Abstracts of Poster Presentations
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Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 loth ktertSic (Qn nsanAtiopuipdAMibocEes PFates 584 ANTIPHOSPHOLIPID ANTIBODIES IN PATIENTS WITH DEVELOPMENT OF ANTIPHOSDPHOLIPID-ANTIBODIES AND INTOLERANCE TO METALS IN THE ORAL CAVITY FACrOR V-INHIBITOR AFTER CIPROFLOXACIN IN TWO CASES H. Kucerova', J. Prochazkovat, I. Janatkova2, J. Bartova', T. Fucikova2 W.Miesbachl, J. Vogte, D. Peez3, B. Buehler', G. Asmelashl', 'Institute ofDental Research, 1st Medical Faculty, 2Institute ofClinical 1. Scharrerl Imnmunology andAllergology, General Faculty Hospital, Prague, 'J W.Goethe-University, Frankfurt, 25t. Vincenz Hospital, Mainz, Czech Republic 3Justus Liebig University, Frankfurt, Germany Dental alloys are not a physiological part of the organism. Metals can cause The development of factor V-inhibitor is very rare, especially in the production of autoantibodies in sensitive individuals as well as other combination with the antiphospholipid (aPL)-antibodies. The present paper undesired side effects. The aim of this presentation is to evaluate the describes two patients with factor V-inhibitor and aPL-antibodies after presence of antiphospholipid antibodies in sera of patients with oral trcatment with ciprofloxacin. Lupus anticoagulants (LA) were assayed discomfort and the occurrence of pathological galvanic currents in the oral according to the criteria of SSC of the ISTH, ACL by an ELISA, factor V- cavity. The patients were diagnosed by measurement of galvanic features inhibitor by the Bethesda method. with the device Odontologic accompanied by the modified method ofblastic Q.Oe.atirti 74 years old was treated with ciprofloxacin for a postoperative transfonnation for metals (Melisa) and by the anamnestic data. A group of septicemia with pneumonia. 4 days after start of treatment PT decreased and with galvanic currents and with diagnosed metal intolerance was a prolonged PTT was observed (PT: 13%, PTT > 120 sec). In addition. patients factor V-inhibitor (17,6 BU) and lupus anticoagulants could be detected. As compared to the group of patients with oral discomfort, with metal clinical correlation a massive muscle bleeding and anemia occurred intolerance and without galvanic currents. This study will present the (hemoglobin 6 g/dl). After terminating treatment with ciprofloxacin, factor importance of metal intolerance exaniinations in patients with V-inhibitor and lupus anticoagulants disappeared and the condition of the antiphospholipid antibodies. patient improved qickly. The study was supported by the Grant of the CzMH No.NK 6285-3. The other Datient, 85 years old was admitted to hospital due to a cerebral infarction. After improvenent of the neurological defects he was treated with ciprofloxacin foT 8 days for an urinary tract infection. Also 4 days after start of treatment, PT began to decrease to 12 % and aPTT was prolonged to 172 sec. In addition, factor V-inhibitor (1156 BU), lupus anticoagulants and anticardiolipin-antibodies could be detected. Factor V-activity could not be normalised even in dilutions up to 640 (Factor V-antigen was not reduced). The patient developed massive muscle and visceral bleedings and died from cardiovascular faihire. Thesc two cases demonstrate that treatment with ciprofloxacin can trigger the development of factor V inhibitor and antiphospholipid antibodies. In both cases ciprofloxacin was the only new taken medication. In one case there was a transient occurrence and normalisation after terminating treatment with ciprofloxavin. The other case ended with massive muscle and visceral bleedings and cardiovasculare failure. Probally the association of treatment with ciprofloxacin and development of factor V-inhibitor and antiphospholipid-antibodies is diagnosed to rarely. These two cases emphasize the necessity of a meticulous clatification of a prolonged PTT and drops of PT during and after treatinent with ciprofloxacin.
AUTOIMMUNE ANTIPHOSPHOLIPID ANTIBODIES IMPAIR THE aPL AFFECTS PROTEIN C ACTIVATION IN VIVO INHIBITION OF ACTIVATED FACTOR X BY S.S. Pierangeli, X. Liu, R.G. Espinola, E.N, Harnis PROTEIN Z / PROTEIN Z DEPENDENT PROTEASE INHIBITOR Morehouse School ofMedicine, Atlanta, GA. USA L.C. Kordich2, G.J. R.R Forastiero', M.E. Martinuzzol, Broze3 Antiphospholipid (aPL) antibodies are associated with thrombosis, have 'Favaloro University, 2School ofExact and Natural Sciences, University of been shown to be thrombogenic in mice and to affect various phospholipid- Buenos Aires, Argentina, 3Washington University School ofMedicine, dependent hemostatic reactions in vitro. Studics have shown significant St. Louis, MO, USA inhibition of conversion of protein C into activated protein C (APC) by aPL in vitro, indicating that aPL may impair an important anticoagulant PZ is the cofactor for the inhibition of factor Xa (FXa) by ZPI in thc mechanism. However, no studies have examined whether aPL antibodies presence of phospholipids (PL) and calcium (Ca). Recent data suggests that affect the activation of protein C in vivo. If confinned in vivo, aPL- defects in PZ/ZPI may increase the thrombotic risk in subjects carrying mediated inhibition of prolein C activation might explain thrombogenic other prothrombotic traits. Low plasma PZ levels were found in association mechanisms of aPL. This study addressed that question and examined how with aPL. In this study we tested whether aPL could impair the FXa infusions of APC affect aPL-mediated enhanced thrombus formation in a inhibition by PZ/ZPI. Purified IgGs from 25 aPL patients (19 with and 6 mouse model of venous thrombosis. CDI mice in groups of eighteen were without APS) wcre evaluated in a FXa inhibition assay usinig purified either injected i.p. twice with 500 jig of hunman affinity purified aPL or IgG components. PZ (2.5ug/ml), ZPI (4ug/ml), Hepes/saline/BSA (lmg/ml) control (lgG-NHS). Seventy-two hours aftcr the first injection, the surgical buffer, and Ca (5mM) with or without IgGs (1.75mg/ml) and with or procedure to expose the femoral vein of treated and control mice and to without beta2-glycoprotein I (b2GPI) (100ug/mI) were constructed at RT. examine dynamics of thrombus formation was carried out in anesthetized Then PL (2OuM) and FXa (250ng/ml) were added. At 5 min, samples were animals. Mean thrombus area was determined as described (Circ 1999; diluted 25-fold and assayed for remaining FXa activity (rFXa). Without 99:1997) and a sample of blood was obtained to detennine anticardiolipin b2GPI and IgGs, rFXa was 2%. Adding b2GPI to the mixture, rPXa was (aCL) levels by ELISA. Subsequently, one group of nine mice treated with slightly higher (10%). IgGs from 2 normal subjects did not inhibit the effect aPL and one treated with IgG-NHS were infused intravenously with 0.1 ml FXa and did not enhance the effect of b2GPI (rEXa, 10% and APC (16ILg/ml) in stenle saline-dextrose solution, while the animals still of PZ/ZPI on under anesthesia. The remaining animals were infused with 0.1 ml sterile 11%). However, IgGs from most patients with aPL decreased the FXa saline solution. Thirty minutes later, a second thrombus was induced in each inhibition by PZ/ZPI when b2GPI is present (rFXa, 10-29% n=9, 30-50% animal and its arca (in micrometes square) determined and compared to n=l 1, and >50% n=5). Only one IgG-aPL showed a significant effect on values obtained before the i.v. infusions. Mean aCL titer of aPI-treated PZ/ZPI in the absence of b2GPI (rFXa 19%). These data suggest that some animals was 92 +/- 28 GPL units. Mean trhombus size in aPL-treated mice aPL impair the FXa inactivation by PZ/ZPI and thus may contribute to was significantly larger when compared to IgG-NHS-treated mice (4719+/- thrombosis in the APS. Ongoing studies may provide firther information. 1609 vs 1162 =/- 454 ). Infusions of APC decreased significantly the thrombus size of of aPL-treated mice (from 4719 +/- 1609 to 624 +/- 392, 88% decrease). As expected, a decrease in thrombus size in IgG-NHS- treated mice was also observed after the infusion of APC (from 1162 +/- 454 to 470 +/-252), but the decrease was significantly lower than in aPL-treated mice. Neither thrombus size nor aCL titers were affected by saline infusions in aPL or IgG-NHS-treated mice. The data clearly indicate for the first time, that aPL antibodies affect protein C activation in vivo and this may explain, at least in part their procoagulant and thrombogenic properties.
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Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 10Ctliir anixQs anAhqtdpdhrloldes P*rPWFset*m 585 CHARACTERIZATION OF IMMUNOGLOBULIN-BOUND ANTICARDIOLIPIN ANTIBODIES IN SERUM SAMPLES OF 'NATIVE'BETA2GLYCOPROTEIN I FROM PLASMA (ISOLATED PATIENTS WITH CHRONIC HEPAT[TIS C INFECTION UNDER MILD CONDITIONS) Z. Mondher, Z. Yusr, L. Lilia, A. Ilhem, S. Maryarn, M. Sondes B.L. Myones', S. Majumdarl, L.I. Fedorova', J.D. Morrisett', A.Y. Volgin' Immunology Department, La Rabta, Tunis, Tunisia 'Department ofPediatrics, 2Department ofMedicine, Baylor College of Infectious agents have been implicated in the induction of antiphospholipid USA Medicine, Houston, TX, antibodies particularly chronic hepatitis C virus which was linked to extra 'Native' beta2GPI was previously isolated from plasma under mild liepatic autoimmune phenomena. Objectives: to study the prevalence of conditions by sequential purification on heparin-Sepharose (hepS), high- antiphospholipid antibodies in chronic hepatitis C infection and the binding hepS, Sephacryl-S200, high-binding hepS. (1) Different binding relationship between viral infection in the induction of such antibodies capacity was noted for anti-beta2GPI or aPL compared with perchloric acid Methods: 82 patients with chronic hepatitis C and 100 healthy controls were isolated beta2GPI ('acid'), (2) pH <4 or >9 converted 'native' to 'acid' investigated for the presence of IgG anticardiolipin antibodies by an ELISA beta2GPI, (3) a 205kDa band was noted in 'native' isolates and identified by method. Rheumatoid factors (RF) were detected by latex technique. IgG immunoblot with anti-beta2GPI and anti-IgG (both 50kDa/205kDa with sera levels were measured by minineph. Chi square test were used for anti-beta2GPI). Purity/physical properties of newly isolated 'native' statistical analysis. Results: IgG anticardiolipin antibodies were detected in beta2GPI were established by SDS-PAGE, UV-spectroscopy, circular patients with chronic hepatitis C in 32% of cases against 5% in healthy dichroism (CD). 'Native' beta2GPI, in 2-chloroethanol, developed 75% controls. 91% ofpatients had RF and 43% had high levels ofIgGinthe sera. alpha-helicity by CD compared to 25% for 'acid' beta2GPI coincident with There was no significant differences between anticardiolipin antibodies and rapid formation of insoluble precipitates resistant to resolubilization with either RF or IgG sera levels in chronic hepatitis C patients. Conclusion: acid/base, urea, methanol, DMSO, sonication. Formation rate was examined despite the polyclonal B lymphocyte activation observed in hepatitis C from 4-26 degreesC with changes noted below 16 degreesC. Immunoppt infection there was no correlation between Anticardiolipin antibodies and with either anti-beta2GPI or anti-Ig yielded both bands from 'native' isolates the hyperglobulinemia However, Anticardiolipin antibodies seems to be but anti-beta2GPI yielded primarily the 205kDa band from whole plasma of associated with chronic hepatitis C and could play a potential role in fibrosis controls and asymptomatic patients (high titer aCL). The 205kDa complex progression and liver disease in these patients. indicates a 11 stoichiometry (beta2GPI: antibody). Binding specificities of IgG isolated from this complex by sequential hepS, S200, proteinA have yet to be determined. ELISAs were developed to assess plasma before/after exposure to acid and showed increased percentage of IgG-free beta2GPI after acid treatment. This finding was diminished in plasma from patients with aPL events with an overall trend toward increased percentage of IgG- free beta2GPI before and with little change after acid treatment. Ig-bound beta2GPI from plasma may imply an influence of aPL on PL:protein interaction.
THE MEASUREMENT OF ANTI-CHOLESTEROL ANTIBODY SERU1 LEVELS IN PATIENTS DURING PATTERNS OF THE PLATELET FUNCTION STATUS IN THE CHRONIC HEPATIIS-C IFN-ALPHA PATIENTS WITH APS TREATMENT Biro', A. Horvath', L. Varga2, E. A. Csepregi3, V.G. Karepovl, Y. Levy, Y. Shocnfcld A. Nemesanszky3, K. David4, G. Tolvaj4, E. Ibranyi5, L. Telegdy5, A. Par6, L. Romics', 'Medical Center 'M.E. TE.O.R, Tel Aviv, 2Department ofMedicine B, Karadil, A. Koles7, T. Balint7, M. Horanyi2, Z. Prohaszka', G. Fust' Chaim Sheba Medical Center, Tel Hashomer, Israel 13rd Department ofMedicine, Semmelweis University, 2National Institute of Background: An evaluation of the platelet function status (PFS) is important Haematology and Immunology, 3BudaiIrgalmas Hospital, 41st Department oJ in patients with APS because of the high frequency of the thromboembolic Medicine, Central Hospital, Ministry ofthe Interior, St. Laszlo Hospital, Budap events and bleeding complications in these patients. Objective: The aim of 61st Department ofMedicine, University Medical School, Pecs, 7Schering-Plou} the study was to determine of the PFS in patients with APS under shear Budapest, Hungary stress conditions using the platelet fimction analyzer (USA). Methods: AIMS: Previously we detected more than 3 times higher anti-cholesterol antib Citrated whole blood samples drawn from 31 patients with APS and from 55 (ACHA) levels in HIV positive patients, than in healthy individuals, but this 1 healthy persons (controls) were studied using PFS analyzer. The principle of significantly decreased during HAART. In our present study we examined if tl the is based on the occlusion of apertures in membranes coated with method findings could be detected also in patients with C (CHC). collagen and different platelet agonists (epinephrine -EPI, and adenosine-5'- chronic hepatitis calculated the correlation between the ACHA levels and the C5b-9 complen diphosphate- ADP). The normal range for the EPI closure time (EPICT) test activation product, too. was 80-170 sec., and for ADP closure time (ADPCT) test was 71-114 sec. PATIENTS AND METHODS: 39 patients with CHC, who were treated with I The results of the PES analysis were defined as "normal" or "abnormal" alpha -2b (Schering-Plough) 5 MU daily for 6 weeks, followed by 5 MU 3 ti (prolonged or shortened closure time) and compared between the group of weekly. The serum levels of ACHA and complement products were measured i patients with APS and the control group, using a chi-square test. Results: Of ELISA, the HCV RNA with PCR before and 3 month after the beginninE the 31 patients with APS, only 3 patients (9.7%) had a normal both (EPICT interferon-alpha-2b therapy. and ADPCT) tests. In the control group the normal tests were registered in RESULTS: 1.) The ACHA level was significantly (p=0.0062) higher in the pati 27 persons (49.1%), (p<0.01). Prolonged both (EPICT and ADPCT) tests (med(IQ range)(40(24-69) AU/ml) before the treatment, than in the 52 hea were found in 7 patients with APS (22.6%) and in 2 healthy persons (3.6%), subjects (26(20-35) AU/ml) with the same age. In the 26 responder patients (p Wu Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 loth ktwaird ngess nArtiohdidArtiboes PostseUi1s 586 THE ROLE OF ANTIPHOSPHOLIPID ANTIBODY IN HEART ANTI-HIGH DENSITY LIPOPROTEIN AND ANTI-B2GPI DISEASES ANTIBODIES INVERSELY CORRELATE WITH PARAOXONASE ACTIVITY AND TOTAL ANTIOXID.NT CAPACITY IN A. Rafinejad, M. Hajiloui, S. Rafiei PATIENTS WITH SLE AND APS Tehran University ofMedical Sciences, Iran J. Delgado Alves', P.R.J. Ames2, S. Donohue', J. Noorouz-Zadeh', It has been claimed that athersclerosis may be an autoimmune disorder. To C.T. Ravirajan', D.A. Isenberg' find a clue for this problem, we looked for the role of autoantibodies such as 'Centre or Rheumatology, University College ofLondon, 2Department of as a agent main Antiphospholipid, causative in heart attack ;the Rheumatology, Whittington Hospital, London, UK consequences of atherosclerosis. Methods: 122 patients with various basic heart problem(50 female and 72 male)were tested for Anti Cardiolipin Atherosclerosis has been recognised as a significant feature of SLE and Antibody(ACA) in their sera 60 nonnal cases were exarnined APS. However, the mechanisms by which auto-antibodies might relate to simultaneously. The patients had some signs of chest pain and were elected atherogenesis are still unknown. This study aims to detennine the for angiography. They had different