Rheumatic Diseases

2005
SERIES 4 (REVISED)

COLLECTED REPORTS ON THE

Published by the Arthritis Research Campaign (arc) Editors: Ade O Adebajo FRCP(Glasgow) D John Dickson MBChB FRCP(Glasgow) FRCP(London) MRCGP

These reports are produced under the direction of the arc Education Sub-Committee. They were first published individually between 2000 and 2003 and were subsequently reviewed for this volume.

RHEUMATIC DISEASES ASSOCIATED WITH ANTINUCLEAR ANTIBODIES

Peter J Maddison Consultant Rheumatologist, North West Wales NHS Trust/Professor of Joint and Muscle Disorders, School of Sport, Health and Exercise Sciences, University of Wales, Bangor

INTRODUCTION
The presence of antinuclear antibodies (ANA) is a hallmark of autoimmune rheumatic diseases. Since rst being detected by indirect immunouorescence (IMF),1 ANA have been the subject of intensive study to understand their origin and role in pathogenesis. Laboratory methods to detect certain of these antibodies have provided the clinician with valuable tools to assist in diagnosis and, to some extent, prognosis in patients with autoimmune rheumatic diseases. Serology is of particular value in situations where clinical expression of a disease such as systemic lupus erythematosus (SLE) is incomplete when the presence of a particular ANA prole can be diagnostic. However, ANA can be found in a variety of clinical settings and their occurrence does not necessarily indicate the presence of disease at all. Therefore it is imperative that ANA tests are planned and the results are interpreted in the light of the clinical ndings. Conversely, to be of most use to the clinician the serology laboratory should have the facility to detect a wide range of relevant antibody specicities. ANA are a diverse group of antibodies, often directed to large cellular complexes containing protein and nucleic acid components. The most frequently occurring ANA react with components of deoxyribonucleic acid (DNA)-protein or ribonucleic acid (RNA)-protein complexes.2,3 A large number of studies indicate that the production of these autoantibodies, which are generally hightitre, high-afnity immunoglobulin G (IgG) antibodies, is T-cell dependent and driven by the host autoantigen.4 Screening tests for ANA generally employ techniques such as IMF using cultured cell lines expressing a wide range of autoantigen targets. As illustrated in Table 1, in addition to autoimmune rheumatic diseases ANA are found in organ-specic autoimmune diseases and in other clinical settings such as infection and lymphoproliferative disorders. About 15% of healthy adults and 8% of children have detectable ANA, usually in low titre.5 The frequency of ANA in normal people is higher in women and increases with age so that at least 25% of women over the age of 60 years are positive. The frequency is 34

Antinuclear antibodies (ANA) are a prominent feature of autoimmune rheumatic diseases Detection of ANA plays an important part in diagnosis and, to some extent, predicting prognosis (best use of serology described below) Immunofluorescence using HEp-2 cells is a sensitive screening test for ANA but has low positive predictive value for diagnosing systemic lupus erythematosus (SLE) When considering the diagnosis of lupus, it is not cost-effective to proceed to specific assays if the ANA test is negative unless the clinical picture dictates If the ANA test is positive it is important to define the ANA profile using specific assays ANA relevant to autoimmune rheumatic diseases are usually present at the time of clinical onset, tend to persist, and may be helpful in predicting the pattern of disease expression

First published September 2001; reviewed April 2005 A fully revised version of this paper is scheduled for publication as Topical Reviews (Series 5) No 8, February 2006.

diseases detected by indirect immunofluorescence. Condition 1. Autoimmune rheumatic disease

Drug-induced lupus Systemic lupus erythematosus Systemic sclerosis Sjgrens syndrome Pauciarticular juvenile idiopathic arthritis Polymyositis/dermatomyositis Rheumatoid arthritis 100% 98% 98% 80% 70% 60% 50% 100% 70% 50% 45% 20% 20% 15% 15% 8% 15%

