Abstract
Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison’s disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.
Highlights
Canine hypoadrenocorticism is characterised by a deficiency in production of corticosteroid hormones by the adrenal gland
Full length coding sequences were cloned into both pTNT and pVAX1 vectors and sequencing revealed each to be consistent with corresponding sequences in the CanFam3.1 dog genome assembly. (See: http://www.ncbi.nlm.nih.gov/ genome/85) Use of recombinant plasmid DNA in the in vitro transcription and translation system demonstrated production of recombinant radiolabelled protein of the anticipated sizes for all constructs, but with an additional smaller band present for canine 17-OH, by autoradiography (Fig 1B)
Reactivity to P450 side-chain cleavage enzyme (P450scc) was seen in a proportion of cases (Fig 2D), using the mean + 3SD CPM of the control values as the threshold for positivity
Summary
Canine hypoadrenocorticism is characterised by a deficiency in production of corticosteroid hormones (usually cortisol and aldosterone) by the adrenal gland. Diagnosis of hypoadrenocorticism relies upon use of the ACTH stimulation test, whereby a deficiency in cortisol secretory capacity is demonstrated [5,10]. Histopathology of adrenal glands from dogs affected with hypoadrenocorticism indicates lymphocytic adrenalitis leading to adrenocortical atrophy [22,23,24,25,26], suggesting an autoimmune pathogenesis similar in nature to human autoimmune Addison’s disease (AAD) [2]. Use of indirect immunofluorescence has demonstrated the presence of adrenal autoantibodies in dogs affected with hypoadrenocorticism [23,27]
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