type of vessels disorder; ranging from prevalence of anti-high density lipoprotein (anti-HDL) antibodies and to one vessel to three vessels disease and normal vessel. To estimate the ACA establish a possible relationship between anti-HDL, anticardiolipin ELISA technique was employed. Results: 41 patient showed a high level of antibodies (anti-CL), anti-Beta2-GPl and paraoxonase (PON) activity in ACA (21 female, 17 male). There was a significant statistical difference patients with SLE and PAPS. Thirty-two SLE patients, 36 PAPS and 20 age between and control groups. Conclusions: AntiCardiolipin Antibody and and sex matched healthy controls were considered. Serum levels of anti-CL, other kind of antiphospholipid antibodies may have a critical role in anti-Beta2-GP I (IgG and IgM) and IgG anti-HDL were assessed by ELISA, inducing heart vascular complications. We suggest that; evaluation of ACA PON activity by nitrophenol formation and Total Anti-oxidant Capacity in individuals who are at risk group for heart attack may be helpful in (TAC) by chemiliuminescence. HDL (Total, 2 and 3) was reduced in patients prognostic and prevention of it. with SLE when compared to controls (p<0.001, p<0.001 and p<0.004, respectively). Patients with SLE and PAPS had higher titres of anti-HDL antibodies (p<0.005 and p<0.05 respectively) and lower PON activity (p<0.001 and p<0.0009, respectively), than controls. In the SLE population PON activity inversely correlated with IgG anti-HDL titres (r--0.48, p<0.OOS) whereas in the PAPS population, IgG anti-Beta2-GPI was the only independent predictor of PON activity (r--0.483 p<0.003). In the SLE group, anti-HDL and PON activity were inversely correlated with TAC (r=-0.40, p<0.02 and r-0.43, p<0.02, respectively). Anti-HDL and anti- Beta2-GP1 IgG antibodies are associated with reduced PON activity in patients with SLE and PAPS. Paraoxonase prevents low-density lipoprotein oxidation, hence its anti-atherogenic effects. The reported interactions may, therefore, be relevant to the development of atherosclerosis in these conditions. CORRELATION BETWEEN P2-GLYCOPROTEIN I AND CRP INCREASED LEVELS OF OXIDIZED-LDL IN SLE: DETE(C1ION LEVELS IN INFLAMMATION BY ELISA USING AN ANTI-B2GP1 MONOCLONAL ANTIBODY K. Dier', C. K. E. L. Lopez' F. Lin', C. Feighery2, j. Jackson' D. Lopez', Fink', Kobayashi2. Matsuura2, 'Department ofBiological Sciences, Dublin Institute of Technology, 'Corgenix, Inc., Westminster, CO. USA, 2Okayama University School of 2Departmene of Immunology, St James'Hospital, Dublin, Ireland Medicine, Okayama, Japan with f32-glycoprotein I (,B2GPI) has been identified as a phospholipid-binding Increased incidence of premature atherosclerosis in patients protein thought to be involved in the regulation of the clotting cascade. autoimmune diseases (SLE) has been recognized. One mechanism may be related to increased oxidation of LDL-cholesterol (oxiLDL). The specific Several studies have described as a negative acute phase protein. 02GPI interaction of oxiLDL with B2GPI provides additional evidence for the However, little is known about the actual levels of during the very 02GPI participation of antiphospholpid autoantibodies in the development of course of the inflammation process. In this study, we measured levels P2GPI atheroesclerosis. Serum levels of oxiLDL were measured by ELISA in SLE (using a sandwich ELISA) in 144 patients with elevation of CRP above 10 patients, with and without APS. 24 healthy blood donors served as controls. mg/L. was also quantified in 269 healthy individuals to act as P2GPI A mouse monoclonal antibody, specific for human B2GPI (WB-CAL-1) controls (mean ± SD = 176.1 ± 45.7 FLg/mL) Separating the samples in when coated onto microwells binds to the B2GPI molecule of of CRP were moderately elevated quartiles, it was observed that when levels oxiLDL/B2GPI complexes present in patient samples. Bound oxiLDL was (betwecn 10.1 and 22.8 mg/L), the levels of P2GPI showed a modest detected by adding biotinylated mouse monoclonal antibody to human Apo elevation (mean ± SD = 186.5 ± 53.5 pg/mL). However, at higher levels of B100 (1D2) and HRP-streptavidin. Results were expressed in ug/mI against CRP (above 105.1 mg/L), P2GPI was found to be significantly reduced a standard curve prepared with Cu++ oxidized human LDL and exogenous (mean ± SD = 133.0 ± 57.7 jLg/mL) (P<0.000l). The overall Spearman rank B2GPI. Cut-off was determined at 120 ug/ml (mean + 3 SD of healthy correlation coefficient was -0.2837 (P= 0.0006). Similar results were found controls). The mean oxiLDL levels, prevalence (% positives), and p value in both genders. 32GPI therefore appears to act as an acute phase reactant versus controls were: SLE+APS (n=40), 177.8 ug/ml (SD 317.2), 28% when CRP is moderately elevated. As the CRP increases to very high positive, p<0.05; SLE Control (n=32), 131.4 ug/ml (SD 224.5), 19% concentrations, ,2GPI levels fall due to reduced synthesis (with P2GPI positive, p<0.05, Healthy Controls (n=24), 34.5 ug/mI (SD 28.2), 0% behaving then as a negative acute phase protein), or increased consumption, positive. The mean oxiLDL level and prevalence of positives in the SLE or a combination of both. As a potential natural anticoagulant, [B2GPI might with APS group were higher than SLE without APS, but this difference did have an important role to play during the course of inflammation which is not reach statistical significance (p=0.487). These results suggest not only characterised by increased risk of thrombosis. Although several studies have an increased oxidation of LDL, but also the association of B2GPI with may be a mechanism for the accelerated described ,2GPI deficiency as not a risk factor for thrombosis, it is oxiLDL in SLE patients. This in autoimmune patients. tempting, with regard to our findings, to consider the decrease in J2GPI development of atherosclerosis levels as a potential risk factor for thrombosis when associated with an inflammatory condition. WuE Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 10hCitmiald Oioss an AMOicipdArIoces pftpsatcmo 587 ANTIBODIES AGAINST BETA 2 GLYCOPROTEIN1 AND HOMOCYSTEINE (HCY) AND THROMBOTIC RISK IN PATIENTS CARDIOVASCULAR DISEASES IN RHEUMATOID ARTHRITIS WITH ANTIPHOSPHOLIPID ANTIBODIES PATIENTS M. M. Martiniuzzo, R.R. Forastiero, G. S. Cerrato, M. L. Iglesias Varela, Y. P. Adamczuk, L. Carreras A. Durazinskal, M. Szmyrkal, B. Jazwiec2, J. Szechinskil 0. 'Department ofRheumatology, 2Department ofHaematology, Favaloro University, Buenos Aires, Argentina Wroclaw University ofMedicine, Wroclaw, Poland Hyperhomocysteinemia (HHcy) is associated with venous and arterial Objective: Cardiovascular diseases are the main cause of the morbidity and thrombosis (VT and AT). Few reports have searched the influence of HHcy mortality in rheumatoid arthritis (RA) patients. The objective of the study in the thrombotic risk of the APS. In this study we evaluated Hcy levels in was to determine, whether the association exists between the levels of anti- 127 consecutive patients with aPL (49 male, 78 female). There were 79 patients with criteria for definite APS (48 with VT and 28 with AT). Ninety- 2 glycoprotein I (anti- 2GP1) antibodies and cardiovascular diseases in RA patients. Study group consisted of 44 RA patients (40 female, mean age- six sex- and age-matched healthy subjects were recruited as normal controls Hcy were by ELISA and results expressed as 59 y., mean disease duration - 9 years), out of which 14 had the history of (NC). Plasma levels measured cardiovascular accident (myocardial infarction, stroke, DVT), 15 had mean (SD) in micromolar. Higher levels of Hcy were found in aPL patients atheromatosis but with no CVA, and 15 had RA without the evidence of [14.3 (7.7)] as compared to NC[11.5 (8.2)], pO0.01.APS but not non-APS cardiovascular disease. Methods: Serum levels of anti- 2 glycoprotein 1 patients had higher levels of Hey [14.4 (7.0)] than NC, p<0.05. Similar IgG and IgM antibodies were detected using commercial ELISA test kits results were obtained considering patients with history of thrombosis [14.6 (EUROIMMUN, Gemiany). The association of anti-2 GP1 antibodies (6.9)] as compared to NC, p<0.05. Prevalence of HHcy (cut-off value=18.2, levels and disease activity, RF seropositivity, serum lipids, steroid or other 95th percentile of NC) was higher in patients with aPL (24.4%) than NC treatment and other factors like concomittant infections were analysed. (5.5%), p<0.00l. This prevalence was slightly higher in patients with Results: IgG anti- 2GPI levels in sera from study group were below the definite APS (26.6%) and particularly AT (32.1%), p ROMOCYSTEINE LEVELS AND ANTIPHOSPHOLIPID ANTICARDIOLIPIN ANTIBODY TITER AND PLASMA ANTIBODIES: IS THERE AN ASSOCIATION? HOMOCYSTEINE LEVEL ARE INDEPENDENT PREDICTORS OF CAROTID INTIMA MEDIA THICKNESS IN PRIMARY APS P. Casais, M.F. Alberto, M J Salviu, L.C. Gennari, S.S. Meschengieser, A.N. Blanco, M.A. Lazzari P.R.J. Ames', A. Margarita2, J. Delgado Alvez5, C. Tommasino4, L. Iannaccone3, V. Brancaccio3 Institute ofHematological Research, National Academy ofMedicine, Buenos Aires, Argentina 'Department ofRheumatology, North Middlesex Hospital, London, UK, 2Angiology and3Coagulation Unit, Cardarelli Hospital and 'Chemical site thrombosis or obstetric Patients with venous, arterial, and unusual Pathology, S. Gennaro Hospital, Naples, Italy, 'Bloomsbury Centrefor complications with homocysteine levels higher than 15 urnol per liter were Rheumatology, University College London, UK evaluated to determine if there was an association between homocysteine levels and antiphospholipid antibodies (APA, i.e: lupus anticoagulant, IgG IgG aCL is considered a risk factor for atherosclerosis in patients with and IgM anticardiolipin antibodies) in patients referred to our Department systemic lupus erythematosus. We evaluated whether IgG anticardiolipin for thrombophilic screening after at least 1 month from the acute event. antibody (aCL) and traditional atherosclerotic risk factors bore any Methylentetrahydrofolate reductase (MTHFR) thermolabile variant relationship with the intimna media thickness (IMT) of carotid arteries of genotype was recorded. A hundred and fifty-seven patients were included. patients with idiopathic antiphospholipid antibodies (aPL). IMT was carotid Median age was 46 years old, 75 (47.8 percent) were female. Clinical determined by high resolution soniography at the comimon carotid, manifestations were (n): venous (47), arterial (57), both (8), unusual site bifurcation and internal carotid in 42 (13M, 29F, mean age 31±10 years) thrombosis fie: retinal vein occlusion, mesenteric or cerebral thrombosis] aPL subjects, 29 with primary antiphospholipid syndrome and 13 with (23) and recurrent fetal losses (12). Median (SD) homocysteine level was: persistence of aPL in the absence of any underlying disorder. The following 22.98 (12.67). APA were detected in 50.3 percent of study population. A were measured: plasma fibrinogen (FNG), von Willebrand factor (vWF), normal MTHFR genotype was found in 23 percent (26/114), heterozygous plasminogen activator inhibitor (PAI), homocysteine (HC), total cholesterol (HDL of termolabile variant was found in 43 percent and 34 percent were (CHO), triglycerides (TG), high density and low density lipoprotein homozygous. Median homocysteine levels were significantly higher in and LDL), platelet numbers and aCL of IgG and IgM isotype. Univariate homozygous patients (p=0.0000) than in heterozygous or normal. There was analysis revealed associations between IMT of the common carotids, carotid statistical significant difference in homocysteine levels between normal and bifurcation and internal carotid and age (p Wus Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1CShktaaicid Ongess an io iidArtibocIes Postsaims 588 FLUVASTATIN INHIBITS INFLAMMATORY AND IMT were age, anti-oxPAPC, DBP and ECLAM >2 at the time of diagnosis THROMBOGENIC PROPERTIES OF ANTIPHOSPHOLIPID (R 0.706). Predictors of plaque were: age, anti-oxPAPC and Tpred. ANTIBODIES IN AN IN VIVO MODEL Conclusions: Anti-oxPAPC antibodies seem to play a minor role as predisposing factors to atherosclerosis in SLE, probably due to the effect of D.E. Ferrara', X. Liu', R.G. Espinolal, K. Labat', P.L. Meroni2, prednisone and other features related to its intake E.N. Harris', S.S. Pierangelil 'Morehouse School ofMedicine, Atlanta, GA, USA, 2University ofMilano, Italy Studies have slhown that antiphospholipid (aPL) antibodies enhance the adhesion of leukocytes to ECs and upregulate the expression of adhesion A POSSIBLE PREDICTIVE ROLE FOR VASCULAR EVENTS OF molecules on ECs in vitro. The induction of a pro-inflammatory and APLA AND PLATELET ACTIVATING FACTOR-ACETYL proadhesive phenotype on ECs is thought to be an important pathogenic HYDROLAZE(PAF-AH) IN PATIENTS WITH CORONARY mechanism Our group has recently shown that aPL activate ECs in vitro and DISEASE in vivo and this correlates with a thrombogenic effect in a mouse model. New lipophilic statins such as fluvastatin have been shown to have anti- C.M. Tanaseanul, E. Moldovean2, T. Kosaka3, S. Tanaseanu4, inflammatory effects, to reduce levels of tissue factor on ECs treated with M. Popescul TNF-alpha, etc. More recently, investigators have shown that fluvastatin 'Hospital Pantelimon, Bucharest, Romania, 2Babes Institute, Bucharest, decreases the aPL-induced adhesion of leukocytes and the expression of Romania, 30saka University, Japan, 'Colentina Hospital, Bucharest, adhesion molecules on ECs in vitro. The aim of this study was to determine Romania whether fluvastatin has an effect on aPL-induced adhesion of leukocytes and Background: Inflammatory reactions within coronary atherosclerotic thrombosis in an in vivo mouse model. Four groups of CD1 male mice (approx weight 30 g) were fed with fluvastatin (10 micromolar) or with plaques are determinants of the clinical course of patients with coronary placebo for 15 days. Each one of these groups was further divided to receive artery disease. APLA are related to active endothelial lesions and PAF-AH either IgG with aPL activity (IgG-APS) or IgG control (2 infusions). is considered to reflect the ongoing inflammatory process in patients with Seventy two hours after the first injections with Igs, two surgical procedures atherosclerosis. Objective: The study was done to determine the activity of were carried out in each mice in order to examine the adhesion of leukocytes PAF-AH and the frequency of APLA in patients with coronary artery on ECs using a mouse model of microcirculation in cremaster muscle and disease, and their relations with the severity of coronary syndromes the thrombus dynamics in a dissected femoral vein, as described (Circ Res: Methods: The study group included 45 patients with ST depression at effort 2001; 88: 245-250). Levels of soluble ICAM-1 and aCL titers were also treadmill test (ETT) and 45 age-, sex-, risk factors-matched controls, determined in the sera of the treated and control mice. Thrombus size was selected from 190 patients with coronary artery disease. Patients underwent significantly increased in mice treated with IgG-APS and fed with placebo clinical examination, ECG, ultrasonographic assessment of atherosclerotic when compared to mice treated with IgG-NHS and fed with placebo (4,959 score, ETF, coronarography, routine laboratory tests, determination of +/- 1142 micrometer square vs 556 +/- 407 micrometer square). Thrombus APLA, PAF-AH assay. Patients were followed up for a mean period of 8 size was significantly reduced when lgG-APS-treated mice were fed with month. Results: From 45 patients with positive ETT 37 patients presented fluvastatin (1,235 +/- 345 micrometer square). Similarly, the number of APLA (82%) and increased activity of PAF-AH (21p), higher ultrasound adhering leukocytes to ECs (in the cremaster muscle) was significantly atherosclerotic score compared with controls. During the follow up period larger in mice infused with IgG-APS and fed with placebo, when compared coronary thrombotic events and need for revascularization was greater in to micc treated with IgG-NHS and fed with placcbo (8.8 +/- 1.6 vs 4.2 +/- patients with APLA and increased PAF-AH activity (27p) than in controls. 