Autoantigen specificity in Juvenile Idiopathic Arthritis

Lay Summary

It is often assumed that arthritis only develops as we get older but in the UK there are approximately 15,000 children and young people affected by arthritis; that’s about 1 in every 1000 children. Juvenile idiopathic arthritis (JIA) is inflammation of one or more joints that develops before 16 years of age. Some children may develop disabling joint disease and have persistent active disease into adulthood. There are different types of JIA and the symptoms and likelihood of outgrowing the disease varies between the different types. Some children with JIA also develop eye involvement known as uveitis. This is symptomless inflammation in the eye that can lead to permanent visual impairment. 

We are trying to identify a simple blood test that could help predict which children are most at risk of JIA associated uveitis. This would allow those children to be more closely monitored so they can receive treatment early. Early treatment is important to prevent sight loss. Children who are not at high risk of developing uveitis would not need to be monitored as regularly freeing up clinical time. 

We and others have shown that the presence of factors in the blood known as autoantibodies are associated with JIA and the development of eye disease in particular. In healthy people the immune system creates antibodies against viruses and bacteria to help fight infection but in patients with diseases like JIA, the immune system mistakenly creates antibodies against substances normally found in our own bodies, these are known as ‘autoantibodies’. Patients with different diseases produce different autoantibodies. While we know patients with JIA can produce autoantibodies and that these are important, we do not know the specific type of autoantibody. Characterising the JIA autoantibody further will allow the development of specific blood tests so that children can be screened for the autoantibody when they are diagnosed and their risk of uveitis and need for monitoring easily established.

Scientific Summary

Background – This project has established that a dense fine speckled/homogeneous antinuclear antibody (ANA) pattern on immunofluorescence in patients with JIA is associated with an increased risk of uveitis. The ANA pattern suggests an antigenic target associated with DNA/chromatin. 

Determine the specificity of ANA – As based on our previous work Hep-2 cells are known to contain the JIA antigen(s) of interest, I have obtained Hep-2 cells for culture. The cells have been successfully grown and an adequate stock for future work stored in liquid nitrogen at the University of Bath. 

The immunofluorescence pattern described above suggests a nuclear location of the antigen. I have optimized the procedure for obtaining a nuclear enriched extract of Hep2 cells that can be used as an antigen source. I have used the Hep2 cell nuclear extract in western blotting experiments with JIA patient sera but have thus far been unable to detect the JIA antigen using a standard blotting technique. Possible reasons for this include, that the antigen has a very low molecular weight, the antigen has a conformational epitope, is rapidly degraded or the antigen is not a protein but RNA or DNA. 

Future directions – Planned experiments include further optimization of the blotting procedure to detect low molecular weight proteins and RNA immunoprecipitation to detect an RNA antigen. If these prove unsuccessful alternative approaches may include protein microarray analysis. 

Other work – In addition to the above project I have prepared a paper on anti-HMGCR autoantibodies in patients with juvenile dermatomyositis (JDM) and this has been submitted for publication to Arthritis and Rheumatology. I have also prepared a paper for submission describing the autoantibody associated clinical phenotype of patients with JDM in the UK JDM cohort (379 patients). 

Impact – The work on JIA was presented as an oral abstract at the British Society of Rheumatology annual conference April 2016. The work on anti-HMGCR in JDM was presented as a poster presentation at the same conference: Sarah L Tansley, Juliet Dunphy, Amelia A C Jobling, Roberto Carrasco, Angela Midgley, Michael Beresford, Andrew D Dick, Lucy R Wedderburn, Wendy Thomson, CAPS, Athimalaipet Ramanan & Neil J McHugh. A dense fine speckle pattern on immunofluorescence is strongly associated with the development of uveitis in children with juvenile idiopathic arthritis. Sarah L Tansley, Zoe E Betteridge, Stefania Simou, Clarissa Pilkington, Mark Wood, Kishore Warrier, Lucy R Wedderburn & Neil J McHugh on behalf of the JDRG. A diagnostic and treatment challenge: The prevalence and clinical associations of anti-HMGCR autoantibodies in a large UK juvenile-onset myositis cohort

The prevalence and clinical associations of anti-HMGCR autoantibodies in a large UK juvenile-onset myositis cohort has been submitted as a paper publication. 

Obtaining control samples has required collaboration with other centres including UCL, the University of Liverpool and the University of Bristol. We have also collaborated with Inova. Such collaborations help to raise our national and international profile as a centre for specialised autoantibody testing in rheumatological diseases. 

Future impact: Identification of an autoantigenic target in JIA will impact on patient investigation and diagnosis particularly with regard to uveitis risk. It may impact on policy in terms of uveitis screening recommendations/guidelines and have cost savings to the NHS and other organisations by better targeting for ophthalmological screening and preventing unnecessary appointments.

Updates

So far we have analysed blood samples from 433 patients with JIA and 48 healthy children. We have found an autoantibody in over half of patients with JIA and typically at high levels. We noticed that the autoantibody seen in most children with JIA had a characteristic pattern looking down the microscope, we called this ‘dense fine speckle/homogenous’. We have been able to show that it is autoantibodies producing this pattern that are strongly associated with the development of uveitis. These findings were presented at the British Society of Rheumatology annual conference in April 2016. The dense fine speckle/homogenous pattern we see down the microscope is an important clue in characterising the JIA uveitis autoantibody further and has helped us to plan the next phase of experiments. 

I have been successful in optimising experimental techniques used in the past to identify autoantibodies in different rheumatological diseases that we think have similar characteristics to the JIA autoantibody. This has allowed us to use the same technique on blood samples from JIA patients but unfortunately this has not allowed us to identify the JIA autoantibody. There are several reasons why this type of experiment may not work and we now plan to these reasons to adapt the technique further and/or choose different experimental strategies.