Downloaded from http://ard.bmj.com/ on November 17, 2015 - Published by group.bmj.com
Letters being highly correlated to disease activity: CXCL10, TNFR2 and Galectin-9.5 We measured these proteins before and at different time points after aSCT to determine kinetics and correlation to disease activity in the context of this intervention. Values of creatine kinase were not elevated in the weeks before aSCT and did not change after, indicating low correlation with actual disease activity. CXCL10, TNFR2 and Galectin-9 were elevated in the patients prior to the conditioning regimen, irrespective of maintenance treatment (figure 1A). After aSCT, these three markers decreased over time in patients 1 and 2 with the most pronounced reduction in levels of Galectin-9 and CXCL10. These levels remained low, even after full reconstitution of the immune system. In parallel, clinical scores improved over time as shown in figure 1B. Pre-existent severe calcinosis disappeared completely in patient 2 after aSCT4 but is still persisting in patient 3. Importantly, only in this patient, CXCL10 and Galectin-9 levels remained elevated after aSCT. In contrast to TNFR2, CXCL-10 and Galectin-9 are produced by immune and non-immune cells under inflammatory conditions.6–9 As circulating immune cells are largely depleted during aSCT, our data suggest that CXCL10 and Galectin-9 are mainly produced by tissue cells or tissue infiltrating cells. Therefore, even during profound immunosuppression these markers may reveal ongoing disease activity in the tissues. This is also supported by the observation that CXCL10 and Galectin-9 levels dropped very gradually following aSCT in patients 1 and 2 and mirrored clinical disease improvement. In conclusion these data demonstrate that aSCT can induce prolonged drug-free disease remission in refractory patients with JDM with regards to the myositis. Furthermore, we show that the proinflammatory signature as measured by TNFR2, CXCL-10 and Galectin-9 leads to a differentiated response after aSCT, with a marked decrease in the two patients with inactive disease but persistent elevation in a patient with skin involvement. Further studies are needed to determine the immunopathogenic role of these proteins in JDM. F Bellutti Enders,1,2 E M Delemarre,1 J Kuemmerle-Deschner,3 P van der Torre,1 N M Wulffraat,1 B P Prakken,1 A van Royen-Kerkhof,1,# F van Wijk1 1
Laboratory of Translational Immunology, Department of Pediatric Immunology, University Medical Centre Utrecht, Utrecht, The Netherlands Division of Allergology, Immunology and Rheumatology, Department of Pediatrics, University Hospital, Lausanne, Switzerland 3 Division of Pediatric Rheumatology, Department of Pediatrics, University Hospital Tuebingen, Tuebingen, Germany 2
Correspondence to Dr A van Royen-Kerkhof, Laboratory of Translational Immunology, Department of Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Centre Utrecht, Utrecht, Room KC 03.063.0, Lundlaan 6, Utrecht 3584 EA, The Netherlands; [email protected] FBE, EMD, AvR-K and FvW contributed equally. Acknowledgements The patients in this study gave informed consent to publication of the data. The authors thank Iris Haugg, Dr Julia Thomas and Dr Sandra Hansmann for providing clinical data and laboratory results. Contributors Substantial contributions to the conception or design of the work, or the acquisition, analysis or interpretation of data; drafting the work or revising it critically for important intellectual content; final approval of the version published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: FBE, EMD, FvW, AvR-K, NW, BP, PvdT, JK-D. Funding Ettore e Valeria Rossi Foundation; Bas Foundation, The Netherlands; ZonMw Veni grant from the Netherlands Organisation of Scientific Research (NWO); ZonMw AGIKO grant from the NWO; Reumafonds, the Netherlands. Competing interests FBE is supported by Swiss Grants (The foundation of Ettore e Valeria Bossi). FBE is supported by the patient’s society (Bas Stichting). FvW is supported by a ZonMw Veni grant from the Netherlands Organisation of Scientific Ann Rheum Dis January 2015 Vol 74 No 1
Research (NWO). EMD is supported by a ZonMw AGIKO grant from the NWO. NW and BP are supported by the Reumafonds. Ethics approval Netherlands, CCMO: NL34124.041.10. Provenance and peer review Not commissioned; externally peer reviewed.