1.3). Fluvastatin decreased the IgG-APS effect on adhesion of leukocytes to Conclusions: APLA and increased PAF-AH activity are present in patients ECs significantly (to 4.6 +/-2.8). The mean aCL titer in the sera of mice with active coronary disease. Both parameters shown a positive correlation infused with IgG-APS was 101.8 +/- 26.4 GPL units and 111.4 +/- 22.4 for with atherosclerotic lesions score, other inflammatory markers, ETT, animals fed with fluvastatin or placebo, respectively. Fluvastatin also coronarography. seemed to decrease levels of sICAM-l in the sera of the mice. Fluvastatin reversed the proadhesive, pro-inflammatory and thrombogenic properties of aPL in an in vivo mouse model. The data provide a rationale for using statins as an additional therapeutic tool in APS. CLINICAL SIGNIFICANCE OF SERUM OXIDIZED LDL IN PATIENTS WITH CHRONIC RENAL DISEASE J. Kasaharal, K. Kobayashi%, Y. Yamasakil, T. Yasudal, T. Koike2, H. Makino', E. Matsuura' DO ANTI-B2GPI AND ANTI-oxPAPC ANTIBODIES HAVE ANY ROLE IN THE DEVELOPMENT OF ATHEROSCLEROSIS IN SLE? 'Okayama University, Graduate School of Medicine and Dentistry, Okayama, 2Hokkaido University, Graduate School ofMedicine, Sapporo, A. Dorial, A. Ghirardellol, R. We2, M. Puatol, D. Bragagnolo', B. Gilburd3, Japan E:. Favaretto' S. Zampieri', Y. Sherer3, P. Paulettol, Pk. (iambari', Y. Shoenfeld3 We recently established a novel enzyme-linked immunosorbent assay for oxidized low-density lipoprotein (oxLDL), based on the 'Speciality Laboratories, Santa Monica, CA, (ELISA) 'University ofPadova, Italy, principle of the interaction among oxLDL, beta2-glycoprotein I (beta2-GPI), USA, 'Chaim Sheba Medical Center, Tel Hashomer, Israel and mouse anti-beta2-GPI autoantibody (auto-Abs, namely, WB-CAL-1). In The aim of our study was to evaluate whether anti-beta 2 glycoprotein I the present study, serum oxLDL was measured in patients with chronic renal (b2GPI) and anti-oxidized Palmitoyl Arachidonoyl Phosphocholine diseases and its clinical significance was evaluated. Serum oxLDL level in (oxPAPC) may represent immunologic risk factors for premature the patients chronic renal failure (CRF) and chronic nephritis (CN), as well atherosclerosis in SLE. Methods: 78 patients with SLE were evaluated (67 as those with antiphospholipid synidrome (APS), systemnic lupus female and 11 male, mean age 32 years). At the time of the study, serum erythematosus (SLE), and diabetes mellitus (DM), was significantly higher anti-oxPAPC (unit) and anti-b2GPI (SGU) were tested by EIA. All patients than that in normal subjects. oxLDL positive did not associated with underwent a duplex carotid sonography (7-10 MHZ, ASPEN ADVANCED positives of total cholesterol (T-C), triglyceride, cholesterol in high-density - ACUSON). Intima-media thickness (IMT) was measured. Plaque lipoprotein (HDL-C), LDL-cholsterol (LDL-C), serum creatinine, and (IMT>l.4 mm), mean IMT (m-IMT) and maximum IMT (M-IMT) were urinary protein excretion (U-Pro) in the patients with CRF. In contrast, considered. We performed utiivariate analysis and a stepwise multiple linear oxLDL positive was significantly associated with low HDL-C (< 50 mg/dl) and logistic regression analysis. Results: We observed carotid plaque in 13 (p=0.001), with renal dysfinction (e.g., 24hr-creatinine clearance, <70 patients (16.6%). M-IMT and m-IMT were (mean±SD) 0.04±0.04 and ml/min) (p=0. 035), with high U-Pro (>lg/day) (p=0.010), with high dietary 0.05+0.01, respectively. In patients with plaque vs those without plaque protein intake (>l.Og/kg/day) (p=0.01 1) and high sodium chloride intake autoantibody mean (±SD) levels were: anti-oxPAPC 7.80±14.91 vs (>7g/day) (p=0.007), in the CN patients. Malondialdehyde-modified LDL 4.64±5.83; anti-b2GPI 14.53+19.70 vs 19.70±38.17. Patients with plaque (MDA-LDL) was significantly associated with T-C and with LDL-C in compared with those without plaque were significantly older (p<0.001), had either group but did not with any renal functions. These results indicate that higher SBP (p=0.004) as well as DBP (p=0.002) and had taken a higher total serum oxLDL, modified by in oxidative stresses such as dietary factors, prednisone dose (Tpred; p=0.001). In decreasing order of statistical weight, might be a novel risk factor and diagnostic marker for development of predictors of higher M-IMT were age, anti-oxPAPC, Tpred, renal chronic renal diseases, rather than MDA-LDL. involvement (RI) at the time of diagnosis (R 0.652); predictors of higher m- Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lOUlrteidiw (is onioFtipdAllnles PwterFin6~~~~~~~~~ --l -t 2 589 ENDOTHELIAL CELL DYSFUNCTION IN ANNTIPHOSPHOLIPID aPLS ACTIVATE NUCLEAR FACTOR KAPPA B ON SYNDROME (APS) ENDOTHELIAL CELLS M.O. Borghil, F. Raschil, C. Testonil, C. Luzzanal, M. Cugno2, D. Man2, S.S. Pierangelil, J.L. THall', R.G. Espinolal, D.E. Ferrara', K. Roye-Green2, A. Tincani3, P.L, Meronil E.N. Harris' 'Department ofInternal Medicine, University ofMilan, IRCCS Istituto 'Morehouse School ofMedicine, Atlanta, GA, USA, 2University Of The West Auxologico Italiano, 2IRCCS Ospedale Maggiore, Milan, 3Spedali Civili, Indies, Kingston, Jamaica Brescia, Italy Studies have shown that antiphospholipid antibodies (aPL) can activate Background: Anti-p2glycoprotein (i2GPI) antibodies lead to both endothelial cells (ECs) in vitro, as demonstrated by increased expression of thrombotic diathesis and obstetrical manifestations so representing adhesion molecules: ICAM-1, VCAM-1 nad E-selectin and this correlated pathogenic markers for APS. The interaction of anti- P2GPI antibodies with with EC activation and enhanced thrombosis in vivo. The intracellular mechanism(s) involved in this process are not understood. The nuclear ,2GPI adhered on the endothelial cell surface induces in vitro a pro- inflammatory and pro-coaugulant phenotype, with increased adhesion factor kappa B is a multisubunit transcription factor whose activation leads molecule expression and tissue factor activity. Comparable effects have to upregulation of gene transcription of adhesion molecules on ECs and to initiation of various signal transduction pathways. The present study vivo in an animal model of pinched-induced thrombosis. been found in the question whether aPL-induced EC activation requires Contraslinig results have been reported in few studies regarding the increase addressed activation of NFkappa B. NFkappa B activity was determined by transiently of plasma levels of several endothelial activation parameters in APS transfecting bovine pulmonary aortic cells (CPAE) utilizing a patients: a) soluble adhesion molecules (sADMs), b) soluble endothelial Thrombomodulin (sTM), c) vonWillebrand factor (vWF). Objective of this lipid base approach (Effectene, Qiagen). Twenty-four hours after transfection, cells were treated with TNF-alpha (40ng/ml), with aPL study was to investigate whether serum markers of endothelial cell damage could be detectable in APS patients. Methods. We studied patients with both antibodies (100 microg/ml) or with control IgG(IgG-NHS) (100 microg/ml) for 5 hours. Luciferase activity and EGFP fluorcscence wcrc measured and primary (PAPS) and SLE-associated APS (SAPS), compared to sex-, age- expressed in arbitrary units as luciferase/EGFP fluorescence. The expression and blood group-matched healthy individuals. Of note, SAPS patients did of E-sel, VCAM-1 and ICAM-1 on aPL-treated ECs was determined by not display disease activity (SELENA-SLEDALI score < 2). Plasma levels of was treated with TNF- sADMs: sICAM-1, sVCAM-l, sE-selectin), sTM, vWF and tissue ELISA. NF-kappa B significantly higher in CPAE alpha or with aPL (16052 +i- 1677 and 18574 +/- 1328 luciferase act/eGFP Plasminogen Activator (tPA-Ag) were measured by commercial (sAdM, units) when compared to EC treated with IgG-NHS or medium alone or home-made (vWF, tPA-Ag) capture ELISAs. Results. Plasma sTM) (9108+/- 594 and 7891 +/- 628 luciferase act/eGFP units, respectively). sADMs and sTM did not significantly differ between APS patients and healthy donors. A statistically significant increase of both vWF and tPA-Ag APL-treated confluent monolayers of ECs expressed 16-fold more VCAM- 1, 2 fold more ICAM-1 and 28 fold more E-sel after 4 hours exposure, when plasma levels was observed in the patients in comparison with healthy compared to IgG-NHS-treated cells. aPL-induced upregulation of VCAM-1, controls (176.7+88.8 vs 115.6+32.3 U/ml for vWF; 10.2+5.7 vs 6.5+3.9 ICAM-1 and E-sel on ECs is associated with significant activation of NF- U/ml for tPA-Ag). Patients with PAPS or SAPS showed comparable plasma levels of vWF and tPA-Ag. Conclusions. These data indicate for the first kappa B. Other signal transduction pathways may be turned on and this would lead to the expression of adhesion molecules and to the activation of time a strong in vivo increase of endothelial activation/damage parameters ECs upon exposure with aPL. Understanding the intracellular mechanism(s) (vWF and PA-Ag) in APS patients. It should be pointed out that an of aPL-mediated EC activation may help establishing new modalities to endothelial damage related to the systemic vasculitic process should be rule revert the prothrombotic state observed in APS patients. out in SAPS patients since all of the included patients displayed inactive disease (SELENA-SLEDAI score < 2). Our' results indicate that an endothelial dysfunction does occur in vivo in APS patients further stressing the pathogeniic role ofthe interaction of anti-phospholipid antibodies and the endothelium. On the other hanid the lack of any abnormality in sADM and PROBING ANTIPHOSPHOLIPID - MEDIATED THROMBOSIS: sTM levels is in line with the absence of a clear inflammatory process in the THE INTERPLAY BETWEEN ANTICARDIOLIPIN ANTIBODIES APS vasculopathy in contrast with the other forms of systemic autoimmune AND ENDOTHELIAL CELLS vasculititis. S.S. Pierangeli, A.E. Gharavi, E.N. Harris Morehouse School ofMedicine, Atlanta, GA, USA IS ANTI ENDOTHELIAL CELL ANTIBODY A SIGN OF The association of antiphospholipid (aPL) antibodies with thrombosis in ANTIPHOSPHOLIPIDSYNDROME IN IDDM ANGIOPATHY? patients with Antiphospholipid Syndrome (APS) is well documented in S. Rafiei, A. Rafinejad, N. Mosafa humans an in animal studies. However, the mechanisms by which aPL antibodies induce thrombosis is the subject of much current study. It has Tehran University ofMedical Sciences, Iran been suggested that aPL may activate endothelial cells (Ecs) thus creating a Is Anti Endothelial Cell Antibody a sign of Antiphospholipidsyndrome in hypercoagulable state that precedes and contributes to thrombosis in patients IDDM Angiopathy? Alterations in immunological finctions play a role both with APS. Several studies have shown that aPL upregulate ECs adhesion in the pathogenesis and secondary complication of IDDM (Insulin molecules (CAMs): intercellular cell adhesion molecule -1 (ICAM-I), Dependent Diabetes Mellitus). Anti phosopholipid antibodies are involved vascular cell adhesion molecule-1 (VCAM-1) and E-selectin (E-sel) or in many manifestations of autoimmune disorders and Anti Endothelial induce tissue factor (TF) in monocytes in vitro. Similarly, the incubation of Antibody (AEA) is known to be a kind of anti phospholipid antibodies It EC with antibodies reacting with B2GPI has been shown to induce EC may be the cause of angiopathy in IDDM. the goal of this study was to look activation with concomitant upregulation of CAMs, IL-6 production and for AEA in Diabetic patients with and without any vascular complications alteration of prostaglandin metabolism. Our group has shown that aPL- .as a co factor for tissue damage we evaluated the CIC (circulating Immune mediated upregulation of adhesion molecules on ECs correlates with an Complexes ) and C3,C4. Methods: 52 subjects suffering from IDDM in increased adhesion of leukocytes to endothelium in the microcirculation of various stagcs of the disease were sampled. 18 nonrnal conltrols were tested mouse cremaster muscle, an indication of EC activation in vivo, and with as well. Indirect Immunofluorescent technique (IFT) was employed to enhanced thrombosis in vivo. In another series of studies, investigators have evaluate the AEA. Rat pancreas frozen sections were used as antigen CIC shown that upregulation of expression of adhesion molecules by some and complement components such C3, C4 were measured by conventional murine monoclonal anti-B2glycoprotein I (anti-B2GPI) antibodies methods. Results :16 patients had AEA. 10 AEA positive patients had correlated with fetal resorption in mice in vivo. More recently, one study vascular complications and six were without any obvious angiopathy. The showed that the anti hypercholesterolemic drug fluvastatin inhibited the CIC level inIDDM in comparison to control shown a significant rises the aPL-mediated enhanced adhesion of monocytes to ECs in vitro. complement components were decreased in patients co ordinate with the Unipublished data from our laboratories indicate that fluvastatin also presence of AEA and retinopathy. Using BemoliTest, the probability for a reverses thrombus formation and activation of EC induced by aPL in an in patient with high CIC levels to demonstrate AEA is 59% and probability for vivo mouse model. As additional support for the hypothesis that aPL the AEA positive cases to show vascular complications is 64%. Conclusion: antibodies activate ECs and may create an hypercoagulable state in APS antiendothelial antibody may be classified as antiphospholipisd antibodies. patients, two recent studies indicated that levels of soluble ICAM-1 and High CIC plus AEA may be the cauative reason for vascular complications VCAM-1 were significantly increased in the plasma of patients with APS in IDD\M. The presence of AEA accompanied by vascular angiopathy in our and recurrent thrombosis. Furthermore, studies utilizing knockout mice and IDDM patients may suggest the idea that IDDM angiopathies would be a specific monoclonal anti-VCAM-l antibodies have demonstrated that sign ofanti-phospholipid syndrome. expression ofICAM-1, P-selectin, E-selectin and VCAM-1 are important in in vivo aPL-mediated thrombosis and EC activation in mice. Recent data bLPs Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lOh kterici Qigassptipd AtibocIes Poft Fsat(s response to TNF was significantly greater in the statin-pretreated cells than 590 suggests that aPL antibodies also induce expression of TF not only in in the control cells. Pretreatment of the cells with mevalonate reversed the monocytes but in ECs. Hence, the interference of aPL with the TF co-stimulatory effect of statins. Our rcsults imply that statins cooperate mechanism may be another important mechanism by which these antibodies rather than interfere with inflammatory cytokines in enhancing the response create a hypercoagulable state and prone patients to thrombosis. of endothelial cells. Specifically, how aPL alters EC activation state and the molecular and intracellular mechanisms involved have not yet been defined. APL may interact with specific cell surface receptors (proteins and/or lipids) induce signals that have consequences downstream, and that ultimately will result in upregulation of cell surface proteins (i.e. CAMs and TF) and that our recently subsequently induce EC activation. In regard, group CERIVASTATIN NEITHER INHIBITS THROMBIN GENERATION showed that aPL-mediated upregulation of adhesion molecules in ECs is NOR DECREASES MARKERS OF INFLAMMATION IN PATIENTS preceded by activation of the nuclear factor kappa B (NFKB). Other WITH APS intracellular mechanisms triggered by aPL are not completely understood Iwaniec and are the subject of current investigation. In conclusion, studies suggest J. Musial, J. Swadzba, D. Rys, W. Sydor, T. that activation of ECs by aPL is an important mechanism that may precede Jagiellonian University, Department ofMedicine, Cracow, Poland thrombus formation in patients with APS. Hence, the interplay between aPL antibodies and ECs is important in the pathogenesis of thrombosis in APS. Beneficial effects of statins in cardiovascular diseases may depend in part on their pleiotropic effects (e.g. anti-inflammatory and antithrombotic). Such combined actions ascribed to statins may be also of potential value in the treatment of anfiphospholipid syndrome. For this reason we treated 45 patients with antiphospholipid syndrome with cerivastatin (0.4 mg per day CIRCULATING ENDOTHELIAL CELLS ARE A NOVEL MARKER for 4 weeks) or placebo. Active drug was administered to 32 subjects while OF ENDOTHELIAL DAMAGE IN ANCA-ASSOCIATED SMALL- placebo to 13 of them (2:1 design). Before and after treatment we measured VESSEL VASCULITIS serum levels of lipid parameters as well as the level of CRP, TNF alpha, assessed Haller, IL6, IL2 soluble receptor, VICAM I and tissue factor (TF). We A. Woywodt, F. Streiber, M. Schroeder, K. de Groot, H. also thrombin generation in blood emerging from skin incisions by M. Haubitz measuring levels of thrombin-antithrombin complexes. While serum total Division ofNephrology, HannoverAfedical School, Hannover, Germany and LDL cholesterol decreased promptly after cerivastatin treatment, there were no differences in serum of inflammation and endothelial Background: Damage to endothelial cells is a salient feature of a variety of markers damage. Acenocoumarol profoundly thrombin generation in such as systemic vasculitis. Yet a simple and specific test to impaired disorders, with oral anticoagulants but indicate ongoing endothelial injury is not readily available at present. clotting blood in subgroup of patients treated had no influence on parameters of thrombin formation in both Objectives: The aim of this study was to evaluate the number of circulating cerivastatin treated and non-treated subjects. conclusion: cerivastatin exerts no endothelial cells as a marker of vascular damage, using ANCA-associated In measurable antithrombotic and anti-inflammatory action in subjects with vasculitis as a model. Methods: Patients with active (n-18) and inactive We speculate that the lack of plciotropic (n=20) ANCA-associated vasculitis, and 20 healthy controls were studied antiphospholipid syndrome. effects of this particular may be responsible for its unfavorable profile of prospectively. Endothelial cells were isolated with anti-CD-146 coated statin action which led to the drug withdrawal from the market, which took place immunomagnetic DynabeadsTM, von Willebrand factor, CD 31 and Ulex just completion of our study. europaeus lectin I staining was perfonned in parallel. The cell phenotype after was also studied with tissue factor immunocytochemistry, TUNEL staining, culture, and annexin/propidium iodide staining. Results: Large numbers of circulating endothelial cells were detected in patients with active vasculitis (20-5700 cells/ml, median 136 cells/ml). Patients in remission had moderately but still significantly elevated cell numbers (0-52 cells/ml, median 16 cells/ml) compared to hcalthy controls (0-20 cells/ml, median 6 PREVALENCE OF ANTICARDIOLIPIN AND ANTUI-BETA2- cells/mI). Cell numbers declined substantially during successful immuno- GLYCOPROTEIN I ANTIBODIES IN GREEK PATIENTS WITH suppressive treatment. Preliminary studies of the cells indicated a necrotic STL tissue-factor positive phenotype. Conclusion: The number of circulating V. Galanopoulou2, G. Papaioannou3, A. endothelial cells is a novel marker of endothelial damage in vasculitis. Our A. Pavlitoul, Giannakoul, A. D. I. data, we believe, suggest that this marker should also be evaluated in M. Speletas3, R. Zilidoul, Fleval, Papadopoulou4, Griveas4, patients with other endothelial disorders, including the antiphospholipid G. Sakellariou4 antibody syndrome. 