L vs R; D vs RI) as well as formation of symmetry-related structures for the chiral isomers (L vs D; R vs RI). CD, therefore, discriminated chiral but not directional isomers. Nanopore analysis, which depends on interaction of the peptides with chiral pores, discriminated all four isomers on the basis of unique signatures of bumping and translocation. Nanopore analysis offered greater opportunity than CD to discriminate the isomers although neither technique provided a definitive biomarker of biological activity. Functionally, the R and RI isomers resist proteolytic degradation and none of the isomers possess hemolytic activity or cellular toxicity. Collectively, this investigation highlights the potentials and limitations of CD and nanopore analysis for investigation of peptide isomers as well as offering insight into the structural criteria to mimic peptide biological activity. For this example, retro-inversion, through undefined contributions of increased stability and maintained biological activity, was best suited to mimic the biological activity of the parent peptide., (Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.)

Monkeypox virus (MPXV) is comprised of two clades: Congo Basin MPXV, with an associated case fatality rate of 10%, and Western African MPXV, which is associated with less severe infection and minimal lethality. We thus postulated that Congo Basin and West African MPXV would differentially modulate host cell responses and, as many host responses are regulated through phosphorylation independent of transcription or translation, we employed systems kinomics with peptide arrays to investigate these functional host responses. Using this approach we have demonstrated that Congo Basin MPXV infection selectively down-regulates host responses as compared with West African MPXV, including growth factor- and apoptosis-related responses. These results were confirmed using fluorescence-activated cell sorting analysis demonstrating that West African MPXV infection resulted in a significant increase in apoptosis in human monocytes as compared with Congo Basin MPXV. Further, differentially phosphorylated kinases were identified through comparison of our MPXV data sets and validated as potential targets for pharmacological inhibition of Congo Basin MPXV infection, including increased Akt S473 phosphorylation and decreased p53 S15 phosphorylation. Inhibition of Akt S473 phosphorylation resulted in a significant decrease in Congo Basin MPXV virus yield (261-fold) but did not affect West African MPXV. In addition, treatment with staurosporine, an apoptosis activator resulted in a 49-fold greater decrease in Congo Basin MPXV yields as compared with West African MPXV. Thus, using a systems kinomics approach, our investigation demonstrates that West African and Congo Basin MPXV differentially modulate host cell responses and has identified potential host targets of therapeutic interest.

The central roles of kinases in cellular processes and diseases make them highly attractive as indicators of biological responses and as therapeutic targets. Peptide arrays are emerging as an important means of characterizing kinome activity. Currently, the computational tools used to perform high-throughput kinome analyses are not specifically tailored to the nature of the data, which hinders extraction of biological information and overall progress in the field. We have developed a method for kinome analysis, which is implemented as a software pipeline in the R environment. Components and parameters were chosen to address the technical and biological characteristics of kinome microarrays. We performed comparative analysis of kinome data sets that corresponded to stimulation of immune cells with ligands of well-defined signaling pathways: bovine monocytes treated with interferon-γ (IFN-γ), CpG-containing nucleotides, or lipopolysaccharide (LPS). The data sets for each of the treatments were analyzed with our methodology as well as with three other commonly used approaches. The methods were evaluated on the basis of statistical confidence of calculated values with respect to technical and biological variability, and the statistical confidence (P values) by which the known signaling pathways could be independently identified by the pathway analysis of InnateDB (a Web-based resource for innate immunity interactions and pathways). By considering the particular attributes of kinome data, we found that our approach identified more of the peptides involved in the pathways than did the other compared methods and that it did so at a much higher degree of statistical confidence.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, represent a unique form of infectious disease based on misfolding of a self-protein (PrP(C)) into a pathological, infectious conformation (PrP(Sc)). Prion diseases of food animals gained notoriety during the bovine spongiform encephalopathy (BSE) outbreak of the 1980s. In particular, disease transmission to humans, to the generation of a fatal, untreatable disease, elevated the perspective on livestock prion diseases from food production to food safety. While the immediate threat posed by BSE has been successfully addressed through surveillance and improved management practices, another prion disease is rapidly spreading. Chronic wasting disease (CWD), a prion disease of cervids, has been confirmed in wild and captive populations with devastating impact on the farmed cervid industries. Furthermore, the unabated spread of this disease through wild populations threatens a natural resource that is a source of considerable economic benefit and national pride. In a worst-case scenario, CWD may represent a zoonotic threat either through direct transmission via consumption of infected cervids or through a secondary food animal, such as cattle. This has energized efforts to understand prion diseases as well as to develop tools for disease detection, prevention, and management. Progress in each of these areas is discussed.