TABLE 1. Antinuclear antibodies (ANA) in various

Frequency of ANA

cells they present antigens only expressed during certain stages of the cell cycle, which are either absent or occur only in small quantities in the resting nuclei of tissue sections. Also, ANA in autoimmune rheumatic diseases, such as anti-Ro antibodies, are directed primarily to the human antigen.9 Interpretation of this assay still depends on the skill of the technician, but with the advent of commercially available cell-culture substrates, easily available positive standards,10 and the requirement for laboratories to participate in quality assurance schemes, the IMF technique is generally reliable and reproducible. Both the ANA pattern and the titre will generally be reported. The pattern of immunouorescence will give some hints to the principal ANA specicity in the serum and may inuence the subsequent approach to determine the antibody specicity.11 Although a low-titre ANA is not necessarily clinically insignicant, higher titres (>1:160) are more likely to indicate the presence of an autoimmune rheumatic disease.12 In some instances, the IMF ANA test gives a false negative result. This may occur if the antigen is located outside the nucleus (e.g. antiJo-1 and anti-ribosomal P , both frequently categorised under the umbrella term ANA) or if it is present in a form not recognised by a particular autoantibody (e.g. when anti-Ro is directed exclusively to determinants on the native Ro molecule not expressed in cultured HEp-2 cells). In these situations the clinical picture will dictate that specic assays need to be undertaken. In order to reduce technician time and expertise by introducing automation, a number of commercial enzymelinked assays for ANA screening are now available. These employ various principles for preparing the substrate, including whole cell extracts and specic mixtures of puried or recombinant autoantigens. These assays vary considerably in their sensitivity and specicity13 and they have not yet taken over from IMF, which is still the gold standard. Autoantibodies binding native, double-stranded DNA (nDNA) and/or denatured, single-stranded DNA (ssDNA) have a central place in the immunology of lupus. It is techniques to detect anti-nDNA antibodies, which are most specic for SLE,14 that are routinely used in the diagnostic laboratory. Indirect immunouorescence using the haemoagellate Crithidia luciliae15 is a frequently used technique for detecting anti-nDNA, combining high sensitivity with high disease specicity,16 but is only semi-quantitative. This microorganism contains a giant mitochondrion which consists of pure nDNA, and it is the uorescence of this which constitutes a positive test. The Farr assay is a uid phase radioimmunoassay in which antibodies combined to 125I-labelled DNA are precipitated by 50% saturated ammonium sulphate.17 35

2. Organ-specific autoimmunity
Primary autoimmune cholangitis Autoimmune hepatititis Myaesthenia gravis Autoimmune thyroid disease

3. Other conditions
Waldenstroms macroglobulinaemia Subacute bacterial endocarditis Infectious mononucleosis Leprosy

4. Normal population
Children Adults

also higher in healthy rst-degree relatives of patients with autoimmune rheumatic diseases.6,7 An important observation is that ANA are directed to very characteristic autoantigen targets in patients with autoimmune rheumatic diseases. Furthermore, in the individual patient the ANA prole is often quite restricted. It is now appreciated that certain autoantibody proles are associated with diagnostic categories of autoimmune rheumatic diseases and sometimes with particular patterns of clinical manifestations.8 Therefore once ANA have been detected with a screening test it is important to determine their specicity. This is now part of the standard operating procedures of serology laboratories but the process is greatly facilitated by the clinician providing adequate clinical information when ANA testing is requested.

