Project description:Application of allergens onto the sublingual epithelium is used to desensitize allergic individuals, a treatment known as sublingual immunotherapy. However, the response of sublingual epithelial cells to house dust mite allergen and potential tolerance-promoting adjuvants such as Toll-like receptor ligands and calcitriol has not been investigated. In order to study this, primary sublingual epithelial cells were isolated from dogs and cultured in vitro. After 24h incubation with Dermatophagoides farinae extract, TLR2 ligands (FSL-1, heat-killed Listeria monocytogenes, Pam3CSK4), a TLR3 ligand (poly I:C), a TLR4 ligand (LPS) and calcitriol (1,25-dihydroxyvitamin D3), viability of the cells was analyzed using an MTT test and their secretion of IL-6, IL-10, CXCL8 and TGF-β1 was measured by ELISA. Additionally, to evaluate its potential effect as an adjuvant, sublingual epithelial cells were incubated with calcitriol in combination with D. farinae extract followed by measurement of CXCL8 secretion. Furthermore, the effect of D. farinae and calcitriol on the transcriptome was assessed by RNA-sequencing. The viability of the sublingual epithelial cells was significantly decreased by poly I:C, but not by the other stimuli. CXCL8 secretion was significantly increased by D. farinae extract and all TLR ligands apart from LPS. Calcitriol significantly decreased CXCL8 secretion and co-administration with D. farinae extract reduced CXCL8 concentrations to levels seen in unstimulated sublingual epithelial cells. Although detectable, TGF-β1 secretion could not be modulated by any of the stimuli. IL-6 and IL-10 could not be detected at the protein nor at the mRNA level. It can be concluded that D. farinae extract and TLR ligands augment the secretion of the pro-inflammatory chemokine CXCL8, which might interfere with sublingual desensitization. On the other hand, CXCL8 secretion was reduced by co-application of calcitriol and D. farinae extract. Calcitriol therefore seems to be a suitable candidate to be used as adjuvant during sublingual immunotherapy.

Project description:Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq proved more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas.

Project description:Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq proved more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas. RNA was extracted from 10 lymphoma fine needle aspirates attained from companion canines. 4 normal lymph node samples were obtained from a Beagle breeding colony at Pfizer, including two samples that were taken from the same dog but different lymph nodes. This Series represents the Affymetrix gene expression only, not RNA-Seq referenced above. RNA-Seq data have been submitted to SRA as SRA059558.

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Canis lupus familiaris

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