Background/Purpose: Approximately 6% of all cases of uveitis arise in children (Nguyen, Foster, 1998). One of the common causes of uveitis is Juvenile Idiopathic Arthritis (JIA). Up to 20% of JIA children have uveitis (Kesen et al., 2008) and often uveitis is the initial clinical manifestation of JIA, the delay of arthritis can be estimated at years which complicates treatment for uveitis and lead to blindness. During the last years the number of proteins identified in tears increased from 200 (Herber et al., 2001) of only 17  (Zhou et al., 2003) different molecular weights (MW) to 491 (de Souza et al., 2006) and  80 chemokines, cytokines, and growth factors (Sack et al., 2005). Objective: to reveal protein markers of JIA-associated uveitis in tears using high-resolution mass-spectrometry.  

Methods: Patients with uveitis were examined by ophthalmologists and rheumatologists and the standard clinical protocol was used to diagnose the disease. Tear samples drawn from 13 JIA-patients and 3 healthy subjects were analyzed. Tears were collected on a filter paper, digested with trypsin and the peptides were purified on Zip tips. The samples were loaded to nano C18 column attached to Shimadzu nano LC coupled in-line to LTQ Orbitrap XL tandem mass spectrometer (Thermo Fischer Scientific, GA, USA). The mass spectra of the peptides were detected with a data-dependent 4-event scan method (a survey FT-MS parent scan followed by sequential data-dependent FT-MS/MS scans on the three most abundant peptide ions from the parent scan). Proteins were identified from the mass spectra results with Proteome Discoverer software for protein database search using the International Protein Index (IPI) Human Protein Database (version 1.79). Quantification was conducted using SIEVE. 

Results: About 3000 proteins were detected in tears. Among those, 236 proteins (MW 7.4 – 466 kDa) were chosen as candidates for early diagnosis of the JIA-associated uveitis. Besides the rheumatoid factors RF-ET12, RF-IP14, RF-IP24, cytokines and T-cell receptor alpha-chain, we identified anti-CD40 single-chain antibody fragment A49, inhibiting protein CD40 which is a member of the TNF-receptor superfamily, lipocalin-1 precursor, associated with immune response and prostaglandin synthesis, clusterin  inhibiting the cytolytic reaction of complement components, anti-TNF-alfa antibody light chain Fab fragment, protein phosphatase, regulatory subunit 15B, which plays a role in cell progression, apoptosis and regulation of membrane receptors and channels, DMBT/8kb.2 protein, which takes part in the interaction of tumor cells and the immune system.

Conclusion:  Our pilot study demonstrates the benefits of high resolution mass- spectrometry for analysis and development of molecular signatures which can be used in future diagnostics. Earlier it was shown that induced anterior uveitis in the eyes of rats can be described in terms of proteins drawn from the anterior chamber (Sobn et al., 2000), which result is close to our terms of description. Our results support the suggestion that it is possible to identify protein markers of JIA-associated uveitis in tears prior to the clinical signs of arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/proteomic-profile-of-tears-can-it-help-to-diagnose-juvenile-arthritis-associated-uveitis-prior-to-the-clinical-signs-of-joints-inflammation/