DETECTION OF ANTINUCLEAR ANTIBODIES

IMF using whole cell preparations detects a wide range of ANA specicities and is the technique used by most serology laboratories as a primary screening test for ANA. Human cell lines particularly HEp-2 epithelial cells, derived from a human laryngeal carcinoma are now used in preference to cryostat sections of mammalian tissues such as mouse or rat liver or kidney. HEp-2 cells have the advantage that, in addition to the easy visualisation of individual cells and their organelles, as rapidly dividing

Modications of this include the use of lters or an antihuman immunoglobulin serum. It is important that the substrate is impeccably pure, double-stranded DNA. The Farr assay detects high-afnity antibody and is at least as specic as IMF using Crithidia, and titres correlate best with disease activity. Increasingly, however, laboratories are turning for convenience to enzyme-linked immunoassays (ELISA). The ELISA is more sensitive but generally less specic than the Farr and IMF assays because it detects low- as well as high-afnity antibody. Different commercial ELISA assay systems are not always comparable18 and are inuenced by important factors such as characteristics of the DNA antigen, how the DNA is presented to antibody in the serum, and the reaction conditions. The presence of antibodies reacting with certain highly conserved, nucleic acid-binding proteins (extractable nuclear antigens ENA) is a very characteristic feature of autoimmune rheumatic diseases. The rst observations were made over 40 years ago when antibodies to what are now called Ro and La were detected by immunodiffusion in the sera of patients with Sjgrens syndrome.19,20 Subsequent clinical interest in these systems results from observations that certain proles of these antibodies are associated with particular patterns of disease. Traditionally these antibodies have been detected by immunodiffusion using buffered saline extracts of mammalian tissue, such as rabbit or calf thymus extract and human spleen extract. A range of prototype sera, available from the Centers for Disease Control and Prevention (CDC), Atlanta, are used to detect a precipitin system. Increasingly, more sensitive methods of antibody detection are being used, such as immunoblotting, protein or RNA immunoprecipitation, and ELISA. The techniques of immunoblotting and immunoprecipitation are described elsewhere.21 They tend to be too labour intensive for the routine laboratory but are the principal ways of identifying many of the myositis- and sclerodermaassociated antibodies. ELISAs using puried antigens have been developed and provide a sensitive, quantitative way of detecting these antibodies. Initially, immunoafnitypuried antigens were used, but recombinant antigens are increasingly used as the substrate. Commercial ELISAs vary in their performance22 but generally show high sensitivity but a corresponding lack of disease specicity compared to immunodiffusion.

using rodent substrates is the increased detection of patients with an immune response predominantly to Ro. The identication of this patient subset can be further enhanced by using HEp-2 cells transfected with human 60 kD Ro antigen gene.23 Thus in the situation where the clinician wishes to exclude the possibility of SLE, IMF is sufcient as a screening test for ANA, and it is not cost-effective automatically to test for anti-DNA or other antibody specicities.24 However, a very small number of SLE patients are ANA-negative even using HEp-2 cells, and proceeding with other techniques to look for SLE-associated antibodies is indicated if the clinical picture dictates. Conversely, since the positive predictive value in an ANA test for SLE is low as low as 11% in some studies25 once the ANA test is positive it is then important to look for antibodies reacting with DNA or nucleic acid-binding proteins. Approximately 70% of untreated patients with active SLE have anti-nDNA detected by IMF or the Farr technique. In some patients, but not in all, a steady increase in antiDNA levels followed by a sharp drop in titre precedes a clinical exacerbation.26 Consequently there is value in monitoring serial serum anti-DNA levels. In studies where the relative proportion of high- and low-avidity anti-DNA antibodies was measured, clinical exacerbation was often heralded by an increase in high-avidity antibodies.26 In SLE, antibodies react most frequently with four groups of RNA-binding proteins, namely Sm, U1RNP , Ro, and La.27 The antigens have been well characterised at a molecular level.28,29 High titres of these antibodies, for example as detected by immunodiffusion, are found frequently and almost exclusively in the context of autoimmune rheumatic diseases. Anti-Sm has the greatest specicity for SLE but there is a marked ethnic variation in the presence of these antibodies, being more commonly found in AfroCaribbeans than in northern European Caucasians.30 These antibodies identify distinctive serological subsets within the spectrum of SLE. Antibodies to Sm frequently occur in association with anti-U1RNP and antibodies to La are virtually always accompanied by anti-Ro. It is apparent that these serological subsets are associated with certain patterns of disease expression (Table 2) in which they may have a pathogenetic role. These antibodies are usually present from the beginning of the clinical presentation and are detectable throughout the course of the disease. Using an ELISA, uctuations in antibody titre can be detected, but there is an inconsistent relationship between titres measured in longitudinal studies and disease activity.31 A variety of other antibody specicities, for example antiPCNA (cyclin),32 SL (Ki),33 and ribosomal P protein,34 can be detected in SLE. They occur in a small proportion of sera and although clinical associations have been reported, such as anti-ribosomal P proteins and CNS lupus, these 36