To cite Enders FB, Delemarre EM, Kuemmerle-Deschner J, et al. Ann Rheum Dis 2015;74:315–317. Received 15 July 2014 Revised 12 September 2014 Accepted 13 September 2014 Published Online First 30 September 2014 Ann Rheum Dis 2015;74:315–317. doi:10.1136/annrheumdis-2014-206287
REFERENCES 1 2
3 4
5
6 7
8 9
Robinson AB, Reed AM. Clinical features, pathogenesis and treatment of juvenile and adult dermatomyositis. Nat Rev Rheumatol 2011;7:664–75. Snowden JA, Saccardi R, Allez M, et al. Haematopoietic SCT in severe autoimmune diseases: updated guidelines of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 2012;47:770–90. van Wijk F, Roord ST, Vastert B, et al. Regulatory T cells in autologous stem cell transplantation for autoimmune disease. Autoimmunity 2008;41:585–91. Holzer U, van Royen-Kerkhof A, van der Torre P, et al. Successful autologous stem cell transplantation in two patients with juvenile dermatomyositis. Scand J Rheumatol 2010;39:88–92. Bellutti Enders F, van Wijk F, Scholman R, et al. CXCL10, TNFR2 and Galectin-9 correlate with disease activity in Juvenile Dermatomyositis. Arthritis Rheumatol 2014;66:2281–9. Antonelli A, Ferrari SM, Giuggioli D, et al. Chemokine (C-X-C motif ) ligand (CXCL)10 in autoimmune diseases. Autoimmun Rev 2014;13:272–80. de Kivit S, Kraneveld AD, Knippels LM, et al. Intestinal epithelium-derived galectin-9 is involved in the immunomodulating effects of nondigestible oligosaccharides. J Innate Immun 2013;5:625–38. Hirashima M, Kashio Y, Nishi N, et al. Galectin-9 in physiological and pathological conditions. Glycoconj J 2004;19:593–600. Imaizumi T, Kumagai M, Sasaki N, et al. Interferon-gamma stimulates the expression of galectin-9 in cultured human endothelial cells. J Leukoc Biol 2002;72:486–91.
Antibodies to domain I of β-2-glycoprotein I and IgA antiphospholipid antibodies in patients with ‘seronegative’ antiphospholipid syndrome The standard serological tests included in the classification criteria1 for antiphospholipid syndrome (APS) are those to detect immunoglobulin G (IgG) and IgM antibodies to cardiolipin (aCL) or β-2-glycoprotein I (anti-β2GPI) and the lupus anticoagulant. It is increasingly recognised, however, that some patients have typical thrombotic and non-thrombotic features of APS but test repeatedly negative in these routinely used assays. It has been suggested that these patients have the so-called seronegative APS (SN-APS).2 In a retrospective study, there were no significant differences in clinical manifestations between 87 patients with seropositive APS and 67 with SN-APS.3 Several authors have suggested that in these ‘seronegative’ patients, clinically relevant antibodies can be detected by looking for different isotypes, particularly IgA2 and/or different antigen specificity4 or by using different techniques4 5 than those of the routine assays. In a recent paper, 79% of 24 patients with SN-APS had serum antibodies detectable by such strategies.5 There is considerable evidence that IgA antiphospholipid antibody tests may be a useful diagnostic tool in APS.6 Antibodies to domain I (DI) of β2GPI have attracted particular interest as they are strongly 317
Downloaded from http://ard.bmj.com/ on November 17, 2015 - Published by group.bmj.com
Letters Table 1 Clinical features of patients in the study
Number in study Gender Male Female Mean age at sample Diagnosis PAPS SLE/APS SN-PAPS SN-APS/SLE Thrombosis Venous Arterial None Pregnancies? Yes No N/A Pregnancy morbidity Miscarriage Stillbirth Prematurity Preeclampsia None LA positivity Yes No
Table 2
APS group
SN-APS group
40
40
4 36 45.3
0 40 46.1
22 18 N/A N/A 34 19 15 6
N/A N/A 35 5 25 12 13 15
33 3 4 23 12 19 4 7 17
39 1 0 35 29 25 7 10 5
27 13
0 40
LA, lupus anticoagulant; PAPS, Primary Antiphospholipid Syndrome; SLE, systemic lupus erythematosus; SN-APS, seronegative antiphospholipid syndrome.
