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Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris) Affected with Hypoadrenocorticism (Addison's Disease).

Boag AM, Christie MR, McLaughlin KA, Syme HM, Graham P, Catchpole B - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, United Kingdom.

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.

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Amplification and expression of canine adrenal autoantigens.(A) Coding regions of selected canine adrenal steroid synthesis enzymes were amplified and cloned. Shown are PCR results of (1) screening primers and (2) cloning primers for canine CYP17A1 and (3) screening primers and (4) cloning primers for canine CYP11A1. M, molecular weight ladder (Hyperladder I, Bioline, London, UK). Image has been cropped for ease of interpretation; the original image, with extra cDNA samples represented by extra lanes, is available as S1 Fig. (B) Autoradiograph of radiolabelled recombinant canine adrenal autoantigens. The coding regions of (1) canine CYP21A2 (21-hydroxylase; 55 kDa), (2) CYP17A1 (17-hydroxylase; 57 kDa), (3) HSD3B2 (3β hydroxysteroid dehydrogenase; 42 kDa) and (4) CYP11A1 (P450 side-chain cleavage enzyme; 60 kDa) were cloned into the pVAX1 vector and 35S-methionine radiolabelled recombinant protein expressed in an in vitro transcription and translation assay. Translates were subjected to SDS-PAGE and exposed to X-ray film for 24 h.
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pone.0143458.g001: Amplification and expression of canine adrenal autoantigens.(A) Coding regions of selected canine adrenal steroid synthesis enzymes were amplified and cloned. Shown are PCR results of (1) screening primers and (2) cloning primers for canine CYP17A1 and (3) screening primers and (4) cloning primers for canine CYP11A1. M, molecular weight ladder (Hyperladder I, Bioline, London, UK). Image has been cropped for ease of interpretation; the original image, with extra cDNA samples represented by extra lanes, is available as S1 Fig. (B) Autoradiograph of radiolabelled recombinant canine adrenal autoantigens. The coding regions of (1) canine CYP21A2 (21-hydroxylase; 55 kDa), (2) CYP17A1 (17-hydroxylase; 57 kDa), (3) HSD3B2 (3β hydroxysteroid dehydrogenase; 42 kDa) and (4) CYP11A1 (P450 side-chain cleavage enzyme; 60 kDa) were cloned into the pVAX1 vector and 35S-methionine radiolabelled recombinant protein expressed in an in vitro transcription and translation assay. Translates were subjected to SDS-PAGE and exposed to X-ray film for 24 h.

Mentions: The selected genes (CYP21A2, CYP17A1, CYP11A1 and HSD3B2) were successfully amplified from the canine adrenal cDNA (Fig 1A). 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).


Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris) Affected with Hypoadrenocorticism (Addison's Disease).

Boag AM, Christie MR, McLaughlin KA, Syme HM, Graham P, Catchpole B - PLoS ONE (2015)

Amplification and expression of canine adrenal autoantigens.(A) Coding regions of selected canine adrenal steroid synthesis enzymes were amplified and cloned. Shown are PCR results of (1) screening primers and (2) cloning primers for canine CYP17A1 and (3) screening primers and (4) cloning primers for canine CYP11A1. M, molecular weight ladder (Hyperladder I, Bioline, London, UK). Image has been cropped for ease of interpretation; the original image, with extra cDNA samples represented by extra lanes, is available as S1 Fig. (B) Autoradiograph of radiolabelled recombinant canine adrenal autoantigens. The coding regions of (1) canine CYP21A2 (21-hydroxylase; 55 kDa), (2) CYP17A1 (17-hydroxylase; 57 kDa), (3) HSD3B2 (3β hydroxysteroid dehydrogenase; 42 kDa) and (4) CYP11A1 (P450 side-chain cleavage enzyme; 60 kDa) were cloned into the pVAX1 vector and 35S-methionine radiolabelled recombinant protein expressed in an in vitro transcription and translation assay. Translates were subjected to SDS-PAGE and exposed to X-ray film for 24 h.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664467&req=5

pone.0143458.g001: Amplification and expression of canine adrenal autoantigens.(A) Coding regions of selected canine adrenal steroid synthesis enzymes were amplified and cloned. Shown are PCR results of (1) screening primers and (2) cloning primers for canine CYP17A1 and (3) screening primers and (4) cloning primers for canine CYP11A1. M, molecular weight ladder (Hyperladder I, Bioline, London, UK). Image has been cropped for ease of interpretation; the original image, with extra cDNA samples represented by extra lanes, is available as S1 Fig. (B) Autoradiograph of radiolabelled recombinant canine adrenal autoantigens. The coding regions of (1) canine CYP21A2 (21-hydroxylase; 55 kDa), (2) CYP17A1 (17-hydroxylase; 57 kDa), (3) HSD3B2 (3β hydroxysteroid dehydrogenase; 42 kDa) and (4) CYP11A1 (P450 side-chain cleavage enzyme; 60 kDa) were cloned into the pVAX1 vector and 35S-methionine radiolabelled recombinant protein expressed in an in vitro transcription and translation assay. Translates were subjected to SDS-PAGE and exposed to X-ray film for 24 h.
Mentions: The selected genes (CYP21A2, CYP17A1, CYP11A1 and HSD3B2) were successfully amplified from the canine adrenal cDNA (Fig 1A). 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).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus