Around 78% of patients with large vessel vasculitis may have antibodies to 14-3-3 proteins, the presence of which may serve as a novel biomarker for the disease, according to researchers at the Cleveland Clinic and University Hospitals Case Medical Center.
Analyses were performed on 11 aorta samples from 11 patients with large vessel vasculitis (LVV) and 11 control participants matched for age, sex and race. Western blot and immunoprecipitation studies were performed on aortic samples from 23 patients with aortitis undergoing thoracic aortic reconstructive surgery, 23 matched control participants with noninflammatory aortic aneurysm, and 41 patients with vasculitis or other diseases. Sera from 23 patients with LVV, 26 matched control participants, 48 control participants with other diseases and 11 healthy participants were analyzed by ELISA-based colorimetric assays developed by the researchers with 100 µg/mL of bovine serum albumen on a 96-well plate.
A significant difference was seen in sera from patients with LVV and immunoglobulin G (IgG) antibodies, which had higher absorbance compared to other samples from patients with other diseases or healthy donor samples.
In 22 frozen aortic samples from patients with LVV, light and heavy chains of IgG were seen and a third band of positive reactivity was observed in 78% of patients with LVV at around 30 kd, which was absent from 89% of matched samples from patients with noninflammatory diseases. The band was also absent from 80% of samples from patients with granulomatosis with polyangiitis and was not seen in samples from healthy participants. Overall, 6.7% of the samples from participants other than patients with LVV showed the band at 30 kd. No differences were observed in patients treated with prednisone.
Mass spectroscopy of the 30-kd region with in-gel trypsin digestion was used to identify the proteins present. Of the 33 proteins identified, 14-3-3 was the most abundant in the lysates from patients with LVV.
Three independent experiments with eight aorta lysates and 12 samples of serum were performed to determine the auto-antigenicity of 14-3-3, the presence of anti-14-3-3 antibodies with antigen pull-down assays. Immunoprecipitation of 14-3-3 from the tissue from patients with LVV was observed on Western blot and mass spectroscopy.
Detailed mass spectroscopy techniques revealed three 14-3-3 protein isoforms of γ, ζ and ε in samples from patients with LVV in two independent experiments.
After additional testing, the researchers wrote that immunoreactivity was confirmed with each of the three isoforms.
“While our findings will need to be confirmed, they do raise important questions for further studies that may address whether targeted 14-3-3 isoforms are native or modified antigens, whether there is a relevant T cell immune response to 14-3-3, and whether circulating antigen concentrations and/or antibodies to these proteins may be useful surrogates for diagnosis and monitoring disease activity,” the researchers wrote. – by Shirley Pulawski
Disclosure: The research is supported by the Vasculitis Foundation (grant VF1301), the Clinical and Translational Science Collaborative of Cleveland (CTSC Cleveland grant T53250), the American Heart Association (Scientist Development grant 23080025 to Chakravarti), and the Konigsberg Family Fund for Vasculitis Research. The acquisition of the Orbitrap Elite instrument was made possible by the NIH Shared Instrumentation Grant Program (grant NIH-1S10-RR-031537-01).