Gene Expression Analysis of the Acute Phase Response Using a Canine Microarray

Abstract

The safety of pharmaceuticals is typically assessed in the dog and rat prior to investigation in humans. As a result, a greater understanding of adverse effects in these preclinical testing species would improve safety assessment. Despite this need, there is a lack of tools to examine mechanisms and identify biomarkers in the dog. To address this issue, we developed an Affymetrix-based oligonucleotide microarray capable of monitoring the expression of thousands of canine genes in parallel. The custom canine array contains 22,774 probe sets, consisting of 13,729 canine and 9045 human-derived probe sets. To improve cross-species hybridization with human-derived probes, the detection region was moved from the variable 3' UTR to the more homologous coding region. Testing of this strategy was accomplished by comparing hybridization of naive dog liver RNA to the canine array (coding region design) and human U133A array (standard 3' design). Although raw signal intensity was greater with canine-specific probe sets, human-derived probes detected the expression of additional liver transcripts. To assess the ability of this tool to detect differential gene expression, the acute phase response was examined in beagle dogs given lipopolysaccharide (LPS). Hepatic gene expression 4 and 24 h post-LPS administration was compared to gene expression profiles of vehicle-treated dogs (n=3/group). Array data was consistent with an acute inflammatory response, with transcripts for multiple cytokines and acute phase proteins markedly induced 4 h after LPS challenge. Robust changes in the expression of transcripts involved with glucose homeostasis, biotransformation, and extracellular matrix remodeling were observed 24 h post-dose. In addition, the canine array identified several potential biomarkers of hepatic inflammation. Strong correlations were found between gene expression data and alterations in clinical chemistry parameters such as serum amyloid A (SAA), albumin, and alkaline phosphatase (ALP). In summary, this new genomic tool successfully detected basal canine gene expression and identified novel aspects of the acute phase response in dog that shed new light on mechanisms underlying inflammatory processes.

Utilization of the Zucker Diabetic Fatty (ZDF) Rat Model for Investigating Hypoglycemia-related Toxicities

“…In dogs, the increased serum ALP in females given 60 or 120 mg/kg/day and in individual males on day 16 and day 22 given 120 mg/kg/day may have resulted from the corticosteroid-induced ALP isoenzyme secondary to stress (Hall 2001). The increased serum triglycerides on day 16 in males given 120 mg/kg/day were possibly part of the acute phase response due to the condition of the dogs (Higgins et al 2003;Burstein 1992). Based on this literature, we hypothesized that the degenerative lesions observed in these normal euglycemic rats and dogs were not a direct toxic effect of BMS-820132 but were rather a secondary effect caused by extended periods of hypoglycemia.…”

Microarray analysis of regional cellular responses to local mechanical stress in acute lung injury

“…This approach is useful for gene products with an adequate degree of homology to their human counterparts, whereas failure to detect an expressed gene cannot distinguish between lack of stimulus effect vs. failure to hybridize because of species-specific sequence differences. The present call rate can be increased using masking techniques that either eliminate poorly functioning probe sets or relax the stringent criteria for detecting all the probe set sequences in Affymetrix-type arrays, since differences between species may be limited to only portions of the sequence (22,23,26). As our purpose was to demonstrate regional differences in gene expression in response to different mechanical stresses, false negatives are irrelevant with the acknowledgment that these results are but a subset of the gene expression changes that have occurred.…”

Insights into Gene Expression Changes Impacting B-Cell Transformation: Cross-Species Microarray Analysis of Bovine Leukemia Virus Tax-Responsive Genes in Ovine B Cells

“…The assumption is that the conservation of gene sequences between species will be sufficient to generate a reasonable amount of good-quality data. While there have been relatively few previously published reports that described the use of microarrays for cross-species hybridization (8,24,26,31,47), this technique is potentially a powerful tool for understanding molecular mechanisms associated with cancer in model organisms such as sheep.The sheep has been of particular interest as a large-animal model for studying aspects of immunology and offers a number of experimental opportunities that are not available in murine systems. Furthermore, sheep develop B-cell leukemia following experimental transmission of bovine leukemia virus (BLV), a complex retrovirus that is structurally and functionally related to human T-cell leukemia virus type 1 (HTLV-1) (6,42,78).…”

“…The assumption is that the conservation of gene sequences between species will be sufficient to generate a reasonable amount of good-quality data. While there have been relatively few previously published reports that described the use of microarrays for cross-species hybridization (8,24,26,31,47), this technique is potentially a powerful tool for understanding molecular mechanisms associated with cancer in model organisms such as sheep.…”