'Departnment ofImmunology, 2bepartment ofRheumatology, 3Department of Heacmatology, 'Department ofNephrology, Papageorgiou General Hospital, Thessaloniki, Greece Objectives: To determine the prevalence of serum anticardiolipin (aCL) and anti-beta 2-glycoprotein I (anti- beta2GPI) antibodies in Greek patients with EFFECT OF STATINS ON ENDOTHELIAL ACTIVATION BY systemic lupus erythematosus (SLE). Correlation with the clinical TUMOR NECROSIS FACTOR manifestations of the antiphospholipid syndrome (APS) was also studied. S. Dunoyer-Geindre, Y. Dimitrova, E.K. Kruithof, G. Reber, Patients and Methods: We studied 72 patients with systemic lupus P. de Moerloose erythematosus; 50 SLE patients without nephritis (group I) and 22 SLE patients with nephritis (group II), 21 patients with primary antiphospholipid 'University Hospital ofGeneva, Division ofAngiology and Hemostasis, syndrome-PAPS (group III) and 30 healthy volunteers (group IV). IgG and Geneva, Switzerland IgM, aCL and anti- beta2GPI antibodies were measured using enzyme The clinical potential of 3-hydroxy-3-methylglutaryl coenzyme A reductase linked immunosorbent assay (ELISA). Serum tests for antinuclear inhibitors (statins) to reduce adverse events in patients with cardiovascular antibodies (ANA) and antibodies to dsDNA were also done. Results. None disease has been well established. Originally the beneficial effect of statins of the autoantibodies were determined in group IV. In the patients with was thought to be due to inhibition of cholesterol biosynthesis and lowering PAPS the prevalence of aCL and anti-beta2-GPI antibodies was 20/21 of blood cholesterol. An alternative explanation for the therapeutic effect of (95%). The presence of aCL antibodies was 18/50 (36%) for the group I and statins has been proposed, i.e. interference with activation of endothelial 7/22 (32%) for group IT, while the presence of anti-beta2GPI was 20/50 cells by inflammatory cytokines. The latter is still a subject of controversy. (40%) and 7/22 (32%) respectively. The prevalence of aCL antibodies only The presenit study was undertaken to investigate to what extent pretreatment at the whole of SLE patients was 25/72 (35%) while the prevalence of aCL of human umbilical cord derived endothelial cells (HUVEC) by different and anti- beta2GPI was 37/72 (51%, p<0.001)). Conclusion: These results statins (simvastatin and pravastatin) had a direct effect on adhesion show no significant differences in the prevalence of aCL and anti-beta2GPI molecule expression or on the adhesion molecule response to tumor necrosis antibodies between Greek SLE patients without nephritis and SLE patients factor (TNF). The expression at the cell surface of the adhesion molecules with nephritis. The deternination of anti- beta2GPI antibodies in addition to the ICAM-1, VCAM-1 and E-selectin was analyzed by flow cytometry and by aCL antibodies might be very useful to increase the sensitivity of an ELISA technique on fixed cells. We observed that adhesion molecule diagnosis of APS in patients with SLE. expression by HUVEC pretreated with the statins was similar to that by control HUVEC. In contrast the expression of adhesion molecules in Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1CUhlanbinfia OrescnbCo,A1ipudbIocies PmtmPms8tadom 591 ANTIPHOSPHOLIPID ANTIBODIES ARE NATURAL THE INFLUENCE OF CALCIUM IONS ON THE DETECTION OF AUTOANTIBODIES PLUS HISTONES ANTI-ANNEXIN V ANTIBODIES D.N. Abakushin, [A.M. Poverennr N. Gaspersic, T. Kveder, B. Rozman, B. Bozic Medical Radiological Research Center, Obninsk, Russia University Medical Center, Ljubljana, Slovenia in a calcium Introduction: The main feature of natural autoantibodies (aAbs) are their Annexin V (ANXV), a protein which binds to phospholipids polyspecificity and ability to interact with various charged cellular dependent manner, is one of the possible protein cofactors for molecules. We supposed that histones released after cell destruction take anitiphospholipid antibodies. We studied the influence of calcium added to diluent and washing buffers on the detection of anti-anncxin V antibodies part in formation ofpolyspecificity of natural aAbs and interactions of aAbs with various phospholipids. Methods: Antigen-binding characteristic of (aANXV) by our in-house ELISA. High binding microtiter plates were coated with human affinity purified ANXV, followed consequently by immunoglobulin (Ig) preparations and tested blood serum were deternined blocking with bovine serum albumin (BSA), incubation of tested sera, by ELISA. Isolation of Ig-histone complexes was carried out by gel addition of alkaline-phosphatase labelled anti-human IgG, the substrate and filtration on Sephacryl S-200. Results: We deternined that natural aAbs, measurement of absorbance at 405 nm. All washing steps were reacting with histones, are widely distributed and they can be detected in final 0.1% Tween in either a) buffered saline (PBS), pool of normal human Igs and blood serum of healthy individuals. Initial perfoined using phosphate preparations do not interact with phospholipids. It is known that for b) Tris buffered saline (TBS) or c) TBS with added calcium (TBS-Ca). Corresponding diluent buffers all contained BSA at 0.1% concentration. interaction of some anticardiolipin Abs with cardiolipin needs a cofactor - Five selected sera with increased levels of aANXV by the PBS/ELISA were beta2-glycoprotein I. Blood serum of healthy donors after incubation with sera retained their histones acquired the properties like APS and SLE serum. Preincubation of retested by the TBS/ELISA and TBS-Ca /ELISA. 3/5 level of positivity when tested by TBS/ELISA and TBS-Ca /ELISA. But histones with Igs was accompanied by forming the complex of Ig-histone the in two cases a marked decrease in absorbance values was noted in the which can be identified by electrophoresis in SDS-polyacrylamide gel by presence of calcium: Serum 1: PBS vs. TBS vs. TBS-Ca: 1.162, 1.184 and presence of different amount of histones absolutely in all fractions of Igs, reacting with phospholipids. Besides, obtained fractions interact not only 0.108, respectively; Serum 2: PBS vs. TBS vs. TBS-Ca: 0.318, 0.301 and with cardiolipin and phosphatidylcholine, but also with another 0.133, respectively. It is known that ANXV undergoes confonnational changes upon binding with calcium, likely resulting in different epitopical phospholipids, such as phosphatidylserine and phosphatidylinositol. Distribution of activity by fractions to all anion phospholipids is presentation. Our results show that the presence of calcium in ELISA abolishes the binding of certain Further approximately identical and differs from the same to zwitterionic detection procedure aANXV. studies should elucidate which - calcium sensitive, calcium resistant or phospholipid - phosphatidylcholine. Conclusion: We supposed that histones both aANXV are of clinical can perform the function of cofactor in polyspecific interactions of natural relevance. aAbs with phospholipids. LUPUS ANTICOAGULANT QUANTIFICATION IN HEPARINIZED LINK BETWEEN ANTI-CD36 ANTIBODIES AND THROMBOSIS PATIENTS USING ADDITION OF POLYBRENE IN THE APS E.M. Jacobsen2, E.J. Trettenes1, F.O. Strekerud', F. Wislofla, Y. Pelogri, G. Corrato, R. Forastiero, M. Maitinluzzo, L.O. Carreras U. Abildgaard' Favaloro Foundation, Buenos Aires, Argentina University Hospital, Oslo, Norway 'Aker University Hospital, 2Ullevaal CD36 is a membrane glycoprotein expressed by platelets and many other In the lupus ratio (LR) test, patient plasma is mixed with normal plasma cells. There are scarce evidence suggesting the involvement of (1:1) and the APTT is performed at low and high cephalin concentrations autoantibodies against CD36 (anti-CD36) in the pathogenesis of APS- with ellagic acid as activator. The ratio APTT (low cephalin): APTT (high related thrombosis. Therefore, we evaluated the prevalence of anti-CD36 in cephalin) is calculated for both patient plasma and normnal plasma. LR is 62 patients with autoimmune aPL but without SLE. There were 38 (F/M defined as the ratio between these two ratios. Although LR is highly 17/21, median age 43 years) aPL patients with thrombosis (27 venous, 14 specific, heparin may result in false positive results. Various basic arterial) and 24 (F/M 15/9, median age 35 years) without thrombosis. polyaminoacids, protamine and polybrene neutralized heparin but also, to Nineteen (FlM 10/9, median age 44 years) patients with thrombosis (16 varying degree, changed the APTT of plasma not containing heparin. When venous, 3 arterial) served as aPL(-) control group and 54 (F/M 28/26, polybrene was added to test plasma (1/50 volume) to a final concentration of median age 40 years) healthy subjects as normal group. Anti-CD36 were 8 ug/ml, LR was not significantly changed in plasma from 30 patients (11 measured by an indirect MAIPA assay. OD above the normal 98th patients with LA and 19 others), irrespective of preaddition of heparin (1.0 percentile were considered positive results. We found that 24.2% (15/62) U/ml). Further experiments showed that this amount of polybrene aPL patients but only 1.8% (1/54) nonnal controls had anti-CD36 neutralized 1.5 U/ml unfractioned (UF) heparin and 1.7 U/ml LMW heparin. (p=0.0003). As compared to the normal group, the prevalence of anti-CD36 than Finally, LR was tested in plasma from 11 patients with previously diagnosed in aPL patients with thrombosis was slightly higher (26.3%, p=0.0005) But the difference between LA. Plasma was taken from these patients before and 5 minutes after in those without thrombosis (20.8%, p<0.01). intravenous injection of 5000 IE UF heparin. The heparin concentration in both aPL groups did not reach statistical significance. Anti-CD36 in aPL postheparin plasma ranged 0.5-1.2 U/ml. APTTs were performed after patients with thrombosis were also significantly more prevalent than in addition of polybrene, and LR was calculated. Mean LR in preheparin thrombosis aPL(-) ones (26.3% vs 0%, p=0.02). Interestingly, anti-CD36 plasma was 1.51 (range 1.05-2.02), in preheparin plasma with polybrene seems to be more frequent in aPL patients with recurrent thrombosis (7/19, 1.59 (range 1.11-2.07), and in postheparin plasma with polybrene 1.48 36.8%) than in those without recurrence (3/19, 15.8%). In conclusion, the (range 1.08-1.92). The mean difference between regular LR and LR with presence of anti-CD36 is highly prevalent in patients with autoimmune aPL, polybrene was 6.3% (SD 4.08) in preheparin samples, and 1.4% (SD 5.16) with a trend to be more frequent in patients with recurrent episodes of in postheparin samples. The SD of repeated determinations of LR was 0.04. thrombosis. Thus, the LR was not significantly changed by heparin and polybrene. Polybrene added to test plasma to a final concentration of 8 ug/ml permits the quantification of LR irrespective of heparin 0.0 - 1.5 U/ml. Wus Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 ktmari Omg a Artitopldid Artibocles PoslmPm6seUi 592 ANTIPHOSPHATIDYLATHANOLAMINE ANTIBODIES IN SLE ASSOCIATION OF AUTOANTIBODIES AGAINST THE PATIENTS AND PRIMARY APS PATIENTS PHOSPHATIDYLSERINE-PROTHROMBIN COMPLEX WITH APS T.M. Reshetnyakl, B. Wojciechowska2, Y. Zabek2, E.S. Mach', D. Pascual-Saleedol, E.M. Delgado', F. Arribas', M.V. Cuesta2, Z.S. Alekberoval, V.A. Nassonoval R. Madero3, M.F.G. Iglesias4, A. Gil Aguado4 'Institute ofRheumatology ofRAMS, Moscow, Russia, 2Institute of 'Department ofImmunology, 2Department ofHematology, 3Department of Rheumatology, Warsaw, Poland Statistics, 4DepartmentofInternal Medicine, 5APS Study Cooperative La Paz Madrid, Aim: To detect antiphosphatidylathanolamine antibodies (aPE) as Group University Hospital, Spain antiphospholipid antibodies (aPL) in 127 patients we investigated the sera of Introduction: Antiphospholipid antibodies (aPL) in APS patients have of these patients for different aPL and estimate the association with clinical broad range of specificities. One of the aPL are directed to prothrombin, a manifestations. Methods: Patients with definite systemic lupus phospholipid-binding protein. The clinical significance of antiprothrombin erythematosus (SLE) (n=99; 52 of them had aPL-associated features) and 38 antibodies has not been established because the nature of the exposed pts with primary antiphospholipid syndrome (PAPS) were investigated for antigen(s) seems to be heterogeneous. Aims of the study: To clarify which different aPL: anticardiolipin antibodies (IgG-, IgM-aCL), antibodies to one of the methods currently in use to detect antiprotrombin antibodies has phosphatidilserine (IgG-aPS), to inositol (aPI), to glycerol (aPG), to more clinical significance in APS. Methods: IgG and IgM antiprothrombin phosphatydilcholine (aPCh) and aPE by ELISA. Lupus anticoagulant (LA) antibodies were detected by ELISA usiIIg irradiated plates coated with was detected in all studied patients. Results: LA was found to have 63 purified human prothrombin (aPT), and non irradiated plates previously studied pts and different aPL had 104 pts. The 48 of 127 patients were coated with phosphatidylserine (aPS/PT). LAC was detennined by the positive for aPE and 10 of 48 patients had aPE alone without other aPL or APTT/KCT and a neutralization test with phospholipid. Standardized LA. We have found significant association of aPE with anemia, transverse ELISA methods were used to detect ab2GPI and aCL antibodies. Results: 1) myelitis and livedo reticularis (p<0,03). Conclusion: Patients whose clinical Prevalence of aPS/PT and aPT was 20.4% and 21.3% respectively. 2) There symptoms suggested antiphospholipid syndrome but whose sera are was a positive correlation between aPS/PT and aCL (ro=0,38 p<0.001 for negative for other antiphospholipid antibodies should be investigated for IgG, ro=0.158 p=0.046 for IgM), ab2GPI (ro=0.436 p<0.001 for IgG, aPE. ro=0.213 p=0.008 for IgM) and IgG aPT levels (ro=0.533 p<0.001). 