CLINICAL ASSOCIATIONS WITH ANTINUCLEAR ANTIBODY PROFILES

Systemic lupus erythematosus
The use of HEp-2 cells enhances the sensitivity of the ANA test in SLE so that ANA can be detected in 95% of active, untreated patients. The main difference from

TABLE 2. Antinuclear antibody (ANA) specificities in diagnosis and disease expression.

Disease
Systemic lupus erythematosus

Antibody
Anti-nDNA Anti-Sm Anti-U1RNP Anti-Ro Anti-La Anti-rRNP

Frequency
70% 1025%* 30% 40% 15% 15% 6090%** 3585%** 30% 4% 25% 20% 5% 5% 2% 30% (3%) 4% 10% ** using sensitive ELISA assays

Clinical association
Lupus nephritis Vasculitis; CNS lupus Raynauds phenomenon, swollen fingers, arthritis, myositis, MCTD Photosensitive rash, SCLE, neonatal lupus, CHB, Sjgrens syndrome As for anti-Ro CNS lupus Extraglandular disease, vasculitis, lymphoma As for anti-Ro Limited cutaneous disease, micro/macrovascular disease, telangiectasia Limited cutaneous disease Diffuse cutaneous disease, interstitial lung disease Diffuse cutaneous disease, renal disease Diffuse cutaneous disease, pulmonary hypertension Scleroderma/polymyositis overlap Scleroderma/polymyositis overlap Antisynthetase syndrome (Antisynthetase syndrome) Severe myositis Dermatomyositis

Sjgrens syndrome Systemic sclerosis

Anti-Ro Anti-La Anticentromere Anti-ThRNP Anti-topoisomerase-1 Anti-RNA-polymerases Anti-U3RNP Anti-PM-Scl Anti-Ku

Dermato/ polymyositis

Anti-Jo-1 (antibodies to other tRNA synthetases) Anti-SRP Anti-Mi2

* higher frequency in blacks and Asians

CHB congenital heart block; CNS central nervous system; MCTD mixed connective tissue disease; SCLE subacute cutaneous lupus erythematosus

associations require conrmation in larger, prospective studies and the role of these antibodies in routine SLE serology is not yet dened.

Sjgrens syndrome
With sensitive techniques, antibodies to Ro and La can be detected in virtually all patients.35 They are a marker for Sjgrens syndrome developing in SLE, systemic sclerosis and primary biliary cirrhosis. Several studies, including that of Pease et al,36 have shown that antibodies to Ro and La identify patients at greatest risk of developing extraglandular complications such as vasculitis.

Systemic sclerosis
Antinuclear and/or antinucleolar antibodies are an almost universal feature of patients with systemic sclerosis (SSc). Several of these antibodies are highly specic for the disease, are rarely found in other clinical settings, and occur as an early feature so that their identication has an important role in early diagnosis.37 Certain of these antibodies can be detected in the routine serology laboratory by observing a typical pattern of IMF (in the case of anticentromere antibodies) or using immunodiffusion or a specic ELISA (in the case of anti-topoisomerase-1). However, many of these systems require techniques such 37

as immunoprecipitation for their detection and it is recommended to have sera from SSc patients analysed by a specialised reference laboratory when possible. An important observation is that there is virtually no overlap between subsets of patients identied by a particular antibody prole and these subsets tend to be associated with certain patterns of clinical expression. Thus anticentromere antibodies38 and antibodies to the nucleolar constituent ThRNP are almost exclusively found in patients with limited cutaneous systemic sclerosis39 and identify patients at risk of micro- and macrovascular disease. By contrast, antibodies to topoisomerase-1, RNA polymerases I, II and III and to U3RNP identify clinical subsets of SSc with severe disease involving extensive scleroderma and visceral organ involvement.40