associated with thrombosis.7–9 No formal analysis of anti-DI antibodies (of any isotype) or IgA antiphospholipid antibodies in patients with SN-APS has been reported. Serum samples from 80 patients with APS (40 with seropositive APS fulfilling classification criteria1 and 40 with SN-APS fulfilling clinical but not serological criteria) from St Thomas’ Hospital (STH) and 200 healthy controls were tested at University College London (UCL) in nine ELISAs—IgG, IgM and IgA for each of aCL, anti-β2GPI and anti-DI. ELISAs were carried out blind to the clinical and serological information from STH using methods published previously10 with appropriate modifications to detect IgA. We defined the cut-off for a positive result in each assay as the 99th centile of the healthy population. Clinical features of the patients are shown in table 1 and results of the ELISAs in table 2. For ease of interpretation, table 2 groups the four criteria tests used in routine clinical practice (IgG aCL, IgM aCL, IgG anti-β2GPI and IgM anti-β2GPI) together at the top and the non-standard ELISAs (all anti-DI, IgA aCL and IgA anti-β2GPI) below. In the seropositive APS group, we found large numbers of samples that tested positive in the five non-criteria ELISAs. Thus 62.5% were positive in at least one of these assays. In the SN-APS group, we found no samples positive in the standard assays (thus 100% agreement with STH in tests at UCL done blind to STH results) but four (10%) were positive in one of the non-standard ELISAs. In conclusion, this blinded serological analysis of seropositive and SN-APS cohorts confirms that anti-DI, IgA aCL or IgA anti-β2GPI antibodies, while present in a significant proportion 318
ELISA results Seropositive APS (n=40)
Standard ELISAs No. (%) testing No. (%) testing anti-β2GPI No. (%) testing No. (%) testing anti-β2GPI Anti-DI ELISAs No. (%) testing No. (%) testing No. (%) testing Other IgA ELISAs No. (%) testing No. (%) testing anti-β2GPI
positive for IgG anti-CL positive for IgG
18 (45%) 6 (15%)
SN-APS (n=40)
0 0
positive for IgM anti-CL positive for IgM
4 (10%) 9 (22.5%)
0 0
positive for IgG anti-DI positive for IgM anti-DI positive for IgA anti-DI
11 (27.5%) 9 (22.5%) 7 (17.5%)
3 (7.5%)* 0 0
positive for IgA anti-CL positive for IgA
12 (30%) 8 (20%)
0 1 (2.5%)*
The cut-off for positive in each assay was defined as 99th centile of the healthy control population. *The titres of IgG anti-DI in the three positive patients were 16 absorbance units (AU), 15.3 AU and 22.2 AU respectively compared with the positive cut-off of 10 AU. The titre of IgA anti-β2GPI in the one positive patient was 16 AU compared with a positive cut-off of 9 AU. anti-β2GPI, anti-β-2-glycoprotein; CL, cardiolipin; DI, domain I; Ig, immunoglobulin; SN-APS, seronegative antiphospholipid syndrome.
of seropositive patients with APS, may also pick up a small proportion of patients with SN-APS. In this study, the IgG anti-DI assay had the highest pick-up rate (despite samples testing negative for anti-β2GPI), which is interesting given the accumulating evidence that IgG anti-DI antibodies are important in the pathogenesis of APS.7–10 Laura Cousins,1 Charis Pericleous,1 Munther Khamashta,2 Maria Laura Bertolaccini,2 Yiannis Ioannou,1,3 Ian Giles,1 Anisur Rahman1 1
Centre for Rheumatology Research, University College London, London, UK Lupus Research Unit, The Rayne Institute, King’s College London School of Medicine, London UK 3 Arthritis Research UK Centre for Adolescent Rheumatology, University College London, London, UK 2
Correspondence to Professor Anisur Rahman, Centre for Rheumatology Research, Division of Medicine, Fourth Floor Rayne Institute, 5 University Street, London WC1E 6JF, UK; [email protected] LC and CP are joint first authors, who contributed equally to the work. Contributors LC and CP carried out the laboratory experiments. MLB and MK recruited the patients and provided the samples and clinical information. AR, IG, CP and YI developed the anti-DI assays and designed the project. LC and AR wrote the final paper. All authors read and commented on the final manuscript. Funding This work was funded by Arthritis Research UK Programme Grant 19423 and supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. YI is also supported by Arthritis Research UK Grant 20164. MLB is funded by the Louise Gergel Fellowship. Competing interests None. Ethics approval London Hampstead National Research Ethics Service Committee. Provenance and peer review Not commissioned; externally peer reviewed.
