Profiling of Differentially Expressed Genes Using Suppression Subtractive Hybridization in an Equine Model of Chronic Asthma

Background :\ud Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma.\ud \ud Objective :\ud To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition.\ud \ud Methods :\ud Eleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay.\ud \ud Results :\ud Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways.\ud \ud Conclusions :\ud Pathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

Background Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. Results In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis. Conclusion Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated

Diverse perspectives on interdisciplinarity from the Members of the College of the Royal Society of Canada

Various multiple-disciplinary terms and concepts (although most commonly “interdisciplinarity”, which is used herein) are used to frame education, scholarship, research, and interactions within and outside academia. In principle, the premise of interdisciplinarity may appear to have many strengths; yet, the extent to which interdisciplinarity is embraced by the current generation of academics, the benefits and risks for doing so, and the barriers and facilitators to achieving interdisciplinarity represent inherent challenges. Much has been written on the topic of interdisciplinarity, but to our knowledge there have been few attempts to consider and present diverse perspectives from scholars, artists, and scientists in a cohesive manner. As a team of 57 members from the Canadian College of New Scholars, Artists, and Scientists of the Royal Society of Canada (the College) who self-identify as being engaged or interested in interdisciplinarity, we provide diverse intellectual, cultural, and social perspectives. The goal of this paper is to share our collective wisdom on this topic with the broader community and to stimulate discourse and debate on the merits and challenges associated with interdisciplinarity. Perhaps the clearest message emerging from this exercise is that working across established boundaries of scholarly communities is rewarding, necessary, and is more likely to result in impact. However, there are barriers that limit the ease with which this can occur (e.g., lack of institutional structures and funding to facilitate cross-disciplinary exploration). Occasionally, there can be significant risk associated with doing interdisciplinary work (e.g., lack of adequate measurement or recognition of work by disciplinary peers). Solving many of the world’s complex and pressing problems (e.g., climate change, sustainable agriculture, the burden of chronic disease, and aging populations) demand thinking and working across long-standing, but in some ways restrictive, academic boundaries. Academic institutions and key support structures, especially funding bodies, will play an important role in helping to realize what is readily apparent to all who contributed to this paper—that interdisciplinarity is essential for solving complex problems; it is the new norm. Failure to empower and encourage those doing this research will serve as a great impediment to training, knowledge, and addressing societal issues

Genomic and non-genomic effects of dexamethasone on equine peripheral blood neutrophils.

[en] BACKGROUND: Glucocorticoids have potent anti-inflammatory properties and are frequently used for the treatment of domestic animal species, including horses. They induce a down-regulation of multiple inflammatory pathways through both genomic and non-genomic effects. Currently, little is known on the effects of glucocorticoids on equine peripheral blood neutrophils. HYPOTHESIS: Dexamethasone (DEX), a potent synthetic glucocorticoid, inhibits the functions of equine peripheral blood neutrophils through both genomic and non-genomic effects. ANIMALS: Six healthy adult mixed breed female horses. METHODS: To assess the genomic effects of DEX, peripheral blood neutrophils were isolated using a gradient technique and incubated 6 h with 100 ng/ml LPS and 10(-6) M DEX alone, or combined with the glucocorticoid receptor (GR) inhibitor RU486 (10(-5) M). Messenger RNA for IL-8, TNF-alpha and TLR-4 were measured using real-time RT-PCR. The non-genomic effects of DEX were studied in neutrophils incubated with 5 microM dichlorodihydrofluorescein (DCF) and 10(-6) M DEX 5, 10 and 15 min prior to being stimulated with 5 ng/ml phorbol myristate acetate. Neutrophils were similarly co-incubated with DEX (10(-6) M, 15 min) and RU486 (10(-5) M) to evaluate the contribution of the GR to these effects. The oxidation of DCF was studied using flow-cytometry. RESULTS: Neutrophils stimulation with LPS resulted in a significant increase in IL-8, TNF-alpha and TLR-4 mRNA expressions (p<0.0001); incubation with DEX significantly down-regulated this process (p<0.0001). DEX significantly reduced oxidation of DCF after 10 and 15 min of incubation (p<0.0001). Those effects were mediated through the GRs. CONCLUSION: DEX exerts anti-inflammatory effects on equine peripheral blood neutrophils through both genomic and non-genomic pathways

Inflammatory stimuli differentially modulate the transcription of paracrine signaling molecules of equine bone marrow multipotent mesenchymal stromal cells

SummaryObjectiveOsteoarthritis (OA) is a degenerative disease of joint tissues that causes articular cartilage erosion, osteophytosis and loss of function due to pain. Inflammation and inflammatory cytokines in synovial fluid (SF) contribute to OA progression. Intra-articular (IA) injections of multipotent mesenchymal stromal cells (MSCs) are employed to treat OA in both humans and animals. MSCs secrete paracrine pro-inflammatory and anabolic signaling molecules that promote tissue repair. The objective of this study was to investigate the effects of OASF on the gene expression of paracrine signaling molecules by MSCs.MethodsThe effects of Lipopolysaccharide (LPS) and interleukin (IL)-1β as well as both normal (N) and osteoarthritis (OA) SF stimulations on the expression of paracrine pro-inflammatory (tumor necrosis factor (TNF)-α, IL-1β, IL-8), modulatory (IL-6) and anabolic (vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β1 and insulin-like growth factor (IGF)-1) signaling molecules by equine bone marrow multipotent mesenchymal stromal cells (eBM-MSCs) was investigated employing reverse transcriptase-polymerase chain reaction (RT-PCR).ResultsIn contrast with NSF, OASF significantly up-regulated the expression of VEGF in eBM-MSCs. Both NSF and OASF significantly down-regulated the expression of IL-1β. LPS and IL-1β significantly increased the expression of pro-inflammatory cytokines (TNF-α, IL-8 and IL-6; and IL-1β and IL-8 respectively).DiscussionWe conclude that the transcription of paracrine signaling molecules in eBM-MSCs is modulated by SF. Furthermore, OA alters the properties of SF and the response of eBM-MSCs. Finally, the effects of LPS or IL-1β stimulation are distinct to that observed following stimulations with OASF