Phosphorylation is the predominant mechanism of post-translational modification for regulation of protein function. With central roles in virtually every cellular process, and strong linkages with many diseases, there is a considerable interest in defining, and ultimately controlling, kinase activities. Investigations of human cellular phosphorylation events, which includes over 500 different kinases and tens of thousands of phosphorylation targets, represent a daunting challenge for proteomic researchers and cell biologists alike. As such, there is a priority to develop tools that enable the evaluation of cellular phosphorylation events in a high-throughput, and biologically relevant, fashion. Towards this objective, two distinct, but functionally related, experimental approaches have emerged; phosphoproteome investigations, which focus on the sub-population of proteins which undergo phosphorylation and kinome analysis, which considers the activities of the kinase enzymes mediating these phosphorylation events. Within kinome analysis, peptide arrays have demonstrated considerable potential as a cost-effective, high-throughput approach for defining phosphorylation-mediated signal transduction activity. In particular, a number of recent advances in the application of peptide arrays for kinome analysis have enabled researchers to tackle increasingly complex biological problems in a wider range of species. In this review, recent advances in kinomic analysis utilizing peptides arrays including several of the biological questions studied by our group, as well as outstanding challenges still facing this technology, are discussed., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Treatment of hematopoietic malignancies often requires allogeneic bone marrow transplantation, and the subsequent graft-versus-leukemia response is crucial for the elimination of malignant cells. Cytotoxic T lymphocytes and NK cells responsible for the immunoelimination express Fas ligand and strongly rely on the induction of Fas receptor-mediated apoptosis for their action. Although cancer cells are removed successfully by graft-versus-leukemia reactions in myeloid malignancies, their efficiency is low in T cell leukemias. This may be partially because of the ability of malignant T cells to escape apoptosis. Our work shows that Eph family receptor EphB3 is consistently expressed by malignant T lymphocytes, most frequently in combination with EphB6, and that stimulation with their common ligands, ephrin-B1 and ephrin-B2, strongly suppresses Fas-induced apoptosis in these cells. This effect is associated with Akt activation and with the inhibition of the Fas receptor-initiated caspase proteolytic cascade. Akt proved to be crucial for the prosurvival response, because inhibition of Akt, but not of other molecules central to T cell biology, including Src kinases, MEK1 and MEK2, blocked the antiapoptotic effect. Overall, this demonstrates a new role for EphB receptors in the protection of malignant T cells from Fas-induced apoptosis through Akt engagement and prevention of caspase activation. Because Fas-triggered apoptosis is actively involved in the graft-versus-leukemia response and cytotoxic T cells express ephrin-Bs, our observations suggest that EphB receptors are likely to support immunoevasivenes of T cell malignancies and may represent promising targets for therapies, aiming to enhance immunoelimination of cancerous T cells.

Cytokines IL-32 and IL-17 are emerging as critical players in the pathophysiology of immune-mediated chronic inflammatory diseases. It has been speculated that the molecular mechanisms governing IL-32- and IL-17-mediated cellular responses are differentially dependent on the TNF pathway. In this study, kinome analysis demonstrated that following stimulation with cytokine IL-32, but not IL-17, there was increased phosphorylation of a peptide target corresponding to TNF-R1. Consistent with this observation, blocking TNF-R1 resulted in a suppression of IL-32-induced downstream responses, indicating that IL-32-mediated activity may be dependent on TNF-R1. In contrast, blocking TNF-R1 did not affect IL-17-induced downstream responses. Kinome analysis also implicated p300 (transcriptional coactivator) and death-associated protein kinase-1 (DAPK-1) as signaling intermediates for both IL-32 and IL-17. Phosphorylation of p300 and DAPK-1 upon stimulation with either IL-32 or IL-17 was confirmed by immunoblots. The presence of common targets was supported by results demonstrating similar downstream responses induced in the presence of IL-32 and IL-17, such as transcriptional responses and the direct activation of NF-κB. Furthermore, knockdown of p300 and DAPK-1 altered downstream responses induced by IL-32 and IL-17, and impacted certain cellular responses induced by TNF-α and IL-1β. We hypothesize that p300 and DAPK-1 represent nodes where the inflammatory networks of IL-32 and IL-17 overlap, and that these proteins would affect both TNF-R1-dependent and -independent pathways. Therefore, p300 and DAPK-1 are viable potential therapeutic targets for chronic inflammatory diseases.

