1. Serological Markers in Rheumatoid Arthritis

2. Rheumatoid Arthritis Systemic autoimmune disease
Characterized by chronic inflammation of the joints resulting in tissue degradation and joint destruction
1-2% of the world population
Diagnosis – primarily on clinical manifestation, with only limited serological support

3. Clinical Manifestation of RA
Morning stiffness
Arthritis of 3 or more joint areas
Arthritis of hand joints
Symmetric arthritis
Rheumatoid nodule
Serum rheumatoid factor
Radiographic change
At least 4 of these criteria(1-4 at least for 6 wks)

4. Radiological Manifestations of RA
Periarticular soft tissue swelling
Osteoporosis
Marginal erosions progressing to severe erosions of subchondral bone
Synovial cyst formation and lack of bone repair
Hand, feet, knee, hips, cervical spine, shoulder, elbow

5. Association of RA with HLA
HLA-DR4, sometimes DR1associated with RA, but association is variable.
Functional similarity : Shared epitope
a.a. sequence in the HVR3 of the  chain (QK/RRAA at positions of the HLA DRB1 chain)

6. Association of HLA with RA in Koreans
Hong, et al. J Rheumatol 1996
DR % vs 29% RR=3.7
Shared epitope 57% vs 22% RR=4.8
DRB1* RR=9.4
DRB1* RR=8.8
Kim, et al. Tissue Antigens 1999
DRB1* RR=6.6
DQA1* RR=5.2
DQB1* RR=3.5
DQCAR RR=3.2
DQCAR RR=3.6
DQCAR 113/115 RR=11.2

7. Serological Markers in RA
1. RA factor (IgM)
IgG/ IgA
2. Filaggrin associated Abs
1) antiperinuclear factor
2) antikeratin Ab
3) anticitrullinated filaggrin Ab
3. Anti-RA 33
4. Anti-Sa
5. pANCA
6. Other autoAb: HSP 60, type II collagen, calreticulin, calpastain, MTOC(?), GiM(?)

8. Rheumatoid Factors (RF)
Ab to directed to the constant region of IgG
Usually IgM type, but IgG, IgA
Agglutination, nephelometry, ELISA based test
Detected in 70-80% of RA, in high % in other autoimmune and infectious diseases, in up to 15% of healthy individuals

9. Anti-perinuclear Ab(1)
Described by Nienhuis and Mandema, in1964
Ab to a component of a number of so-called keratohyaline granules surrounding the nucleus in IIF using buccal mucosa cells

10. Anti-perinuclear Ab(2)
Profilaggrin, precursor of filaggrin colocalize with PF
Keratohyalin granule of epidermis contains profilaggrin.
Sensitivity 49-91%, specificity 73-99%
APF positive buccal mucosa donor ~5%
Correlated with bone erosion in short term

11. Anti-keratin Ab(1) Described by Young in 1979
Determined by IIF on cryosection of rat esophagus – stratum corneum of rat eosophagus epithelium
High correlation with APF

12. Anti-keratin Ab(2) Not cytokeratin
A neutral/acidic variant of human epidermis filaggrin, a terminal differentiation protein involeved in the aggregation of cytokeratin filament during cornification
Sensitivity 36-59%, Specificity 88-99%

13. Anti-filaggrin Ab
Filaggrin, cytokeratin filament aggregating protein – the target of the so called antikeratin antibodies (Simon et al., 1993, J Clin Invest)
APF, AKA, anti-filaggrin extracted from human epidermis have similar diagnostic value… partially overlap, not totally identical (Vincent et al., 1999, Ann Rhem Dis)

14. Vincent, et al. Ann Rheum Dis 1999
Distribution fo RA and control serum samples with regard to AKA, AFA and APF positivity
Vincent, et al. Ann Rheum Dis 1999
AFA immunoblot on filaggrin enriched epidermis extract

15. Filaggrin associated Ab
Comparison of Serological Markers in RA RF
Filaggrin associated Ab
APF
AKA(AFA)
AFA
Sensitivity
(%)
70-80
40-80
35-55
Similar to APF/AKA
Specificity
80-90
>90
Good
Good ass /w prognosis
Specific, Appear early, Less change of titer
Stable Ag, ELISA or IB methods
Bad
Seronegative RF, Appear late, Titer fluctuation
Less correl/w
prognosis
Lee available substrate,
IF method
IF method, Less sensitive than APF
Less sensitive than APF

16. Anti-Citrullinated Filaggrin Ab
RA associated Ab to filaggrin label the fibrous matrix of cornified cells but not the profillagrin containing keratohyalin granules in human epidermis (Simon et al., 1995, Clin Exp Immunol)
RA specific autoAb recognize citrulline, posttranslational modified arginine residue (Simon et al., 1998, J Clin Invest)

17. Epidermis Cornification
Profilaggrin: an acidic phosphorylated protein,
Consisting of 10 –12 tandemly repeated filaggrin
units separated by linker peptides
Epidermis
Dephophorylated
Proteolytically cleaved
Filaggrin: basic/neutral
intermediate filament associated protein
Citrullination by
peptidyl arginine deaminase
Cornification
Arginine
Citrulline
Citrullinated Filaggrin

18. Simon et al., 1998, J Clin Invest

19. Simon et al., 1998, J Clin Invest

20. Anti-citrullinated Filaggrin Antibody on ELISA(MBL)
87 sera tested on Hallym University Medical Center
Sensitivity 64%, Specificity 94%, Efficiency 76%

21. Specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: Relevance to antifilaggrin autoantibodies
Baeten et al., Arthritis Rheum 2001
Objectives: investigate the presence of citrullinated proteins in synovial membrane of RA Pt
Methods: stain synovial tissue section of 88 RA pts and 52 other control pt with anti citrulline polyAbs(ACA) and affinity purified antifilaggrin autoantibody(AFA)
Results: intracellularly staining of the lining and sublining layers of RA synovial tissue with ACA and AFA, which were colocalized

22. Prognostic factors in early RA
Important clinical outcomes
joint damage and functional disability
Prognostic factors
Joint involvement, RF positivity, ESR & CRP
IgA RF, RF + AKA or AFA : more active
Genetic predictors over RF : inconclusive
Guideline for aggressive treatment

23. Juvenile RA
Chronic inflammatory disease of unknown etiology and pathogenesis
1. Polyarticular onset JRA (=>5 joints) 30%
1) younger children RF(LA) negative
2) older girl RF(LA) positive
2. Pauciarticular onset JRA (1-4 joints) 50%
1) early onset, young girl /w iridocyclitis, positive ANA
2) late onset, boy /w HLA B27, ankylosing spondylitis
3. Systemic onset JRA ( fever, arthritis, skin rash, etc) 20%

24. Serological Markers for Juvenile RA(1)RF
RF_LA % JRA, late onset polyarticular
ELISA IgM RF 22-35%
IgG RF 4-6%
IgA RF 30-60%
Hidden IgM RF
not detected by conventional methods
using IgM containing fraction from sephadex
chromatography
65(systemic onset) – 85% (polyarticular)

25. Serological Markers for Juvenile RA(2)
2. ANA
50-70% positive in JRA
young girl with pauciarticular onset, iridocyclitis
95-100% positive
 ANA positivity well known risk factor for
development of ant. uveitis
Speckled and homogeneous
No association with Sm, RNP, Ro, La, Scl70
Ab to Histone % of JRA

26. Serological Markers for Juvenile RA(3)
3. Other antibodies
anticardiolipin antibody 7-42%
anticollagen antibody %
antiperinuclear factor non – 40%