Dermatomyositis and polymyositis

Multiple antibody systems are also found in polymyositis or dermatomyositis. These include a number of myositisspecic antibodies.41 Each specicity occurs in a small proportion of patients and is associated with a characteristic pattern of clinical expression (Table 2). Antibodies to Jo-1 (histidyl-tRNA synthetase) is the most common specicity, occurring in approximately 20% of adult patients with polymyositis. These patients frequently

develop additional clinical features such as interstitial lung disease, polyarthritis, Raynauds phenomenon and mechanics ngers (the antisynthetase syndrome). AntiJo-1 antibodies can be detected in the routine serology laboratory by ELISA but other myositis-specic antibodies require the input of a specialised reference laboratory.

7. Maddison PJ, Stephens C, Briggs D et al. Connective tissue disease and autoantibodies in the kindreds of 63 patients with systemic sclerosis. Medicine (Baltimore) 1993;72(2):103-12. 8. Harley JB, Gaither KK. Autoantibodies. Rheum Dis Clin North Am 1988;14(1):43-56. 9. Reichlin M, Rader M, Harley JB. Autoimmune response to the Ro/SSA particle is directed to the human antigen. Clin Exp Immunol 1989;76(3):373-7. 10. Feltkamp TE. Standards for ANA and anti-DNA. Clin Rheumatol 1990;9(1 Suppl 1);74-8. 11. Homburger HA. Cascade testing for autoantibodies in connective tissue diseases. Mayo Clin Proc 1995;70(2):183-4. 12. Tan EM, Feltkamp TE, Smolen JS et al. Range of antinuclear antibodies in healthy individuals. Arthritis Rheum 1997;40(9):1601-11. 13. Emlen W , ONeill L. Clinical signicance of antinuclear antibodies: comparison of detection with immunouorescence and enzyme-linked immunosorbent assays. Arthritis Rheum 1997;40(9):1612-8. 14. Aarden LA, Lakmaker F, Feltkamp TE. Immunology of DNA. I. The inuence of reaction conditions on the Farr assay as used for the detection of anti-ds DNA. J Immunol Methods 1976;10(1):27-37. 15. Aarden LA, Smeenk R. Measurements of antibodies specic for DNA. In: Lefkovits I, Pernis B (ed). Immunological methods. Vol 2. New York: Academic Press; 1981. p.75-82. 16. Smeenk R, van der Lelij G, Aarden L. Measurement of low avidity anti-dsDNA by the Crithidia luciliae test and the PEG assay. Clin Exp Immunol 1982;49(3):603-10. 17. Smeenk R, Brinkman K, van den Brink H, Swaak T. A comparison of assays used for the detection of antibodies to DNA. Clin Rheumatol 1990;9(1 Suppl 1):63-72. 18. Avina-Zubieta JA, Galindo-Rodriguez G, Kwan-Yeung L, Davis P, Russell AS. Clinical evaluation of various selected ELISA kits for the detection of anti-DNA antibodies. Lupus 1995;4(5):370-4. 19. Jones BR. Lacrimal and salivary precipitating antibodies in Sjgrens syndrome. Lancet 1958;2(7050):773-6. 20. Anderson JR, Gray KG, Beck JS, Kinnear WF. Precipitating autoantibodies in Sjgrens disease. Lancet 1961;2(7200):456-60. 21. Verheijen R, Salden M, van Venrooij WJ. Protein blotting. In: van Venrooij WJ, Maini RN (ed). Manual of biological markers of disease. Section A: Methods of autoantibody detection. Dordrecht: Kluwer Academic Publishers; 1993. p.4.1-25. 22. Tan EM, Smolen JS, McDougal JS et al. A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of dened specicities. I. Precision, sensitivity and specicity. Arthritis Rheum 1999;42(3):455-64. 23. Fritzler MJ, Miller BJ. Detection of autoantibodies to SS-A/Ro by indirect immunouorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells. J Clin Lab Anal 1995;9(3): 218-24. 24. Clough JD, Calabrese LH, Valenzuela R. The ANA prole; quality and cost-effective laboratory utilization [erratum in Cleve Clin J Med 1989;56(5):548]. Cleve Clin J Med 1989;56(3):245-8. 25. Slater CA, Davis RB, Shmerling RH. Antinuclear antibody testing: a study of clinical utility. Arch Intern Med 1996;156(13):1421-5. 26. ter Borg EJ, Horst G, Hummel EJ, Limburg PC, Kallenberg CG. Measurement of increases in anti-double-stranded DNA antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus: a long-term, prospective study. Arthritis Rheum 1990;33(5):634-43. 27. Reichlin M, Harley JB. Immune response to the RNA protein particles in systemic lupus erythematosus: a distinctive dichotomy. Am J Med 1988;85 Suppl 6A:35-7. 28. van Venrooij WJ, Sillekens PT. Small nuclear RNA associated proteins: autoantigens in connective tissue diseases. Clin Exp Rheumatol 1989;7(6):635-45. 29. Scoeld RH, Farris AD, Horsfall AC, Harley JB. Fine specicity of the autoimmune response to the Ro/SSA and La/SSB ribonucleoproteins. Arthritis Rheum 1999;42(2):199-209. 30. Arnett FC, Hamilton RG, Roebber MG, Harley JB, Reichlin M. Increased frequencies of Sm and nRNP autoantibodies in American blacks compared to whites with systemic lupus erythematosus. J Rheumatol 1988;15(12):1773-6.