3) LAC and/or venous thrombosis association with aPS/PT was higher, with statistical significance, than with aPT. 4) The difference in specificity and sensitivity between aPS/PT and aPT for LAC in APS patients was statistically significant, and it was nearly significant for venous thrombosis (CI 95% disjointed). Conclusion: The aPS/PT showed a strong correlation with the presence of LAC and venous thrombosis. We suggest that aPS/PT are a better marker for APS than aPT. THE VALUE OF ANTI-B2-GPI IN SLE PATIENTS AUTOANTIBODIES AGAINST PHOSPHOLIPID PROFILE IN PATIENTS WITH SLE S Sokolovic', M. Seremet2 C. M. Drahosova 'Clinic for Heart and Rheumatic Diseases, 2Institutefor Microbiology, Andrys, University Clinical Center Sarajevo, Bosnia Herzegovina 'Department of Clinical Immunology, Charles University, University Hradec Kralove, Czech Introduction: B2-GPI serve as glycoprotein in a serum that inhibits the Hospital, Republic conversion of prothrombin into thrombin. In SLE patients this glycoprotein Aim of Study: To evaluate the presence of autoantibodies against various becomes antigen and it is not known why that happens. Antiphospholipid phospholipids different fiom anticardiolipin in SLE patients with the serum antibody is then binded to B2-GP1 causing the blood clots. Objective: 'Ihe positivity for anti - cardiolipin antibodies. Patients and Methods: Totally, 24 purpose of this study is to determine the clinical importance of anti-B2-GPl anti-cardiolipin IgG or IgM (Dialab, Austria) positive serum samples from in patients suffering from definite SLE and to follow up them in terms of patients with SLE were analyzed for presence the other antiphospholipid eventual thrombosis and other manifestations of antiphospholipid syndrome. antibodies using TromboCombo lgG/IgM kit (Orgentec, Germany): Material and Methods, Total of 33 patients SLE patients, majority females, cardiolipin, phosphatidyl inositol, phosphatidyl serine, phosphatidic acid, B- have been included in this study. The other group served as a control one. 2-GP I and mixture of all phospholipid with and without B-2-GP I. Results: The open clinical randomized controlled study has been designed. Comparison of Dialab versus Orgentec kits for detennination of anti- Results: The preliminary results obtained in this study showed the presence cardiolipin antibodies. There was a consensus in 21 cases (87.5%) in isotype of anti-B2-GPI in about twenty percent of patient's sera correlataing with IgG and in 19 cases (79%) in IgM. Relationship between various antigens: the clinical manifestations. A great majority were female where prevalence All of IgG anti - cardiolipin positive samples were positive in the other tests is higher than in men. Conclusion: This investigation is the first one of this with other phospholipid antigens. The same situation was obtained in IgM type in our region. Our results shows higher sensitivity rnther than isotype antibodies. The only exceptions were antibodies against specificity which help us in searching for more thrombotic events and for phosphatidyl inositol and B-2-GP I in IgM isotype, where the agreernent more clear future therapeutic perspectives. 96% was reached. The antibody response to mixture of phospholipid antigens with B-2-GP I is significantly higher (IgG p<0.05, IgM p<0.001) in comparison to response to mixture of phospholipids without B-2-GP I. Conclusions: The significance of B-2-GP I cofactor is more pronounced in IgM isotype anti-phospholipid antibodies in comparison to IgG isotype. There is a good agreement in the positivity of anti-phospholipid antibodies using various molecular targets. It seems likely that the determination of anti-phospholipid antibodies directed to single target is enough to identify patients with the presence of these autoantibodies. This approach is cost- benefit. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 flirttmdiG Qress nonmhdipd rioies PoPresta CLINICAL SIGNIFICANCE OF ANTI- ANTIPHOSPHOLIPID (HUGHES) SYNDROME IN PATIENTS 593 PHOSPHATIDYLSERINE/PROTHROMBIN (PS/PT) COMPLEX WITH INFLAMMATORY BOWEL DISEASES ANTIBODIES IN PATIENTS WITH SLE A.E. Dorofeyev J. Kaburakil, M. Kuwana2, Y. Ikeda2 Medical University, Donetsk, Ukraine 'TEPCO (Tokyo Electric Power Company) Hospital, 2Keio University, Antibodies to phospholipids are important factors in pathogenesis of Shinjuku-ku, Tokyo, Japan inflammatoxy bowel disease (IBD). High level of these antibodies resulted The diversity of so-called antiphospholipid antibodies has been found in ift development of extra bowel manifestations of IBD and antiphospholipid patients with SLE and related disorders. In this study, wc clarificd thc (Hughes) syndrome (APS). 172 patients with IBD were observed. Among clinical significance of anti-phosphatidylserine/prothrombin (PS/PT) them 148 patients suffered from ulcerative colitis (UC), and 24 had Crohn's complex antibodies in patients with SLE and lupus-like disease. The study disease (CD) of large bowel. Extra bowel manifestations, vasculitis of skin population consisted of 125 patients with SLE and lupus-like disease. IgG (piodermia gangrenosa and livedo reticularis), central nerve system, anti-PS/PT complex antibodies were examined by ELISA. IgG anti-PS/PT thrombosis and microthrombosis, myocarditis symptoms were study in all complex antibodies were found in 38 (30%) out of 125 patients, whereas patients. Antibodies to cardiolipine, lupus anticoagulant were studied in IBD beta2-GPI dependent anticardiolipin antibodies were positivc in 20% and patients. Clinically antiphospholipid syndrome was found in 21 (12,2%) lupus anticoagulants were detected in 15%. Arterial or venous thrombosis patients with IBD. The frequency of antiphospholipid syndrome in patients was present in 53% of patients with IgG anti-PS/PT complex antibodies, with ulcerative colitis and Crohn's disease was equal, but clinical symptoms although the frequency of thrombosis was 17% in patients without these were different. In CD patients the symptoms of hypercoagulations with antibodies. This differecnce was significant (p<0.005). The frequency of vasculitis, endothelial dysfunction brain thrombosis and thrombocytopenia pregnancy morbidity, which are adopted as clinical criteria in the were found. In 4 (2.7%) patients with UC piodermia gangrenosa, in 4 preliminary classification for antiphospholipid syndrome, 'Sapporo criteria', (2.7%) patients livedo reticularis, in 5 (3.4%) patients cerebrovasculitis, in 6 was significantly (p<0.05) higher in patients with IgG anti-PS/PT complex (4.0%) venathrombosis and in 12(8.1%) patients microthrombosis of colon antibodies (23%) than in those without IgG anti-PS/PT complex antibodies mucosa vessels, in 7 (4.7%) patients with UC myocarditis were found. Only (8%). Moreover, IgG anti-PS/PT complex antibodies were positive in 6 out in patients who had 3 ore more signs of APS serological markers were of 20 patients with thrombosis or pregnancy morbidity who did not have found. But for releasing APS is necessary not only antiphospholipid beta2-GPI dependent anticardiolipin antibodies or lupus anticoagulants, and antibodies. The inflammatory cytokines levels (TNF-alfa and IL-6) were did not satisfy 'Sapporo criteria. The above findings indicate that IgG anti- higher more than 2.3 times in patients with clinical symptoms of APS than PS/PT complex antibodies are useful in the diagnosis of antiphospholipid in patients without APS. syndrome. APS AND ITS CLINICAL MANIFESTATIONS APL AND HEPARIN INDUCED THROMBOCYTOPENIA TYPE II (HIT) IN CHRONIC THROMBOEMBOLIC PULMONARY Z. Macejoval, J. Szilasiova2, D. Trejball HYPERTENSION 'Department of Internal Medicine, 2Department OfNeurology, 3Department C. Colorio, R. Forastiero, M. Martinuzzo, A. Macchia, G. Pombo, ofInternal Medicine, Medical Faculty, Safarik University, Kosice, D. Puente, A. Rossi, L. Carreras Slovak Republic, Favaloro Foundation, Buenos Aires, Argentina Objectives: To assess the most frequent organ manifestations in patients with anticardiolipin antibodies (aCLA). Methods: In a retrospective study of Chronic thromboembolic pulmonary hypertension (CTE-PH) is an patients with positive anticardiolipin antibodies (aCLA) hospitalized at the infrequent cause of pulmonary hypertension. It develops in 0.1 to 0.5% of Intcmal and Neurological Clinic between 1995 and 2001 we analyzcd the patients who survive after an acute venous thromboembolic event in which correlation between these antibodies and thrombotic organ manifestations. thrombus does not solve completely. In our recently published series of For assessment of aCLA we used the sandwich method of enzyme patients with diagnosis of CTE-PH, an underlying acquired or congenital immunoanalysis (ELISA) in Cebecauer modification, for assessment of hypercoagulable state was detected in almost 70% of CTE-PH patients. The lupus anticoagulant (LA) we used neutralization and coagulation tests. presence of antiphospholipid antibodies (aPL), including lupus Results: Anticardiolipin antibodies (aCLA) positivity in suspected anticoagulant and/or anticardiolipin antibodies, was the abnormality most thromboembolic events was found in 79 patients (20,2%). 20 patients (25%) frequently found. In the present study we also evaluated the presence of had apparent thrombotic event. The mean age of patients was 47,4+/-10,0 heparin-platelet factor 4 induced antibodies (HPIA) which have been years. Positivity of aCLA isotypes IgG and IgM was recorded in 4 patients, frequently found in association with aPL. The population included 29 (12 12 patients had only the IgG isotype. All sub-classes of aCLA male, 17 female) consecutive patients fulfilling criteria for CTE-PH. 27 (IgG,IgM,IgA) were found in two patients. Four patients had positive LA. patients had dyspnea Im or IV at diagnosis. Most of them (93%) had a Thrombocytopenia was confirmed in 14 patients. The following thrombotic previous history of venous thromboembolism in other territories. Results: events were recorded in patients: thrombosis ofthe vein oflower limbs (9x), 19/29 (65.5%) patients disclosed abnormal results in the screening for thrombosis of the vein of upper limbs (2x), thrombosis of v.cava inferior thrombophilia. aPL were detected in 13/29 (44.8%). There were 7 patients (lx), thrombosis of abdominal aorta (lx), thrombosis of a. mesenterica (lx), with antibodies anti-beta2 glycoprotein I and 5 with antiprothrombin myocardial infarction (2x), thromboembolism of a. pulmonalis (3x), cerebral antibodies. HPIA were assayed in 25/29 patients. HPIA were positive in infarction (8x), cerebrovascular attack type TIA (lx), monocular visual 4/13 aPL patients and in 3112 patients without aPL. Three of the 4 aPL disorder caused by thrombosis of the central retinal vein (lx), thrombosis of patients with HPIA (75%) developed HIT, while none of the 3 aPL(-) the cerebral sinuses (lx), transient global amnesia (lx), reccurent fetal patients with 1PIA developed HIT. Conclusion: HPIA were found in about losses (2x). Conclusion: Thrombotic events, particullary in younger patients 30% of our patients with CTE-PH treated with heparin but in this population who suffer at the same time from thrombocytopenia, should be an indication the development of HIT was associated with HPIA only in aPL patients. for examination of aCLA antibodies as their permanent presence increases the risk offurther thromboembolic attacks. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1Ohhtamicrd Ongmss ainsldip d Artibocies postwPvmsfttms 594 ANTICARDIOLIPIN ANTIBODIES AND STILL DISEASE DIFFUSE PULMONARY HEMORRHAGE IN APS: IMPORTANCE OF ANTICOAGULATION C.C. Belizna, M.A. Pistorius, B. Planchon M. Silverberg, D. Erkan, M.D. Lockshin CHU Hotel Dieu Nantes, France 'Hospitalfor Special Surgery, Weill Medical College of Cornell University, Anticardiolipin antibodies have been demonstrated in a large spectrum of New USA autoimmune disease. Several authors reported anticardiolipin antibodies in York, juvenil idiopathic arthritis (Clin Exp Rheumatol 1999;17: 375-80). We Background: Pulmonary hemorrhage occurs in patients with APS and present a 23 years old patient admitted with fever, arthralgia, headache and catastrophic APS. The prognosis is usually poor, the underlying mechanism temporary rash, diagnosed one week later as Still disease. The patient was is not clear, and the management of anticoagulation is challenging. dismissed with non steroidian antiinflammatory drugs. Two days later he Methods: We report a patient who presented with pulmonary hemorrhage was admitted with dyspnoea and pulmonary scintigraphy concluded at and was found to have pulmonary capillaritis in the presence of high titer pulmonary thromboembolism. Doppler ultrasound revealed left tibial and anticardiolipin antibodies (aCL). He improved with immunosuppressives right femoral thrombosis and lupus anticoagulant and anticardiolipin and plasmapheresis but developed right subclavian vein thrombosis 10 days antibodies were found. This is the first case of detection of anticardiolipin after the initial event. Results: A 69-year-old man with a history of antibodies in Still disease patients. Our report suggest that prophilactic myocardial infarction (4 yea prior) and chronic rcnal insufficiency antithrombotic treatment has to be considered when confronted with Still presented with fever and worsening shortness of breath. He was found to disease patients. have von Willebrand's disease, and a positive lupus anticoagulant and high- titer aCL (IgG>80U) during a preoperative evaluation three years prior to admission. He had no histoTy of bleeding or clotting, and was on aspirin. On admission, he was febrile, in respiratory distress, and had bilateral rhonchi. Ilis admission hematocrit of 34% dropped to 26% and platelet count of 118x109,L fell to 27 x109AL rapidly. A chest x-ray showed bilateral alveolar infiltrates. Cultures of broncoalveolar lavage were negative. On the fifth hospital day he was intubated for worsening respiratory distress. An open lung biopsy showed diffuse intra-alveolar hemorrhage with rare foci of neutrophilic capillaritis and hemosiderin laden macrophages. No granulomas were seen and stains for immunoglobulins and complement were negative. Larger pulmonary vessels showed no evidence of vasculitis. The patient was treated with pulse corticosteroids, cyclophosphamide, plasmapharesis, and intravenous immunoglobulin. The clinical symptoms resolved over the next three days and the patient was successfully extubated. Antinuclear antibodies, c-ANCA, p-ANCA, and anti-GBM were negative. All plasma factor levels were normal. Two days after extubation the patient had right upper extremity swelling. Ultrasound revealed right subclavian vein thrombosis (not in the area of central line placement). The patient was treated with heparin and warfarin without further bleeding or thrombotic events. Conclusion: Pulmonary capillaritis without microthrombosis can occur in APS and suggest a pathophysiology. Despite hemorrhage, these patients are still at risk for thrombosis. Anticoagulation should be started as soon as the bleeding is controlled in antiphospholipid antibody positive patients with pulmonary hemorrhage. SOLUBLE ADHESION MOLECULES IN SLE PATIENTS WITH/WITHOUT ANTIPHOSPHOLIPID ANTIBODIES D.V. Reshetnyak', E.N. Alexandrova2, A.A. Novikov2, T.M. Reshetnyak, N.G. Kljukvinal, M.U. Samsonov2, E.L. Nassonov3 HAEMOLYTIC-UREMIC SYNDROME IN A PATIENT WITH APS 'Moscow Medical Academy, 2Cardiology Research Center, 31nstitute of AND AUTOIMMUNE HEPATITIS Rheumatology, Moscow, Russia E. Nomikoul, G. Theodossiades', P. Raphailidis2, K. Maragos1, M. Bellial, Aim: To study clinical importance of soluble cellular adhesion molecules S. Katsudas2, E. Digenopouloul, l. Kontopoulou-Grival, S. Dourakis2 (sCAM) in patients with primary and secondary antiphospholipid syndrome (APS) Methods: levels of soluble P-selectin (sP-selectin), sE-selectin and '1stRegional Transfusion and Haemophilia Center, 22ndAcademic sVCAM-I were determined by enzyme-linked immunosorbent assay (R&D, Department ofMedicine, Hippokration Hospital, Athens, Greece USA) in sera from 23 SLE+APS pts, 15 SLE pts and 19 pts with PAPS. HUS has rarely been reported in patients with APS. We present a patient Results: mean levels of sE-selectin and sVCAM-l were significantly with autoimmune hepatitis type I and APS ,who developed acute renal increased in all studied group versus control (sE-selectin - 52,3+14,1 in failure due to thrombotic microangiopathy. Case report. A 36 year-old SLE+APS pts, 78,3+61,2 in PAPS pts and 70,5=46,9 in SLE pts vs woman was admitted to our hospital with fever, epigastric and back pain and 38,0±13,4 in donors; sVCAM-1 - 1189,0+691,003,0+552,6, 1227,2±680,2, diarrhea. She had a history of autoimmune hepatitis which was in remission 594,7±63,3, respectively). In contrast the mean level of sP-selectin was for the last 7 years, and APS diagnosed 3 years ago because of a late fetal increased only in pts with SLE without APS. There was found the different loss. Laboratory studies demonstrated haemolytic anemia, mild level of sE-selectin in