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Letters Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 3.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/3.0/
3
4
5 6
To cite Cousins L, Pericleous C, Khamashta M, et al. Ann Rheum Dis 2015;74:317–319.
7
Received 15 August 2014 Revised 7 October 2014 Accepted 12 October 2014 Published Online First 30 October 2014
8
Ann Rheum Dis 2015;74:317–319. doi:10.1136/annrheumdis-2014-206483 9
REFERENCES 1
2
Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. Cervera R, Conti F, Doria A, et al. Does seronegative antiphospholipid syndrome really exist? Autoimmun Rev 2012;11:581–4.
Ann Rheum Dis January 2015 Vol 74 No 1
10
Rodriguez-Garcia JL, Bertolaccini ML, Cuadrado MJ, et al. Clinical manifestations of antiphospholipid syndrome (APS) with and without antiphospholipid antibodies (the so-called ‘seronegative APS’). Ann Rheum Dis 2012;71:242–4. Ortona E, Capozzi A, Colasanti T, et al. Vimentin/cardiolipin complex as a new antigenic target of the antiphospholipid syndrome. Blood 2010;116: 2960–7. Conti F, Capozzi A, Truglia S, et al. The mosaic of “seronegative” antiphospholipid syndrome. J Immunol Res 2014;2014:389601. Bertolaccini ML, Amengual O, Andreoli L, et al. 14th International Congress on Antiphospholipid Antibodies Task Force. Report on antiphospholipid syndrome laboratory diagnostics and trends. Autoimmun Rev 2014;13:917–30. de Laat B, Pengo V, Pabinger I, et al. The association between circulating antibodies against domain I of beta2-glycoprotein I and thrombosis: an international multicenter study. J Thromb Haemost 2009;7:1767–73. Ioannou Y, Romay-Penabad Z, Pericleous C, et al. In vivo inhibition of antiphospholipid antibody-induced pathogenicity utilizing the antigenic target peptide domain I of beta2-glycoprotein I: proof of concept. J Thromb Haemost 2009;7:833–42. Pericleous C, Ruiz-Limon P, Romay-Penabad Z, et al. Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of beta2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis. Rheumatology (Oxford) 2014. doi:10.1093/ rheumatology/keu360 Ioannou Y, Pericleous C, Giles I, et al. Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43. Arthritis Rheum 2007;56:280–90.
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Antibodies to domain I of β-2-glycoprotein I and IgA antiphospholipid antibodies in patients with 'seronegative' antiphospholipid syndrome Laura Cousins, Charis Pericleous, Munther Khamashta, Maria Laura Bertolaccini, Yiannis Ioannou, Ian Giles and Anisur Rahman Ann Rheum Dis 2015 74: 317-319 originally published online October 30, 2014
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Letters being highly correlated to disease activity: CXCL10, TNFR2 and Galectin-9.5 We measured these proteins before and at different time points after aSCT to determine kinetics and correlation to disease activity in the context of this intervention. Values of creatine kinase were not elevated in the weeks before aSCT and did not change after, indicating low correlation with actual disease activity. CXCL10, TNFR2 and Galectin-9 were elevated in the patients prior to the conditioning regimen, irrespective of maintenance treatment (figure 1A). After aSCT, these three markers decreased over time in patients 1 and 2 with the most pronounced reduction in levels of Galectin-9 and CXCL10. These levels remained low, even after full reconstitution of the immune system. In parallel, clinical scores improved over time as shown in figure 1B. Pre-existent severe calcinosis disappeared completely in patient 2 after aSCT4 but is still persisting in patient 3. Importantly, only in this patient, CXCL10 and Galectin-9 levels remained elevated after aSCT. In contrast to TNFR2, CXCL-10 and Galectin-9 are produced by immune and non-immune cells under inflammatory conditions.6–9 As circulating immune cells are largely depleted during aSCT, our data suggest that CXCL10 and Galectin-9 are mainly produced by tissue cells or tissue infiltrating cells. Therefore, even during profound immunosuppression these markers may reveal ongoing disease activity in the tissues. This is also supported by the observation that CXCL10 and Galectin-9 levels dropped very gradually following aSCT in patients 1 and 2 and mirrored clinical disease improvement. In conclusion these data demonstrate that aSCT can induce prolonged drug-free disease remission in refractory patients with JDM with regards to the myositis. Furthermore, we show that the proinflammatory signature as measured by TNFR2, CXCL-10 and Galectin-9 leads to a differentiated response after aSCT, with a marked decrease in the two patients with inactive disease but persistent elevation in a patient with skin involvement. Further studies are needed to determine the immunopathogenic role of these proteins in JDM. F Bellutti Enders,1,2 E M Delemarre,1 J Kuemmerle-Deschner,3 P van der Torre,1 N M Wulffraat,1 B P Prakken,1 A van Royen-Kerkhof,1,# F van Wijk1 1
