Limits...
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.

Show MeSH

Related in: MedlinePlus

Serum antibodies to canine P450scc in radioimmunoprecipitation assay.(A) Scatter plot from a representative 96-well plate showing P450scc immunoreactivity (counts per minute; CPM, mean of triplicate wells) in serum samples from hospital control dogs (Control; n = 11) and dogs affected with hypoadrenocorticism (HypoAd; n = 19). Negative standard (triangle) and positive standard (square) serum samples were identified from initial autoantibody screening experiments and were used on every plate for normalisation purposes. (B) Relative P450scc autoantibody reactivity in control dogs (n = 80), dogs affected with spontaneous hypoadrenocorticism (HypoAd; n = 213), dogs affected with hyperadrenocorticism (HAC; n = 58) and dogs with iatrogenic hypoadrenocorticism (iHAd; n = 4). Circles represent normalised values for each individual serum sample in the P450scc radioimmunoprecipitation assay. Mean + 3SD line represents the threshold positive value established from a reference population of 30 canine control patients.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664467&req=5

pone.0143458.g003: Serum antibodies to canine P450scc in radioimmunoprecipitation assay.(A) Scatter plot from a representative 96-well plate showing P450scc immunoreactivity (counts per minute; CPM, mean of triplicate wells) in serum samples from hospital control dogs (Control; n = 11) and dogs affected with hypoadrenocorticism (HypoAd; n = 19). Negative standard (triangle) and positive standard (square) serum samples were identified from initial autoantibody screening experiments and were used on every plate for normalisation purposes. (B) Relative P450scc autoantibody reactivity in control dogs (n = 80), dogs affected with spontaneous hypoadrenocorticism (HypoAd; n = 213), dogs affected with hyperadrenocorticism (HAC; n = 58) and dogs with iatrogenic hypoadrenocorticism (iHAd; n = 4). Circles represent normalised values for each individual serum sample in the P450scc radioimmunoprecipitation assay. Mean + 3SD line represents the threshold positive value established from a reference population of 30 canine control patients.

Mentions: There was evidence for increased P450scc autoantibody reactivity in the dogs affected with hypoadrenocorticism (Fig 3A). Due to the large number of plates assessed, inter-assay variability was normalised using negative and positive control sera. There was significantly greater P450scc immunoreactivity in the dogs affected with hypoadrenocorticism compared with the hospital control population (p < 0.001; Fig 3B). There was no significant difference in autoantibody prevalence comparing the group of dogs selected on the basis of ACTH stimulation test results (n = 150) and those with a prior diagnosis of hypoadrenocorticism that were undergoing sampling for monitoring purposes (n = 63) and these two populations were combined for subsequent analysis. There was no significant difference comparing the hospital controls and the other two disease groups. Fifty one (24%) of the dogs affected with hypoadrenocorticism were considered to be positive for P450scc, based on the threshold set. One dog within the hospital control group, an 8 year old male Rottweiler affected with a sinonasal epithelial cell tumour, was considered to be weakly positive. There was one dog in the hyperadrenocorticism group that was considered positive, an 8 year old female neutered miniature poodle.


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)

Serum antibodies to canine P450scc in radioimmunoprecipitation assay.(A) Scatter plot from a representative 96-well plate showing P450scc immunoreactivity (counts per minute; CPM, mean of triplicate wells) in serum samples from hospital control dogs (Control; n = 11) and dogs affected with hypoadrenocorticism (HypoAd; n = 19). Negative standard (triangle) and positive standard (square) serum samples were identified from initial autoantibody screening experiments and were used on every plate for normalisation purposes. (B) Relative P450scc autoantibody reactivity in control dogs (n = 80), dogs affected with spontaneous hypoadrenocorticism (HypoAd; n = 213), dogs affected with hyperadrenocorticism (HAC; n = 58) and dogs with iatrogenic hypoadrenocorticism (iHAd; n = 4). Circles represent normalised values for each individual serum sample in the P450scc radioimmunoprecipitation assay. Mean + 3SD line represents the threshold positive value established from a reference population of 30 canine control patients.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143458.g003: Serum antibodies to canine P450scc in radioimmunoprecipitation assay.(A) Scatter plot from a representative 96-well plate showing P450scc immunoreactivity (counts per minute; CPM, mean of triplicate wells) in serum samples from hospital control dogs (Control; n = 11) and dogs affected with hypoadrenocorticism (HypoAd; n = 19). Negative standard (triangle) and positive standard (square) serum samples were identified from initial autoantibody screening experiments and were used on every plate for normalisation purposes. (B) Relative P450scc autoantibody reactivity in control dogs (n = 80), dogs affected with spontaneous hypoadrenocorticism (HypoAd; n = 213), dogs affected with hyperadrenocorticism (HAC; n = 58) and dogs with iatrogenic hypoadrenocorticism (iHAd; n = 4). Circles represent normalised values for each individual serum sample in the P450scc radioimmunoprecipitation assay. Mean + 3SD line represents the threshold positive value established from a reference population of 30 canine control patients.
Mentions: There was evidence for increased P450scc autoantibody reactivity in the dogs affected with hypoadrenocorticism (Fig 3A). Due to the large number of plates assessed, inter-assay variability was normalised using negative and positive control sera. There was significantly greater P450scc immunoreactivity in the dogs affected with hypoadrenocorticism compared with the hospital control population (p < 0.001; Fig 3B). There was no significant difference in autoantibody prevalence comparing the group of dogs selected on the basis of ACTH stimulation test results (n = 150) and those with a prior diagnosis of hypoadrenocorticism that were undergoing sampling for monitoring purposes (n = 63) and these two populations were combined for subsequent analysis. There was no significant difference comparing the hospital controls and the other two disease groups. Fifty one (24%) of the dogs affected with hypoadrenocorticism were considered to be positive for P450scc, based on the threshold set. One dog within the hospital control group, an 8 year old male Rottweiler affected with a sinonasal epithelial cell tumour, was considered to be weakly positive. There was one dog in the hyperadrenocorticism group that was considered positive, an 8 year old female neutered miniature poodle.

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