serum of pts with arterial and venous thrombosis thrombocytopenia and acute renal failure. Lupus anticoagulant was positive (67,1±10,9 versus 50,8+8,1, p<0,05). Furthermore the level of sE-selectin and anticardiolipin antibodies were elevated at a moderate titer. Kidney was significantly lower in pts treated with prednisone. Concentration biopsy was consistent with thrombotic microangiopathy. The patient sVCAM 1 was significantly higher in pts with renal involvement underwent simultaneous courses of hemodialysis and plasma (1446,7±775,7 versus 992,5+506,3, p Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 JflOiltrin id Oxesoniioqitolp dMi e-s pfpS8t3m 595 APS IS A MAJOR CAUSE OF CHRONIC DAMAGE IN PATIENTS APS - THE EFFECT OF THE PENDULUM WITH SLE M.P. Baleval, K.V. Nikolov2 J. Ugalde, V. Egurbide, G. Ruiz-Irastorza, C. Aguirre 'Laboratory ofClinical Immunology, Centre ofAllergology, 2Department of Servicio de Medicina Interna, Hospital de Cruces, Bizkaia, Spain Dermatovenerology, University Alexander Hospital, Sofia, Bulgaria AIM: To determine whether antiphospholipid syndrome (APS) is a cause of Antiphospholipid syndrome (APS) is associated with the presence of chronic damage in patients with systemic lupus erythematosus (SLE). antibodies directed against negatively charged phospholipids and!or lupus METHODS: The study group was a prospective cohort of 75 patients with anticoagulant. It is clinically manifested with recurrent arterial and/or SLE according to ACR criteria (65 women and 10 men). All patients have venous thromboses (thrombophylia), recurrent miscarriages, thrombo- completed a follow-up of at least 5 years after diagnosis. APS was defined cytopeny and livedo reticularis. After the description of APS in 1983 the according to Sapporo criteria. The SLICC index for chronic damage was following questions emerged: What is the nature of antiphospholipid calculated for every patient within 6 months after diagnosis and at 1, 3 and 5 antibodies (APL) and the cause of their heterogenity? What is the role of years of follow-up. Comparisons between patients with and without APS APL? What are the relationships between APL and otlier autoantibodies- were made by using paired, one tailed t-student test, ANCOVA (using antinuclear, antiendothelial etc.? Are there other forms and manifestations of "SLICC at diagnosis" as covariate) and repeated measures ANOVA. APS? What is the relationship between APL and other coagulation RESULTS: The mean (SD) SLICC score at diagnosis was 0.65 (1.6). The disorders? The treatnent of APS. The similarities and dissimilarities mean increase in the SLICC score at 5 years was 0.6 for the whole group (p between the primary and the secondary APS. Whether patients with primary < 0.0001). This increase was more pronounced in the 19 patients with APS APS would develop another autoimmune condition in the future. Is APS a (mean 1.1, p = 0.0005) than in the remaining 56 patients (mean 0.4, p < separate disease entity or merely an epiphenomenon? Just a few years ago 0.0001). The presence of APS was a significant predictor of a higher SLICC APS was well recognized by a small number ofspecialists but recently there score at 5 years after adjusting for the SLICC score at diagnosis (p = 0.002). has been a great interest towards this disease and its clinical and laboratory Repeated measures ANOVA also recognised APS as a significant predictor features. Furthermore, the significance and prevalence of APS has been of increasing SLICC score over the time (p = 0.0046). overestimated by many and this has frequently led to misjudgments in the CONCLUSIONS: APS is a major cause of chronic damage in patients with therapeutic approach. The diagnosis of APS is based on the joint work of SLE. Therefore, significant morbidity and mortality are expected from APS many specialists-rheumatologist, dermatologist, neurologist, gynecologist, manifestations during the course of lupus. hematologist and clinical immunologist. The importance ofAPS as a disease entity requires profound clinical knowledge in all stated fields. ANTIPHOSPHOLIPID THROMBOSIS SYNDROME (APS) THE ALTERATION OF PLATELET FUNCTION IN APS M. Hulikova OM. Serbic', I.V. Elezovic2, V.B. Lazarevic', N.D. Suvajdzic' University IHospital, Kosice, Slovak Republic 'University Hospital Zvezdara, 2Institute ofHaematology, KCS, Belgrade, Yugoslavia Antiphospholipid thrombosis syndrome is acquired hypercoagulable state caused by the presence of antiphospholipid antibodies, characterized by Objective: Investigation of platelet functions in patients with thromboembolic events, recurrent fetal wastage or thrombocytopenia. antiphospholipid antibodies and thrombotic clinical features, to determinate Clinical presentation is depended on localization of thrombosis, might the alteration of platelet functions and to evaluate the role of disorders of potentially be observed in almost every medical discipline. Patients and platelet finctions in the pathogenesis of thrombosis in antiphospholipid Mcthods: Wc present forty-four patients with antiphopholipid sylndrome syndrome (aPLS). A group of 17 patients with aPLS and thrombosis was with different clinical manifestations who have positive tests for Lupus studied. Venous thrombosis was found in II, arterial in, and two patients Anticoagulans (screening, mixing studies and confirmatory tests) and had both types of thrombosis. All patients had lupus anticoagulant and/or detectable Anticardiolipids antibody. We excluded other possible causes of anticardiolipin antibodies in repeated tests. Fifteen healthy volunteers were thrombophilia. Different clinical presentations seen in our patients with used as controls. Platelet counts, bleeding time, platelet adhesion in vitro, Antiphospholipid thrombosis syndrome and venous/arterial thrombosis spontaneous platelet aggregation, platelet aggregation induced by ADP and (ten), pulmonary embolism (one), fetal wastage syndrome (five), collagen, platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) were autoimmune collagen disease with thrombocytopenia (three) with Deep performed. The patients had a lower platelet count, but in normal range venous thrombosis (one), neurological symptoms arterial retinal occlusive 208000 (p Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lot Itimi o(fssmarfilSpliddAriboces Pft Setm( IN PATIENTS WITH 596 BLEEDING SYMPTOMS IN PATIENTS WITH ANTI-PHOSPHOLIPID ANTIBODY TITERS ANTIPHOPHOLIPID SYNDROME SUCCESSFUL RENAL TRANSPLANTS S. Vaidya, J. Daller, K. Gugliuzza W. Miesbach, B. Buehler, G. Asmelash, I. Scharrer Branch, Galveston, TX, USA J. W.Goethe-University, Frankfurt, Germany University of Texas Medical Antiphospolipid antibody syndrome (APAS) has been identified in patients Arterial or venous thrombosis, fetal loss in women and thrombocytopenia with various renal pathologies, including end-stage renal disease (ESRD). characterize the clinical trias of the antiphospholipid syndrome (APS). development of renal is only mild, rarely combined with ESRD patients with APAS are at high risk for the Usually, the thrombocytopenia they are treated with anti-oagulation therapy prior to and does not require any special therapy. Thc thrombosis unless symptoms of hemorrhage, operation. In this study, anticardiolipin antibody (ACA) titers of renal paper describes patients with APS and a hemorrhagic diatheses. their present recipients successfully transplanted under coumadin coverage were From 1996 until 2001 36 patients with antiphospholipid syndrome consulted transplant fulfilled the evaluated. Of approximately 800 ESRD patients awaiting renal the hospital due to hemorrhagig symptoms. All patients with APAS based on their APS according to the SSC of the since June of 1996, 25 have been diagnosed proposed classification criteria for the and ACA titers. These patients were treated the anticardiolipin autoantibodies of 35 patients were history of clotting disorders ISTH. In addition, with coumadin upon diagnosis of APAS. Nine of these patients received one the paticnts an APS had been known prcviously, measured. For part of 6 of which also received heparin at the time of their was diagnosed while investigating the symptoms kidney transplanits, for the other part, the APS Three patients transplanted without heparin lost their renal of hemorrhage. 22 patients were suffering from a primary APS, 14 from a transplant. patients transplant due to thrombosis within 24 to 48 hours despite coumadin secondary APS. There were 14 patients with recurrent hematoma, 9 well at 6 months to 4.5 with massive visceral bleedings and coverage. Of the remaining 6 patients, 5 are doing with striking nose bleedings, 6 patients One patient was lost to follow up. ACA titers of one patients with extraordinarily strong vaginal years post transplant. cerebral hemorrhages, 4 patient declined from 150 units to 35 units of lgM, there was no change in and 2 with petechial hemiiorrhages, 2 patienits withi hematuria patients 4 patients. The coumadin coverage was cardiovascular failure and bleeding due to ACA titers of the remaining bleedings. One patient died from in 2 patients due to bleeding complication; two patients are cause of hemorrhagic symptoms was discontinued factor V inhibitor. The most frequent on coumadin while one patient is maintained on low molecular von Willebrand-syndrome in 8 patients, 3 maintained thrombocytopenia in 14 patients, wcight bcparin. In conclusion thesc data suggest ESRD patients with APAS anticoagulant drugs, 3 bleedings were caused by an bleedings from oral continue to produce ACA. For ESRD patients with APAS, heparin coverage against clotting factors. In 7 patients a reason could not be found. inhibitor at the time of their transplant may be necessary. None ofthe examined patients suffered from an enhanced fibrinolysis. 10 of the 14 patients, suffering from hemorrhagic symptoms with thrombocytopenia, had more frequently a secondary APS. with high ANA titers and an already diagnosed or suspected lupus erythematosus. The APS is rarely combined with distinct hemorrhagic symptoms, which are mostly due to a thrombocytopenia. Also other causes may be found. The thrombocytopenia is more frequent in patients with secondary APS. Therefore patients with hemorrhagic symptoms and a systemic lupus erythematosus should be examined for antiphospholipid antibodies. THROMBOTIC EVENTS IN PATIENTS WITH APS, SLE AND ANTICARDIOLIPIN AND ANTI-beta 2 GLYCOPROTEIN I RELATED DISEASES ANTIBODIES IN MONOCLONAL GAMMOPATHIES F. Ingenito, N. Coraggio, L. Diez Porres, A. Gil Aguado, P. Lavilla, D. Pascual, M.V. Cuesta, S. Ramos, G. Bierfass, G. Caiballo, JM. Bemardino, M. Mora, T. Cobo, F. Gaya A.M. Di Lonardo On BehalfofThe APS Study Cooperative Group Immunology Unit, Hospital Dr. C. G. Durand, Buenos Aires, Argentina La Paz University Hospital, Madrid, Spain The incidence of anticardiolipin (aCL) and anti-beta 2 glycoprotein I in patients (abeta2GPI) antibodies in the sera of 36 patients (18 women, 18 men; 61 Objective: I) To deterninate the prevalence of thrombotic events studied. 2) to evaluate the influence of plus/minus 13 years) with monoclonal gammopathies (MG) was with APS, SLE and related diseases; ELISA (isotypes antiphospholipid antibodies (aPL), lupus anticoagulant (LAC) and other risk These autoantibodies were measured by commercial methods: 46 patients with SLE, 83 with IgG,M,A) and the results were expressed in units. Positive results were factors for thrombosis. Material and From 60 with primary APS (PAPS), 14 with secondary APS (SAPS) defined as higher than 80 UPL for aCL and 40 U/ml for abeta2GPl. SLE+aPL, 4 monoclonal gammnopathy of other than SLE, and 32 related conditions were included. LAC and aPL the 36 patients, 23 had Multiple Myeloma; 6 and 3 Non-Hodgkin Lymphoma. were determinated by standard methods. Results: The prevalence of undetermined significance; Waldenstrom by SPE and IFE (Paragon Systems). None of the patients thrombotic events was 15.2 % in SLE (arterial 13%; venous 22%), 44.5% in MG were detected in PAPS (50%; 28.8%), 71.4% in SAPS had a clinical history of thromboembolism, thrombocytopenia, or fetal loss. SLE+aPL (34.9%; 19.3%), 68.3% No patient and 6.2% in related diseases. The prevalence of From the 36 patients, 6 (17 percent) possessed autoreactivity. (64.3%; 14.3%) aCL and abeta2GPI; 1/12 (8 percent) loss was statistically correlated with arterial, but not with from the 15 IgG-MG was positive for abortion/fetal for aCL-IgA (321 APL) and 3/12 (25 percent) venous thrombosis. In the total patients, a strong association between LAC with IgA-MG was positive and higher than 158 APL); 3/9 (33 percent) of arterial and venous thrombosis was found. Venous thrombosis was also for abeta2GPI-IgA (158, 123 and for (155, 85 and 90 MPL) and 0/9 for associated to aCL-IgG and aBeta2GPI-IgG/IgM, but arterial thrombosis was the IgM-MG were positive aCL-IgM, In SLE subset, aPL was correlated with abeta2GPI-IgM. We havent confirmed yet the incidence of monoclonal only related to aBeta2GPI-IgG. that aCL and was significantly associated to LAC autoantibodies in these patients. Therefore we consider thrombotic events. Total thrombosis and might be thrombosis was associated to PS/PT-IgM. abeta2GPI in high titers might be monoclonal autoantibodies and aBeta2GPI-IgG. Venous In this and oral contraceptive use were important risk factors regarded as an expansion of clones producing natural autoantibodies. Arterial hypertension patients with MG frequently possessed for arterial events; immobilization, pregnancy and factor V Leiden /mutation study we have shown that sera from to CL and beta2GPI and that in many cases this could be 1120210 for venous thrombosis. Conclusions: Patients with APS have a autoreactivity higher risk of thrombosis, especially if they present LAC and some isotypes attributed to the paraprotein. of aPL. Another risk factors should be taken in account for the prevention of arterial anid venous thrombosis in these patients. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1alr1iraicidOxss onAdpdhdtocIes Posatricm 597 SLE AND ACQUIRED INHIBITOR OF THE FACTOR VIII OCCURRENCE OF ANTIPHOSPHOLIPID ANTIBODIES IN PATIENTS WITH INCREASED LEVELS OF SERUM IgM AND D.S. Fernanez Romero, U.J. Scher, M.V. Paolini, M.E. Prada, G. Bierfass, NON-SPECIFIC CLINICAL SYMPTOMS. PRELIMINARY REPORT A.M. Di Lonardo D. Jilek, V. Kral, I. Stiborova, J. Richter Immunology Unit, Dr. C. G. Durand, Buenos Aires, Argentina Institute ofPublic Health, Usti nad Labem, Czech Republic We present a 30 years old female with history of puhnonary (PTE) in 1999 (on warfarin for 6 months), diagnosis of Proposed study concerns with group of patients (n=25; 23 females; 2 males) thromboembolism myalgia, SLE since September 2000 and illness manifestations from 1996 whose was investigated for some non-specific complains (fatigue, (poliarthritis, malar erythema, ANA positive, anti-DNA positive; arthralgia, headaches) and simultaneously presented elevated lcvcl of serum leucopenia) treated with oral prednisone and hydroxicloroquine. She IgM (>3,5g/L). Characteristic autoimmune disorders (SLE, RA, APS) have been excluded. Methods: Basic spectrum of autoantibodies including aPL is consults to emergency room by the middle of 2001 presenting spontaneous investigated: ANA, ASMA, RF and aPL (aCL, a-beta-2-GPI, aPS, bruits and infrapatelar edema in right inferior limb. Laboratory was AMA, aPI). Results: Patients are according to serum IgM levels divided into two requested: platelets 270000/mm3; PT 94 percent; aPTT 149 sec.; TT 19; I groups: A (IgM >5,0g/L; n=15) and B (IgM 3,5-5,0g/L; n=10). In the group 438; VIII absent. It is interpreted as Lupus Anticoagulant and Factor VI A we have found markedly more positive results of aPL: a-beta-2-GPI-IgM Acquired Inhibitor. Deep venous thrombosis (DVT) is ruled out by Doppler x group B aPI-IgM (group A 11/15 x group B 2/10). In and she begins treatment with oral prednisone 1mg/kg/day and Azathioprine (group A 7/15 1/10), are positivity of ANA-IgG (52%, titres 40- 100 mg/day. Without requiring substitute treatment with coagulation factors, both groups there comparable aPTT stabilizes and hematoma decrease considerably. In October 2001 she 2560), ANA-IgA (68%, titres 40-80), ANA-IgM are positive in all sera (titres 40-320) and presence of heterophile antibodies on rat tissue sections is hospitalized presenting multiple bruits after fall from their own height. Preliminary results show probably significant positivity of at least The laboratory control showed aPTT prolongation. Recombinant Human (40%). certain IgM-aPL (a-beta-2-GPI, aPI) in the group A. Both groups are further Factor VIIa (rVIIa) is administered. Due to lack of response, characterized with ANA positivity especially in IgM class