Laboratory of Translational Immunology, Department of Pediatric Immunology, University Medical Centre Utrecht, Utrecht, The Netherlands Division of Allergology, Immunology and Rheumatology, Department of Pediatrics, University Hospital, Lausanne, Switzerland 3 Division of Pediatric Rheumatology, Department of Pediatrics, University Hospital Tuebingen, Tuebingen, Germany 2
Correspondence to Dr A van Royen-Kerkhof, Laboratory of Translational Immunology, Department of Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Centre Utrecht, Utrecht, Room KC 03.063.0, Lundlaan 6, Utrecht 3584 EA, The Netherlands; [email protected] FBE, EMD, AvR-K and FvW contributed equally. Acknowledgements The patients in this study gave informed consent to publication of the data. The authors thank Iris Haugg, Dr Julia Thomas and Dr Sandra Hansmann for providing clinical data and laboratory results. Contributors Substantial contributions to the conception or design of the work, or the acquisition, analysis or interpretation of data; drafting the work or revising it critically for important intellectual content; final approval of the version published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: FBE, EMD, FvW, AvR-K, NW, BP, PvdT, JK-D. Funding Ettore e Valeria Rossi Foundation; Bas Foundation, The Netherlands; ZonMw Veni grant from the Netherlands Organisation of Scientific Research (NWO); ZonMw AGIKO grant from the NWO; Reumafonds, the Netherlands. Competing interests FBE is supported by Swiss Grants (The foundation of Ettore e Valeria Bossi). FBE is supported by the patient’s society (Bas Stichting). FvW is supported by a ZonMw Veni grant from the Netherlands Organisation of Scientific Ann Rheum Dis January 2015 Vol 74 No 1
Research (NWO). EMD is supported by a ZonMw AGIKO grant from the NWO. NW and BP are supported by the Reumafonds. Ethics approval Netherlands, CCMO: NL34124.041.10. Provenance and peer review Not commissioned; externally peer reviewed.
To cite Enders FB, Delemarre EM, Kuemmerle-Deschner J, et al. Ann Rheum Dis 2015;74:315–317. Received 15 July 2014 Revised 12 September 2014 Accepted 13 September 2014 Published Online First 30 September 2014 Ann Rheum Dis 2015;74:315–317. doi:10.1136/annrheumdis-2014-206287
REFERENCES 1 2
3 4
5
6 7
8 9
Robinson AB, Reed AM. Clinical features, pathogenesis and treatment of juvenile and adult dermatomyositis. Nat Rev Rheumatol 2011;7:664–75. Snowden JA, Saccardi R, Allez M, et al. Haematopoietic SCT in severe autoimmune diseases: updated guidelines of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 2012;47:770–90. van Wijk F, Roord ST, Vastert B, et al. Regulatory T cells in autologous stem cell transplantation for autoimmune disease. Autoimmunity 2008;41:585–91. Holzer U, van Royen-Kerkhof A, van der Torre P, et al. Successful autologous stem cell transplantation in two patients with juvenile dermatomyositis. Scand J Rheumatol 2010;39:88–92. Bellutti Enders F, van Wijk F, Scholman R, et al. CXCL10, TNFR2 and Galectin-9 correlate with disease activity in Juvenile Dermatomyositis. Arthritis Rheumatol 2014;66:2281–9. Antonelli A, Ferrari SM, Giuggioli D, et al. Chemokine (C-X-C motif ) ligand (CXCL)10 in autoimmune diseases. Autoimmun Rev 2014;13:272–80. de Kivit S, Kraneveld AD, Knippels LM, et al. Intestinal epithelium-derived galectin-9 is involved in the immunomodulating effects of nondigestible oligosaccharides. J Innate Immun 2013;5:625–38. Hirashima M, Kashio Y, Nishi N, et al. Galectin-9 in physiological and pathological conditions. Glycoconj J 2004;19:593–600. Imaizumi T, Kumagai M, Sasaki N, et al. Interferon-gamma stimulates the expression of galectin-9 in cultured human endothelial cells. J Leukoc Biol 2002;72:486–91.