ROLE OF ANTINUCLEAR ANTIBODIES IN DISEASE

The current view is that while most ANA are the result rather than the cause of disease, certain ANA specicities may be directly involved in pathogenesis of tissue injury. For example, there is evidence that subsets of anti-DNA antibodies cause lupus nephritis. This includes observations that high titres of anti-DNA predict exacerbations of nephritis,26 the elution of anti-DNA antibodies from affected kidneys,42 and, more recently, the demonstration that some, but not all, monoclonal anti-DNA antibodies, of both mouse and human origin, can cause renal pathology when infused into nonautoimmune mice.43 There is similar evidence44,45 suggesting that antibodies to Ro can also be pathogenic. The striking association between anti-Ro and anti-La and neonatal lupus and congenital heartblock is particularly persuasive for a role of these antibodies in pathogenesis.46 To what extent tissue injury results from direct binding of antibodies to autoantigens expressed in the target organ or from the presence of immune complexes is still not known. Additional mechanisms for antibody-mediated injury may also be operating. There is some evidence, for example, that certain autoantibodies such as antiDNA, anti-ribosomal P and anti-U1RNP can penetrate living cells and inuence the process of apoptosis,47,48 thereby inducing tissue damage or dysregulation of immune functions.

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31. de Rooij DJ, Habets WJ, van de Putte LB, Hoet MH, Verbeek AL, van Venrooij WJ. Use of recombinant RNP peptides 70K and A in an ELISA for measurement of antibodies in mixed connective tissue disease: a longitudinal follow up of 18 patients. Ann Rheum Dis 1990; 49(6):391-5. 32. Asero R, Origgi L, Crespi S, Bertetti E, DAgostino P, Riboldi P. Autoantibody to proliferating cell nuclear antigen (PCNA) in SLE: a clinical and serological study. Clin Exp Rheumatol 1987;5(3):241-6. 33. Sakamoto M, Takasaki Y, Yamanaka K, Kodama K, Hashimoto H, Hirose S. Purication and characterization of Ki antigen and detection of anti-Ki antibody by enzyme-linked immunosorbent assay in patients with systemic lupus erythematosus. Arthritis Rheum 1989; 32(12):1554-62. 34. Elkon KB, Bonfa E, Weissbach H, Brot N. Antiribosomal antibodies in SLE, infection, and following deliberate immunization. Adv Exp Med Biol 1994;347:81-92. 35. Harley JB, Alexander EL, Bias WB et al. Anti-Ro (SS-A) and anti-La (SS-B) in patients with Sjgrens syndrome. Arthritis Rheum 1986;29 (2):196-206. 