and heterophile immunosuppressive therapy was changed to Cyclophosphamide 100 be in relation to higher partial improvement. After abandoning treatment, she is antibodies. There is possibility these findings could mg/day, reaching (natural autoantibodies?). However, readmitted in December, this time presenting disseminated bruits, level of IgM autoantibodies synthesis some abnonnalitites, recalcitrant epistaxis and melena. She received four plasmapheresis sessions problem is, if these results signalising immunological are only secondary findings or not, in respect of clinical symptoms of surely and cryoprecipitate transfusions. Later on we prescribed IV non-healthy persons. ln literature we have not met any useful information cyclophosphamide (pulses) and prednisone 1 mg/kg orally. Clinical about such patients in connection with the increased serum IgM level. This improvement was achieved until the present time. We decided to report study was supported by grant IGA-MZ-CR NI/5357-4. this clinical case given the rare association between SLE-APS (antiphospholipid syndrome) and acquired inhibitor of the factor VIII, which leads to clotting mechanism alterations (including prothrombotic states and bleeding disorders alternatively) that respond to immunosuppressive therapy. SEX RATIO OF RHEUMATIC DISEASE MAGIC SYNDROME AND ANTIPHOSPHOLIPID ANTIBODIES M.D. Lockshin, C.C. Belizna', B. Faller2, P. Brignon2 Hospitalfor Special Surgery, Weill Cornell Medical College, New York, 'CHU Hotel Dieu, Nantes, 2Hopital Colmar, France USA MAGIC syndrome represent a rare entity associating mouth and genital The female/male (F/M) ratios of SLE, APS, and other autoimmune diseases ulcers with inflamed cartilage. Antiphospholipid syndrome has been reported as beeing found exceptionnaly in this patients (Med Clin 1998 are unexplained. Explanations for sex discrepancy of illness occur at different biological levels: molecular (imprinting, X-inactivation), cellular 16:678-9). We present four cases of MAGIC syndrome associated with The initial manifestations in three patients were (sex-specific receptor activity), organ (endocrine influences), whole antiphospholipid antibodies. organism (size, age), and environmental-behavioral, including intrauterine recurrent venous thrombosis which occured prior to chondritis. One patient with mouth and genital ulcers diagnosed initially as Behcet influences. The F/M ratios in autoimmune diseases are imprecise; presented years later he developed chondritis and in the same year he definitions and classifications vary; papers on the topic of sex ratio confuse disease. Two antibodies the concept of disease incidence with that of disease severity. The Institute presented bilateral superficial leg thrombosis. Anticardiolipin of of the National Academy of Sciences (USA) study, Exploring were found at elevated titers (IgG and IgM isotypes) in three patients and at Medicine in the Biological Contributions to Human Health: Does Sex Matter? examines low titers in one patient. Antiphosphatydilserine antibodies were elevated one case anti-beta 2 glycoprotein I in one other. Our report is the largest causes for high F/M ratios in many human diseases. Most autoimmune and our of recurrent thrombosis related to antiphospholipid diseases with high ratios occur in young adulthood. Gonadal hormones, if serie of knowledge MAGIC syndrome patients. they play a role, do so through a threshold or permissive mechanism and not antibodies in through quantitative immunomodulation. High F/M ratios can be caused by environmental exposure, X-inactivation, imprinting, X or Y chromosome genetic modulators, and intrauterine influences, all alternate, theoretical, and largely unexplored explanations for differences. The epidemiology of autoimmune diseases suggests that an explanation lies in differential exposure, vulnerable periods, or thresholds. The F/M ratio of lupus is the most obvious and least understood clue to its pathogenesis, and is also an important clue to the role sex differences play in human physiology. Wu Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1OOiktamakadOni ss eninAioplphidAniboces PosterPeseta*ins 598 ANTIPHOSPHOLIPID ANTIBODIES ASSOCIATED WITH DOES SUBCUTANEOUS ADMINISTRATION OF LOW HEMORRHAGIA RATHER THAN THROMBOSIS: A CASE MOLECULAR WEIGHT HEPARIN CAUSE FALSE POSITIVE REPORT TESTS FOR LUPUS ANTICOAGULANT? Z. Mondher, C.R. Rim, S. Maryam, M. Sondes S. Liestoel, F. Wisloff Immunology Department, La Rabrta Hospital, Tunis, Tunisia Haematological Research Laboratory, Ulleval University Hospital, Oslo, Antiphospholipid antibodies represent a diverse group of antibodies directed Norway against phospholipids. Patients with antiphospholipid may be free of Treatment with unfractionated heparin may give false positive tests for symptoms but can have thrombotic complications including stroke, placental Lupus Anticoagulant (LA). Commercial tests often contain heparin infarction, and fetal loss. Rarely hemorrhagic symptoms have been reported. neutralizing agents. To our knowledge, there are no reports on the effect of We report a case of a woman with systemic lupus erythematosus since 1993 low molecular weight heparins (LMWH) on LA testing. We examined including arthritis, skin lesions, photosensitivity, anemia and leucopenia. whether subcutaneous administration of dalteparin with a therapeutic level Antinuclear antibodies were present at high titres with anti-Sm and anti- of anti-factor Xa activity caused false positive tests for LA. Six healthy RNP antibodies bhut without anti-DNA antibodies. Anticardiolipin and subjects without LA were given 100 IE/kg dalteparin subcutaneously. Blood antibeta2glycoproteinl antibodies show high persistent titers. In 2001 she samples were taken before and 4 h after the injection. Mean anti-factor Xa had meningeal hemorrhagic with neurological complications. Recently she activity in plasma after 4 h was 0,67 IU (range 0,45-0,86 IU). We used iu- was hospitalised for uterine hemorrhagia. Laboratory evaluation revealed house LA tests based on the dAPTT, dRVVT and dPT and commercial positive results for anticardiolipin and antibeta 2 glycoprotein 1 antibodies assays (Staclot LA and DVV-Test/Confirm) with a heparin neutralizing in repeated tests and reduced factor II levels. The hemorrhagic agent. Staclot LA was repeated without heparin neutralization. Results were antiphospholipid syndrome should be considered in front of bleeding which calculated as normalized ratios or as recommended by the manufacturers. A could probably represents a distinct pathogenic mechanism from thrombotic small, statistically significant (p<0,05) but clinically irrelevant 0-4h antiphospholipid syndrome. difference in normalized ratio (maxiimum increase< 5%) was seen with Staclot LA, DVV-Test/Confirm, in-house dAPTT and in-house dRVVT No significant differences were found with Staclot LA without heparin neutralization or the in-house dPT. One plasma was weak positive in the Staclot LA both before and after dalteparin injection when the results were calculated as differences in seconds, but negative as nornalized ratios. None of the plasmas became positive after dalteparmn administration. In conclusion, this study suggests that the therapeutic use of LMWH does not cause false positive tests for LA, and heparin neutralization seems to be unnecessary in this setting. POLYARTERITIS WITH ANTICARDIOLIPIN ANTIBODIES CAN TRANSIENT ACL-IGM-TITER BE A MARKER OF A M.L. Ciompi, A. Delle Sedie PREVIOUS THROMBOTIC EVENT? Maria Laura Ciompi, Rheumatology Unit, Internal Medicine, University of W. Miesbach. B. Buehler, G. Asmelash, I. Scharrer Pisa, Italy J. WGoethe-University, Frankfurt, Germany Case Report: In September 2001 a 78 year old woman was hospitalized for a Anticardiolipin (aCL) antibodies are considered as specific markers for the necrosis of the distal tract of III and IV finger of left hand. Positivity to antiphospholipid syndrome (APS). The aCL-ELISA is found to be the most anticardiolipin antibodies was found (IgG- 140, IgM= 76) and she was frequent performed test in patients with suspected APS. While IgG-aCL is discharged with a diagnosis of antiphospholipid syndrome. In November she considered as an independent risk factor especially for cardiac and cerebral was hospitalized again with a thrombophlebitis in the right saphena and a thrombotic disorders, IgM-aCL is found to be more likely associated witli worsening of the necrosis of her fingers. She was treated with lloprost, infections. The present paper describes 40 patients with transient presence of Immunoglobulins and Urokinase. Due to an accidental trauma she IgM-aCL, 32 women and 8 men (median age 42 years). suspended the therapy causing the extension of the necrotic lesions. She was Patients 19/40 patients had a history of venous thrombotic disorders, 5/40 hospitalized again in March 2002. She showed necrosis of the distal phalanx patients of myocardial infarction, 5/40 patients of cerebral infarction, 4/40 of II, III and IV finger of left hand. Iloprost in continuous infusion for 5 patients of abortion or fetal death and 2/40 patients of arterial thrombosis. days did not show to be effective and Solumedrol (1 g) and Endoxan (500 10/40 patients were suffering from secondary APS and in 6/40 patients with mg) were administered. For anti-HBs and anti-HBc antibodies positivity, elevated aCL antibodies a thrombembolic disease could not be found. iperazotemia, ipercreatininemia and neurological alterations it was decided Results In 14/40 patients (35 %) IgM-aCL normalized 7.5 month (median) to make an arteriography of epaortic trunk, upper limbs, mesenteric vessels after diagnosis of the thrombotic event and start of treatment. In 4/40 and celiac trunk. The arteriography showed signs of vasculitis in both renal patients IgM-aCL normalized during pregnancy. In 3/40 patients IgM-aCL arteries ('in the shape of a rosary) and absence of bilateral ulnar artery. On normalized one month after infection. One patient had normalized IgM- the basis of 1990 ACR classification criteria she was diagnosed with PAN. aCL-titer 4 years after treatment of a non-Hodgkin lymphoma. The levels of Conclusion: the presence of anticardiolipin antibodies needs to he taken into 5/40 patients (including 3 patients with secondary APS) ranged several consideration in the diagnosis of systemic vasculitis since it has been times between normal and elevated titers. The values of the other patients pointed out, even though rarely, nont only in PAN but also in Horton normalized without any identified course. Especially in 43 % of patients arteritis and Behcet syndrome. with normalisation of IgM-aCL after the thrombotic event, IgM-aCL was the only thrombotic marker and disappeared after anticoagulant tratment. Conclusion The transient presence of IgM-aCL was associated in 85 % of the patients with venous or arterial thrombosis and in 41 % of these patients the values normalized in median 7,5 month after the thrombotic event. IgM-aCL may be considered as a marker of previous thrombotic event. Association to infection was only found in 5 % ofthe patients. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 Postrestio.llhliktmn inirV(s fonipdP'dfocIes SUSTAINED ELEVATED ANTI-BETA 2 599 IGG ANTIANTICARDIOLiPIN ANTIBODIES TITER IS THE SIGNIFICANCE OF GLYCOPROTEIN 1 ANTIBODIES AND THEIR RELATIONSHIP STRONGLY ASSOCIATED WITH NEW THROMBOEMBOLIC SYNDROMES EPISODES IN PRIMARY ANTIPHOSPOLIPID SYNDROME: A WITH ANTIPHOSPHOLIPID FIVE-YEAR PROSPECTIVE STUDY K Hampton, S. Kitchen, L. Williams, C. Brookfield F. Atzenil, P. Sarzi-Puttinil, E. Rossi2, M. Turiel3, S. Muzzupappa3 Department ofHaematology, Royal Hallamshire Hospital, Sheffield, UK 'Rheumatology Unit, 'Blood Transfusion Center, L. Sacco University Antiphospholipid syndromes are diagnosed by the co-incidence of clinical Hospital, 3Department ofCardiology, Istituto Ortopedico Galeazzi, Milan, features such as arterial and venous thrombosis and recurrent miscarriage Italy and abnormal laboratory tests such as the lupus anticoagulant or anti- antibodies. It has been reported that antibodies to beta 2 Background: Thromboembolic events are observed in primary cardiolipin 1 are more sensitive and specific markers for thromboemnbolic syndrome (PAPS). The risk factors for new thrombotic glycoprotein antiphospholipid in APS and we have been measuring them using an assay Methods: A group of 56 PAPS patients (8 / 48 M/F, complications episodes were assessed. validated in the European collaborative exercise for several years. We have mean age 37±10 years) were investigated during a 5-year follow-up period. to a cohort of subjects with persistently elevated antibodies Main endpoints were: a) the occurrence of thrombosis with any identified b2GPI, without any other laboratory features ofAPS. Of 950 subjects tested complications lcading to hospitalisation or dcath; b) the clinical and/or 125 (13%) had raised anti b2GPI. 5.5% had a transient elevation while serological risk factors for new thrombotic events. Results: At enrolment, 16 73(7.7%) had presistent elevation on two or more tests at least 6 weeks (28.6 %) patients has at least one venous or arterial thrombosis, 27 (48 %) apart. Of these 52% had raised ACA or LA in addition, while 16% fulfilled more than one thrombotic event (often associated to fetal loss) and presented the clinical criteria for APS without other laboratory evidence. In this cohort 13 patients (23.4 %) had fetal losses. During the follow-up period 26 with we found that isolated persistently raised anti-b2GPl is associated patients (46.4 %) showed new thrombotic events or fetal loss; 3 patients (5.4 It is arterial and venous thrombotic events but not recurrent miscarriage. died. Patients with IgG aCL >40 GPL titer presented new thrombotic %) clear that anti-b2GP1 can occur as a transient phenomenon and repeat events in 43.3% of the cases in comparison to those with IgG aCL?40 testing is necessary. We feel that isolated positivity is an indicator of APS. GPL-U with 7.7 % of events. Univariate analysis identified a history of recurrent clinical events (p= 0.004), aCL titer highly positive (p = 0.007) anid the presence of cardiac abnornalities (p = 0.036) as significant risk factors. Multivariate regression model confirmed that IgG aCl titer >40 GPL-U is an independent risk factor for thrombosis (odds ratio 9.17; 95%CI, 1.83 to 46.05). Conclusions Long term follow-up showed that elevated aCL titer is a predictor ofthrombotic events in patients with PAPS. ANTIBODIES AND CLINICAL CORRELATIONS BETWEEN ANTI-BETA2 SERUM LEVELS OF ANTICARDIOLIPIN 78 DISEASE ACTIVITY IN SLE GLYCOPROTEIN I AND ANTICARDIOLIPIN ANTIBODIES IN PATIENTS I.M. Manoloval, M.M. Dancheva2, M.I. Ivanova2, KS. Halacheva' A. Bondanza, M. lannacone, G. Ciboddo, S. Panza, E. Baldissera, 'Department ofImmunology, Laboratory of Clinical Immunology, 2Clinic of L. Praderio, R. len, M.G. Sabbadini Division ofInternal Medicine, University Hospital, Medical Rheumatology, Divisione di Medicina, Faculty, Thracian University, Stara Zagora, Bulgaria Unita de Reumatologia e Immunologia Clinica, IRCCS Hospital San Raffaele, Milano, Italy The objective of this study was to examine the relation between disease IgM and IgA anticardiolipin antibodies (aCL) in Antiphospholipid (aPL) antibodies associates with thrombotic events activity and levels of lgG, and obstetric complications paired samples obtained during active disease and at remission from patients (arterial and/or venous thrombosis) one these clinical events along with aPL with systemic lupus erythematosus (SLE). 22 consecutive patients with SLE (poliabortivity): the presence of of defines the antiphospholipid syndrome (APS) (Wilson et al, were included in the study between January 2000 and December 2001. 