Antibodies to domain I of β-2-glycoprotein I and IgA antiphospholipid antibodies in patients with ‘seronegative’ antiphospholipid syndrome The standard serological tests included in the classification criteria1 for antiphospholipid syndrome (APS) are those to detect immunoglobulin G (IgG) and IgM antibodies to cardiolipin (aCL) or β-2-glycoprotein I (anti-β2GPI) and the lupus anticoagulant. It is increasingly recognised, however, that some patients have typical thrombotic and non-thrombotic features of APS but test repeatedly negative in these routinely used assays. It has been suggested that these patients have the so-called seronegative APS (SN-APS).2 In a retrospective study, there were no significant differences in clinical manifestations between 87 patients with seropositive APS and 67 with SN-APS.3 Several authors have suggested that in these ‘seronegative’ patients, clinically relevant antibodies can be detected by looking for different isotypes, particularly IgA2 and/or different antigen specificity4 or by using different techniques4 5 than those of the routine assays. In a recent paper, 79% of 24 patients with SN-APS had serum antibodies detectable by such strategies.5 There is considerable evidence that IgA antiphospholipid antibody tests may be a useful diagnostic tool in APS.6 Antibodies to domain I (DI) of β2GPI have attracted particular interest as they are strongly 317
Downloaded from http://ard.bmj.com/ on November 17, 2015 - Published by group.bmj.com
Letters Table 1 Clinical features of patients in the study
Number in study Gender Male Female Mean age at sample Diagnosis PAPS SLE/APS SN-PAPS SN-APS/SLE Thrombosis Venous Arterial None Pregnancies? Yes No N/A Pregnancy morbidity Miscarriage Stillbirth Prematurity Preeclampsia None LA positivity Yes No
Table 2
APS group
SN-APS group
40
40
4 36 45.3
0 40 46.1
22 18 N/A N/A 34 19 15 6
N/A N/A 35 5 25 12 13 15
33 3 4 23 12 19 4 7 17
39 1 0 35 29 25 7 10 5
27 13
0 40
LA, lupus anticoagulant; PAPS, Primary Antiphospholipid Syndrome; SLE, systemic lupus erythematosus; SN-APS, seronegative antiphospholipid syndrome.
associated with thrombosis.7–9 No formal analysis of anti-DI antibodies (of any isotype) or IgA antiphospholipid antibodies in patients with SN-APS has been reported. Serum samples from 80 patients with APS (40 with seropositive APS fulfilling classification criteria1 and 40 with SN-APS fulfilling clinical but not serological criteria) from St Thomas’ Hospital (STH) and 200 healthy controls were tested at University College London (UCL) in nine ELISAs—IgG, IgM and IgA for each of aCL, anti-β2GPI and anti-DI. ELISAs were carried out blind to the clinical and serological information from STH using methods published previously10 with appropriate modifications to detect IgA. We defined the cut-off for a positive result in each assay as the 99th centile of the healthy population. Clinical features of the patients are shown in table 1 and results of the ELISAs in table 2. For ease of interpretation, table 2 groups the four criteria tests used in routine clinical practice (IgG aCL, IgM aCL, IgG anti-β2GPI and IgM anti-β2GPI) together at the top and the non-standard ELISAs (all anti-DI, IgA aCL and IgA anti-β2GPI) below. In the seropositive APS group, we found large numbers of samples that tested positive in the five non-criteria ELISAs. Thus 62.5% were positive in at least one of these assays. In the SN-APS group, we found no samples positive in the standard assays (thus 100% agreement with STH in tests at UCL done blind to STH results) but four (10%) were positive in one of the non-standard ELISAs. In conclusion, this blinded serological analysis of seropositive and SN-APS cohorts confirms that anti-DI, IgA aCL or IgA anti-β2GPI antibodies, while present in a significant proportion 318
ELISA results Seropositive APS (n=40)
