Cytokine mRNA quantification in duodenal mucosa from dogs with chronic enteropathies by real-time reverse transcriptase polymerase chain reaction.

The pathogenesis of inflammatory bowel disease (IBD) and antibiotic-responsive diarrhea (ARD) in dogs likely involves an interaction between the intestinal immune system and luminal bacterial or food antigens. German Shepherd Dogs (GSD) are particularly predisposed to both IBD and ARD. CD4+ T cells are important for the regulation of immune responses in the mucosa, and they exert their effects through the secretion of cytokines. The present study examined the role of cytokines in the pathogenesis of canine chronic enteropathies by quantification of mRNA encoding interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, interferon gamma, tumor necrosis factor-alpha, transforming growth factor-beta, and glyceraldehyde-3-phosphate dehydrogenase by real-time reverse transcriptase polymerase chain reaction in duodenal mucosal biopsies obtained from 39 dogs with chronic diarrhea and 18 control dogs. Contemporaneously collected biopsies were assessed for histologic changes with a 4-point grading system. No significant difference in the expression of cytokine mRNA (P > .01) was detected between dogs with and those without chronic diarrhea. Similarly, no significant differences in cytokine mRNA expression were observed between GSD and other breeds with chronic diarrhea, or between histologically normal duodenal mucosa and that with evidence of inflammatory change. Failure to detect a difference in mRNA expression does not rule out the possibility of a defect downstream at the level of translation or protein function. No conclusion can be drawn from these data as to the predominant CD4+ cell type in the pathogenesis of these canine chronic enteropathies.

Measurement of messenger RNA encoding the alpha-chain, polymeric immunoglobulin receptor, and J-chain in duodenal mucosa from dogs with and without chronic diarrhea by use of quantitative real-time reverse transcription-polymerase chain reaction assays.

OBJECTIVE: To examine the difference in expression of messenger RNA (mRNA) transcripts for polymeric immunoglobulin receptor (plgR), alpha-chain, and J-chain determined by use of quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) assays in duodenal biopsy specimens obtained from dogs with and without chronic diarrhea. SAMPLE POPULATION: Biopsy specimens of the proximal portion of the duodenum were obtained endoscopically from 39 dogs evaluated because of chronic diarrhea (12 German Shepherd Dogs and 27 non-German Shepherd Dog breeds); specimens were also obtained from a control group of 7 dogs evaluated because of other gastrointestinal tract diseases and 2 dogs that were euthanatized as a result of nongastrointestinal tract disease. PROCEDURE: Dogs were anesthetized, and multiple mucosal biopsy specimens were obtained endoscopically at the level of the caudal duodenal flexure by use of biopsy forceps; in 2 control dogs, samples were obtained from the descending duodenum within 5 minutes of euthanasia. One-step QRT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase, plgR, alpha-chain, and J-chain in duodenal mucosal tissue. RESULTS: There was no significant difference in the level of expression of any transcript among non-German Shepherd Dog breeds without diarrhea (control group), non-German Shepherd Dog breeds with chronic diarrhea, and German Shepherd Dogs with chronic diarrhea. Conclusions and Clinical Relevance-Results indicated that the susceptibility of German Shepherd Dogs to chronic diarrhea is not a result of simple failure of transcription of the key genes that encode molecules involved in mucosal IgA secretion.

Identification of four allelic variants of the dog IGHA gene.

Multiple IgA subclasses have been identified in humans, primates and lagomorphs, whereas in mice, cattle and dogs only a single subclass has been identified. The two human subclasses (IgA1 and IgA2) are defined by a difference in the length of the hinge region of the alpha chains between the CH1 and CH2 domains. The single IgA subclass so far identified in dogs has an alpha-chain hinge region with a predicted amino-acid sequence similar to that of the human alpha1 chain. Allelic variants that differ in the coding sequence of the hinge region have been identified in mice and pigs. In order to investigate whether allelic variants are present in dogs, a portion of the IGHA gene from eight individual dogs was cloned and sequenced. Four sequence variants were identified, and these differed in the coding region of their hinge. A major difference between the variants was the presence of a base polymorphism in the splice acceptor site for the second exon, which resulted in shortening of the hinge in two of the variants. Individuals expressed one or two of the variants identified, suggesting they may be heterozygous or homozygous. Further work is required to determine the effect of the variation on the biological activity of dog IgA and any relationship to susceptibility to mucosal disease.