The intestinal immune system influences responses to both enteric pathogens and commensal microflora but few models are available to analyze mucosal immune responses to either enteric pathogens or commensal microflora. We surgically isolated ileal segments in 2-3 week old calves, infused antibiotics, and subdivided each segment into three compartments. Following a 6-8 week period the isolated ileal segments appeared grossly normal in 4 of 5 calves, retained compartmentalization, and contents were culture positive for either Enterococcus spp. or Escherichia coli. In a second experiment, isolated ileal segments were examined following a 9-11 month period and appeared grossly normal with compartmentalization retained in 8 of 11 animals. Streptococcus spp or Escherichia coli were cultured from segment contents collected from 3 of these 8 animals. Histology revealed a marked reduction in villus height in isolated ileal segments despite sustained crypt epithelium proliferation. Lymphoid follicles in ileal Peyer's patches were reduced in size but remained sites of active lymphoproliferation within segments. Significant mucosal T cell, macrophage, and dendritic cell depletion was observed in isolated ileal segments and T cell and NK cell depletion increased significantly in the absence of culturable bacteria. Finally, Toll-like receptor (TLR)-4 expression was decreased but TLR-5 and -6 expression increased in ileal segments. Thus, isolated ileal segments remained relatively stable for prolonged periods and significant changes in mucosal leukocyte populations were correlated with the presence or absence of culturable microflora. Stable, as opposed to sterile, isolated ileal segments provide an opportunity to analyze bovine mucosal immune responses in the presence or absence of commensal microflora.

Host defense peptides (HDPs) contribute to immune defense through direct antimicrobial activity as well as modulation of host immune responses. While the antimicrobial activity of HDPs has been successfully exploited as topical antibiotics, their use as systemic immunomodulatory antimicrobials has been limited by their toxicity and biological instability. Peptide modification strategies to address these characteristics, while maintaining biological activity, are likely essential to capture the full therapeutic potential of HDPs. Here we investigate the stability, toxicity, and biological activity of the L, inversed (D), and retro-inversed (RI) isomers of BMAP28. The D and RI isomers both form symmetrically related structures to L BMAP28 and resist proteolytic degradation. With respect to toxicity, the considerable hemolytic activity of L BMAP28 is approximately halved with the D isomer and eliminated with RI BMAP28. Furthermore, while D BMAP28 maintains the same cytotoxicity profile against epithelial cells and monocytes as the natural peptide, RI BMAP28 is markedly less toxic against these cell types. As prophylactic antimicrobials, all three isomers significantly reduced bacterial loads [99.99% bacterial clearance by each peptide at the highest dose (20 mg kg(-1) )], when administered 18 h prior to challenge in a mouse model of peritoneal infection. This protection appears to be mediated through neutrophil recruitment and activation of macrophages for bacterial clearance. Collectively, the increased stability and retained biological activity of D and RI BMAP28 make these isomers attractive as antimicrobial therapeutics. In particular, the protection conferred by RI BMAP28, combined with its reduced toxicities, make it a strong candidate for further consideration., (Copyright © 2010 Wiley Periodicals, Inc.)

Background: Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages., Methodology/principal Findings: An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model., Conclusions/significance: Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

Host defence peptides (HDPs) are multi-functional inducers and effectors of host immunity. Through their direct antimicrobial activity HDPs have for been successfully utilized for many years as topical antibiotics and food preservatives. The more recent appreciation of HDP immunomodulatory activities offers additional opportunities for application as systemic antimicrobials, anti-inflammatory agents and vaccine adjuvants. HDPs have demonstrated proof-of-principle success in each of these applications. Optimization of HDPs for these objectives will benefit from a greater comprehension of the structural basis of their various activities. Such an understanding will facilitate rational design and/or selection of peptides with enhanced properties. This is complicated, however, by the diversity of HDP sequences, structures and mechanisms of action. Furthermore, while the ability of HDPs to undergo template-driven formation of bioactive structures enables these small peptides to perform a diverse range of actions it also complicates efforts to understand contributions of particular structural features to specific activities. With recognition of these limitations, but consideration of the emerging importance of this exciting class of molecules, we review the current understanding of the structural basis of select HDP activities as well as present strategies for HDP selection and optimization.

Host defence peptides (HDPs) have a variety of potential therapeutic applications, including as vaccine adjuvants, energizing efforts for modification strategies to address their toxicity and instability. Here we compare l, d and RI-Bac2A as vaccine adjuvants. d and RI-Bac2A are equally resistant to proteolytic degradation with no increases in toxicity, however, only RI-Bac2A maintains adjuvant activity of the natural peptide through conserved induction of a Th2 immune response. As HDPs potentiate the adjuvant activity of CpG ODNs, the isomers were also evaluated as co-adjuvants. l-Bac2A has no significant co-adjuvant activity while CpG/RI-Bac2A induces antibody titres significantly higher than CpG (P<0.01), CpG/l-Bac2A (P<0.01) or CpG/d-Bac2A (P<0.01). None of the isomers influence ODN duration or distribution but l and RI-Bac2A promote ODN uptake into B cells and antigen presenting cells. The enhanced adjuvant and co-adjuvant of RI-Bac2A is hypothesized to result from an undefined combination of increased stability and retained biological activity supporting application of retro-inversion to this, and potentially other HDPs., (Copyright 2010 Elsevier Ltd. All rights reserved.)