Standard ELISAs No. (%) testing No. (%) testing anti-β2GPI No. (%) testing No. (%) testing anti-β2GPI Anti-DI ELISAs No. (%) testing No. (%) testing No. (%) testing Other IgA ELISAs No. (%) testing No. (%) testing anti-β2GPI
positive for IgG anti-CL positive for IgG
18 (45%) 6 (15%)
SN-APS (n=40)
0 0
positive for IgM anti-CL positive for IgM
4 (10%) 9 (22.5%)
0 0
positive for IgG anti-DI positive for IgM anti-DI positive for IgA anti-DI
11 (27.5%) 9 (22.5%) 7 (17.5%)
3 (7.5%)* 0 0
positive for IgA anti-CL positive for IgA
12 (30%) 8 (20%)
0 1 (2.5%)*
The cut-off for positive in each assay was defined as 99th centile of the healthy control population. *The titres of IgG anti-DI in the three positive patients were 16 absorbance units (AU), 15.3 AU and 22.2 AU respectively compared with the positive cut-off of 10 AU. The titre of IgA anti-β2GPI in the one positive patient was 16 AU compared with a positive cut-off of 9 AU. anti-β2GPI, anti-β-2-glycoprotein; CL, cardiolipin; DI, domain I; Ig, immunoglobulin; SN-APS, seronegative antiphospholipid syndrome.
of seropositive patients with APS, may also pick up a small proportion of patients with SN-APS. In this study, the IgG anti-DI assay had the highest pick-up rate (despite samples testing negative for anti-β2GPI), which is interesting given the accumulating evidence that IgG anti-DI antibodies are important in the pathogenesis of APS.7–10 Laura Cousins,1 Charis Pericleous,1 Munther Khamashta,2 Maria Laura Bertolaccini,2 Yiannis Ioannou,1,3 Ian Giles,1 Anisur Rahman1 1
Centre for Rheumatology Research, University College London, London, UK Lupus Research Unit, The Rayne Institute, King’s College London School of Medicine, London UK 3 Arthritis Research UK Centre for Adolescent Rheumatology, University College London, London, UK 2
Correspondence to Professor Anisur Rahman, Centre for Rheumatology Research, Division of Medicine, Fourth Floor Rayne Institute, 5 University Street, London WC1E 6JF, UK; [email protected] LC and CP are joint first authors, who contributed equally to the work. Contributors LC and CP carried out the laboratory experiments. MLB and MK recruited the patients and provided the samples and clinical information. AR, IG, CP and YI developed the anti-DI assays and designed the project. LC and AR wrote the final paper. All authors read and commented on the final manuscript. Funding This work was funded by Arthritis Research UK Programme Grant 19423 and supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. YI is also supported by Arthritis Research UK Grant 20164. MLB is funded by the Louise Gergel Fellowship. Competing interests None. Ethics approval London Hampstead National Research Ethics Service Committee. Provenance and peer review Not commissioned; externally peer reviewed.
Open Access Scan to access more free content
Ann Rheum Dis January 2015 Vol 74 No 1
Downloaded from http://ard.bmj.com/ on November 17, 2015 - Published by group.bmj.com
Letters Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 3.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/3.0/
3
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To cite Cousins L, Pericleous C, Khamashta M, et al. Ann Rheum Dis 2015;74:317–319.
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Received 15 August 2014 Revised 7 October 2014 Accepted 12 October 2014 Published Online First 30 October 2014
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Ann Rheum Dis 2015;74:317–319. doi:10.1136/annrheumdis-2014-206483 9
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Antibodies to domain I of β-2-glycoprotein I and IgA antiphospholipid antibodies in patients with 'seronegative' antiphospholipid syndrome Laura Cousins, Charis Pericleous, Munther Khamashta, Maria Laura Bertolaccini, Yiannis Ioannou, Ian Giles and Anisur Rahman Ann Rheum Dis 2015 74: 317-319 originally published online October 30, 2014
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