36. Pease CT, Charles PJ, Shattles W , Markwick J, Maini RN. Serological and immunogenetic markers of extraglandular primary Sjgrens syndrome. Br J Rheumatol 1993;32(7):574-7. 37. Weiner ES, Hildebrandt S, Senecal JL et al. Prognostic signicance of anticentromere antibodies and anti-topoisomerase I antibodies in Raynauds disease: a prospective study. Arthritis Rheum 1991;34(1): 68-77. 38. Steen VD, Powell DL, Medsger TA Jr. Clinical correlations and prognosis based on serum autoantibodies in patients with systemic sclerosis. Arthritis Rheum 1988;31(2):196-203. 39. Falkner D, Wilson J, Medsger TA Jr, Morel PA. HLA and clinical associations in systemic sclerosis patients with anti-Th/To antibodies. Arthritis Rheum 1998;41(1):74-80.

40. Bunn CC, Denton CP , Shi-Wen X, Knight C, Black CM. Anti-RNA polymerases and other autoantibody specicities in systemic sclerosis. Br J Rheumatol 1998;37(1):15-20. 41. Targoff IN. Polymyositis and dermatomyositis in adults. In: Maddison PJ, Isenberg DA, Woo P , Glass DN (ed). Oxford Textbook of Rheumatology. 2nd edn. Oxford: Oxford University Press; 1998. p.1249-87. 42. Wineld JB, Faiferman I, Kofer D. Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus: association of high avidity antinative DNA antibody with glomerulonephritis. J Clin Invest 1977;59(1):90-6. 43. Ehrenstein MR, Katz DR, Grifths MH et al. Human IgG anti-DNA antibodies deposit in kidneys and induce proteinuria in SCID mice. Kidney Int 1995;48(3):705-11. 44. Maddison PJ, Reichlin M. Deposition of antibodies to a soluble cytoplasmic antigen in the kidneys of patients with systemic lupus erythematosus. Arthritis Rheum 1979;22(8):858-63. 45. Lee LA, Gaither KK, Coulter SN, Norris DA, Harley JB. Pattern of cutaneous immunoglobulin G deposition in subacute cutaneous lupus erythematosus is reproduced by infusing puried anti-Ro (SSA) autoantibodies into human skin-grafted mice. J Clin Invest 1989;83(5): 1556-62. 46. Kitridou RC. The neonatal lupus syndrome. In: Wallace DJ, Hahn BH (ed). Dubois Lupus erythematosus. 5th edn. Baltimore: Williams & Wilkins; 1997. p.1023-35. 47. Reichlin M. Presence of ribosomal P protein on the surface of human umbilical vein endothelial cells. J Rheumatol 1996;23(7):1123-5. 48. Alarcon-Segovia D, Llorente L, Ruiz-Arguelles A. The penetration of autoantibodies into cells may induce tolerance to self by apoptosis of autoreactive lymphocytes and cause autoimmune disease by dysregulation and/or cell damage. J Autoimmun 1996;9(2):295-300.

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