83 positivity of aPL is frequently detected by ELISA test using a were analyzed for IgG, IgM and IgA autoantibodies against 1999). The presence serum samples aPL antibodies associated with APS by commercially available ELISA cardiolipin substrate (CL). Actually beta2-glycoprotein-Ilcardiolipin complex cofactor, the disease activity was assessed using don't recognize directly phospholipid but a 50 kD plasmatic kits (Binding Site, UK). Corresponding Antibodies specifically directed towards the SLE disease activity index (SLEDAI). All study patients were at least beta2-glycoprotein I (beta2GPI). are indeed present in infectious and neoplastic diseases, and once for IgG-aCL, IgA-aCL or IgM-aCL during the observation phospholipids positive with clinical features of APS. In this work we was weak correlation between SLEDAI and IgG-aCL levels they are not clearly associated period. There IgM, IgG and IgA antibodies for (r=0.30, p=0.009, Spearman rank correlation test). Changes in disease evaluated the predictivity ofanti-beta2GPI APS. To this aim we collected 78 consecutive patients who resulted activity status were not related to changes in IgA-aCL or IgM-aCLlevels. In clinical a titer for anticardiolipin antibodies. We then verified the conclusion our results showed that serial quantification of aCL does not positive at high clinical features (retrospectively evaluated) and anti- contribute to the monitoring of disease activity in SLE patients. correlation between beta2GPI positivity. Our results show that anti-beta2GPI IgG are associated with thrombotic events with a much higher relative risk than the one for aCL. In addition they are associated with hematologic manifestations: hemolitic anemia (IgG) and thrombocytopenia (IgA). Antiphospholipid antibodies, and particularly anti-beta2GPI, were associated also with other signs of systemic autoimmunity, such as antinuclear antibodies (ANA). Taken together these results agree with those of other groups; in particular they apply to a group of patients not a priori selected for the clinical features of APS. Wu Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1GIhkt8naicxd eOnss (n hididAfibocIes PoestP 1ss 600 THE PERSISTENCE OF ANTICARDIOLIPIN ANTIBODIES OVER DIVERSITY OF SO-CALLED ANTIPHOSPHOLIPID ANTIBODIES TIME IS ASSOCIATED WITH A HIGHER LIKELIHOOD OF THE IN PATIENTS WITH SLE AND RELATED DISORDERS PRESENCE OF LUPUS ANTICOAGULANT OR ANTI-BETA2- J KaburakiP'2, T. Sugi2, S. Goto2, J. Okada2, M. Nakarnura2, T. Makino2 GLYCOPROTEIN I ANTIBODIES 'TEPCO (Tokyo Electric Power Company) Hospital, Shinjuku-Ku, Tokyo, P.R. Fortin', J. Kassis3, C. Neville2, E. Chang', L. Joseph2, J. Rauch2 20n Behalfofthe 'Japan Antiphospholipid Project Group, Isehara, 'Toronto Western Hospital Research Institute, University of Toronto, Kanagawa, Japan 2McGill University Health Centre Research Institute, McGill University, It has been widely accepted that so-called antiphospholipid antibodies are 3Hopital Maisonneuve-Rosemont, Universite de Montreal, Quebec, Canada directed against specific phospholipid-binding plasma proteins as well as We defined two exposures to antiphospholipid antibodies (aPL): 1) phospholipids. In this regard, beta2-GPI dependent anticardiolipin persistence of anticardiolipin (aCL) over time and 2) presence of multiple antibodies were adopted as serological marker in the preliminary criteria for aPL (aCL, lupus anticoagulant [LAC] and/or anti-beta2-glycoprotein I the classification of antiphospholipid syndrome, 'Sapporo criteria'. However, [aB2GPI] antibodies). We hypothesized that persistently positive aCL are various kinds of so-called antiphospholipid antibodies have been reported to more likely to be associated with LAC and/or aB2GPI. We collected be positive in sera fiom these patients. In this study, we examined the information on demographic/health characteristics, comorbiditv, and diversity of these so-called antiphospholipid antibodies and clarified the familial history of cardiovascular disease (famCVD) from 318/416 persons clinical significance of these antibodies in patients with SLE and related with three yearly assessments from an ongoing study. We measured disorders. The study population consisted of 97 patients with SLE and IgG/IgM aCL and aB2GPI by ELISA, and LAC by dilute APTT with related disorders. IgG or IgM anticardiolipin antibodies, IgG beta2-GPI confinnation. We calculated the risk difference (RD) and 95% confidence dependent anticardiolipin antibodies, IgG anti-phosphatidyl- interval (Cl) for LAC and/or aB2GPI (LAC/aB2GPI) positivity between serine/prothrombin (PS/PT) complex antibodies, lgG or lgM kininogen those individuals with and without aCL. We performed a logistic regression dependent or independent antiphosphatidylethanolamine (PE) antibodies with number of positive aCL tests over time as independent variable and were assayed by ELISA. Lupus anticoagulants were detected by diluted presence of LAC/aB2GPI as dependent variable. Fifty-five persons had at Russell viper venom time and inhibition test by phospholipids. The least one aCL, while 263 were persistently negative. The two groups were frequency of IgG anticardiolipin antibodies, IgG beta2-GPI dependent similar for all baseline characteristics except for a higher proportion of anticardiolipin antibodies and lupus anticoagulants was 47%, 31% and 32%, patients reporting famCVD (67% vs 28%), previous thrombosis (38% vs respectively. Moreover, IgG anti-PS/PT complex antibodies were found in 13%), and SLE (46% vs 19%) in the aCL positive group. RD [95%CI] for 31%, and IgG kininogen dependent anti-PE antibodies were detected in LAC/aB2GPI between the two groups was 44% [29%, 58%]. The odds of 33%. It was suggested that IgG anti-PS/PT complex antibodies and IgG having LAC/aB2GPI more than doubled (OR=2.3, 95%CI=1.7, 3.1) for each kininogen dependent anti-PE antibodies were associated with thrombosis additional positive aCL. Persistence of aCL over time is associated with a and pregnancy morbidity which were listed in clinical criteria of 'Sapporo higher likelihood of having LAC/aB2GPI antibodies. The persistence and criteria'. Above findings indicate that these anti-PS/PT complex antibodies presence of multiple aPL should be studied to determine if they are better and kininogen dependent anti-PE antibodies may be useful in the diagnosis predictors of thrombotic risk than periodic measurements of individual aPL. ofantiphospholipid syndrome. Registration confirnation number: 4.APS.000115. ANTIPHOSPHOLIPID ANTIBODIES AT THE TIME OF VALIDATION OF SAPPORO CRITERIA IN APS PATIENTS WITH DIAGNOSIS OF SLE ARE A PERSISTENT MARKER OF DISEASE ANTICARDIOLIPIN/LAC POSITIVITY VS. OTHER SEROLOGIC SEVERITY SUBSETS J. Ugalde, V. Egurbide, G. Ruiz-Irastorza, C. Aguirre G.A. McCarty, T.E. Cason Servicio de Medicina Interna, Hospital de Cruces, Bizkaia, Spain 'Division ofRheumatology And Immunology, UVA Health Systems, Charlottesville, VA, USA Aim: To determine whether the presence of antiphospholipid antibodies at the time of diagnosis of systemic lupus erythematosus is a marker of disease Objective: Sapporo Criteria invoke 'gold standards' of IgG/M aCL/LAC severity. Methods: A prospective cohort of 100 patients with SLE according positivity and selected clinical criteria, and one major validation study has to ACR criteria (87 women and 13 men) was analysed. Antiphospholipid been published. Expanding serologic studies and non-criteria APS findings antibodies were determined at the time of diagnosis of lulpus in 77 patients. are evolving and have not been assessed in APS pts. with other stable They were considered positive when anticardiolipin antibodies (IgG and/or serologic profiles. Methods: 167 pts diagnosed with clinical primary or lgM) at medium/high titres and/or lupus anticoagulant were detected in at secondary APS & proven thromboses who exhibited consistent serologic least two occasions separated six weeks. The SLICC index for chronic profiles over 2 yrs with q3mo follow-up were classified by standardized damage was used to quantify disease severity. It was calculated for every ELIZAS and LAC testing (aPTT, DRVVT) as aCL (N=23), aPC (N=15, Z Brahmi aPE patient within 6 months after diagnosis (n = 100) and at 1 (n = 97), 3 PhD), positive(J McIntyre PhD, N=53), or seronegative (n = 92) and 5 (n = 75) years of follow-up. Multivariant ANOVA, using (SNAPS, N=53), ANA, ENA, & dsDNA. 2x4 contingency tables were antiphospholipid, anti-DNA, anti-Ro, anti-La, anti-Sm and anti-UIRNP compared using S-Plus 6 (Insightfuil Corp.), Seattle, WA) by Pearson's Chi- antibodies as indepenident variables and SLICC score at diagnosis, 1, 3 and square (3df with Yates' Continuity correction where 5 years after diagnosis as dependent variables was performed. Results: Only applicable(>7.8 147needed for alpha=0.05 level) Results: Demographic the presence of antiphospholipid antibodies was independently associated characteristics were NS different among the 4 groups(83-95% white F); with higher SLICC scores. This association persisted over the time (p values mean age 39-41 yrs. Significant criteria among the 4 groups by Chi square at diagnosis, 1, 3 and 5 years were 0.002, 0.010. 0.018, 0.0037, and P<.0001 were: arterial thrombosis(AT), LAC, thrombo- cytopenia, respectively). Conclusions: The presence of anticardiolipin antibodies at livedo, migraine, and presence of livedo & migraine & mitral valve medium/high titers and/or lupus anticoagulant at the time of diagnosis of prolapse. Sapporo criteria for >3 fetal losses was 00/n(aCL), lupus is a marker of disease severity for at least 5 years after diagnosis. 8%(aPE)%(aPC), 160/%(SNAPS); >3 pregnancies occurred in 30%(aPC), 42%(aCL), 43%(aPE), 53% (SNAPS); AT 51%(aPE), 67%(aPC),70%(aCL), 92%(SNAPS); VT 23%(SNAPS), 26%(aPE), 33%(aPC), 54% (aCL): LAC 8%(aPE), 15%(SNAPS), 40%(aPC),100%(aCL). Between 2-24% of pts. fiulfilled full ACR criteria. Conclusions: Some Sapporo criteria need to be expanded and reconsidered in light of continued data re: other aPL profiles, and direct comparability to ACR criteria in classification may not be as valid as previously thought. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lahirimiMaOrsonAtosiopdAtfoces PoFrIpsator 601 ISCHEMIC STROKE IN A BOY WITH PRIMARY APS ANTIPHOSPHOLIPID ANTIBODIES IN PATIENTS WITH NEUROPSYCHIATRIC SYSTEMIC LUPUS A. Attilakos', S. Fessatoul, H. Tsapral, E. Katsarou2, V. Tzavara3, C Dracou' L. Stojanovichl, I. Elezovich2, R. Stojanovich3, V. Mirchetich3, V. Kostich4 12nd Department ofPediatrics, 2Department ofNeurology, 'P&A.Kyriakou' Children's Hospital, 3Department ofPathology, 'G. Gennimatas' 'KBC Bezhanyska Kosa, 21nstitute ofHematology, 3Institute of General Hospital, Athens, Greece Rheumatology, 'Institute ofNeurology, Belgrade, Yugoslavia We present a boy who had an ischemic stroke at the age of 20 months. He Objective: A modest progressive neurological process exists in patients with acutely developed a right hemiparesis affecting the upper and lower systemic lutpus erythematosus (SLE). It is well known that antiphospholipid extremities. Magnetic resonance imaging revealed an infarct in the lett basal antibodies (aPL) in patients with SLE may have many causes, including ganglia. Serum immunologic examination revealed a high titer of IgG neuropsychiatric lupus (NL) manifestations. However the association anti-beta2GPI antibody (first titer: 132 U, second titer six weeks later: 350 between brain abnormalities on magnetic resonance imaging (MRI) and aPL U, normal range: 0-100 U). Anti-cardiolipin antibodies (IgG and IgM) were remains controversial. Patients and methods: Our study was comprised of 60 nornal and lupus anticoagulant test was negative. Laboratory studies patient (54 female and 6 male, median age 44.5 years) fulfilling > 4 of the showed negative ANA and anti-DNA. All other causes of cerebral infarction 1982 ACR criteria for SLE at same stage of the diseasc. Only patients with were excluded. The patient received long-term treatment with aspirin NL of the 1999 ACR criteria for NL were included. The secondary NL (50 mg daily). Anti-beta2GPI antibodies have remained elevated; follow-up patients were excluded. Anticardiolipin antibodies (aCL) were tested by the laboratory study done at the 6 months showed anti-beta2GPI IgG levels standard ELISA. A prolonged aPTT and aRVVT, and their neutralisation 164 U. The patient recovered completely 4 weeks after the stroke and has were taken as evidence of lupus anticoagulant (LA). Results: aPL were been well during the 2 years of follow-up after the onset. This is the first present in 20/60 patients (33.3%): aCL were present in 12/52 (23.0%) reported case of childhood ischemic stroke secondary to antiphospholipid patients and LA was detected in 8154 (14.8%) patients. Of these, 4 patients syndrome with anti-beta2GPI antibodies, but no antibodies detectable in presented LA along with aCL. There were no statistically significant standard antiphospholipid assays, in Greece. This case emphasizes the differences between aPL and the following NL syndromes: headaches, importance of evaluating antiphospholipid antibody levels, including anti- movement disorder, cranial neuropathy, seizures, anxiety and mood disorder beta2GPI titer, in children suffering from cerebral ischemic accidents. with depressive or manic features, psychosis, and polyneuropathy. There was a significant association between LA and cerebrovascular accidents (p=0.0111), and between LA and MRI changes (p=0.048). There was no correlation between aCL and NM changes, but 37 patients in this group had focal cerebral disfunction. We found a significant correlation between focal cerebral disfunction and aCL (p=0.0148), and especially between IgG and aCL (p=0.0420). Conclusion: This study shows no association of aPL with the presence of various neurological symptoms in SLE patients, except a correlation between LA and CVI, LA and MRI changes, and aCL and focal cerebral disfunction. 1. Jarec MJ, West SG, Baker MR, Rak MM. Magnetic resonance imaging in SLE patients without a history of neuropsychiatric lupus erythematosus. Arthritis Rheum 1999; 37: 1609-1613. 2. The antiphospholipid antibodies in Stroke Study (APASS) Group. Anticardiolipin antibodies are on independent risk factor for first ischaemic stroke. Neurology 1993; 43: 2069-2073. 3. Hachulla E, Michon-Pasturel U, Leys D et al. Cerebral magnetic resonance imaging in patients with or without antiphospholipid antibodies. Lupus 1998; 7: 124-131. NEUROLOGICAL MANIFESTATIONS IN PATIENTS WITH ANTIPHOSPHOLIPID ANTIBODIES J. Szilasiova, E. Klimova, Z. Macejova Safarik University, Department ofNeurology, Medical Faculty, Kosice, FACTOR VIII (FVIII) ACTIVIY IN PATIENTS WITH SNEDDON'S Slovak Republic SYNDROME (SS) Hypercoagulation states conditioned by the antiphospholipid syndrome L. Kalashnikova, A. Berkovski, L. Dobrynina, E. Sergeeva, A. Kozlov, (APS) may be the cause of serious damage of CNS. OBJECTIVE: To assess E. Alexandrova, E. Nassonov the most frequent manifestation of CNS damage in patients with anticardiolipin antibodies (aCLA). METHODS: In a retrospective study of Institute ofNeurology, Moscow, Russia at the patients with positive anticardiolipin antibodies hospitalized Clinical manifestations of SS and PAPS are similar, but not all SS pts have Neurological and Internal Clinic between 1995 and 2000 the authors aCLJLA. Increased VIIIF is a marker of hypercoagulation. The aim of the evaluated the extend of their neurological manifestations. For assessment of work - to study FVII activity in SS. Material and Methods. We studied 28 we used the enzyme aCLA sandwich method of immunoanalysis (ELISA) in pts with SS (26 f, 2 m, mean age-48.1 ± 9.1 years). FVIHI activity was Cebecauer modification. RESULTS: aCLA positivity in suspected measured by a one-step-clotting assay with the use of FVIII deficient plasma thrombembolic disease was found in 76 patients (21,1%). Neurological (nonnal level < 150 IU/dl). ACL were studied in all pts (ELISA), LA - in manifestations were recorded in 11 patients (14,5%). The mean age of the 26 pts, protein C activity (PCA) - in 22 pts (coagulation assay). was years. aCLA IgG and patients 48,2 +/- 10,5 Positivity of isotypes IgM Results: Increased FVIII activity was found in 21 of 28 pts (75%), in was recorded in 4 patients, 7 patients had only the IgG isotype. All sub- 680%>2001U/dl. Many clinical manifestations connected with thrombosis classes of were 1 aCLA (IgG,IgM,IgA) found in female patient. were only or more often observed in pts with increased then normal FVIII Thrombocytopenia was confirmed in ten patients. The following activity: ischemnic strokes (IS) (91% vs 57%), TIA (76% vs 57%), vascular neurological manifestations were recorded: cerebrovascular attack type TIA dementia (43% vs 0%), IHD (43% vs 0%), PVT (24% vs 0%), cardiac valve (lx), thrombosis of the cerebral sinuses (lx), monocular visual disdorder thickening (91% vs 57%), p>0,05. Recurrent IS occutred only in pts with caused by thrombosis of the central retinal vein (lx), syndrome of benign increased FVIII (71%, p=0,0014). Epilepsy and thrombocytopenia which are intracranial hypertension (lx), cerebral infarction in 8 patients. Relapses of not due to thrombosis occurred less frequently in pts with increased than cerebral infarction occured in 5 patients. CONCLUSION: Manifestations of normal FVIII (24%, 14% vs 43%, 29%, respectively). 9 out of 21 pts (43%) ischaemia of the CNS, particulary in younger patients who suffer at the with increased FVIII did not have aCL and/or LA. PCA was normal in all same time from thrombocytopenia, should be an indication for examination pts. Conclusion. Increased FVUI activity is one of the factors which of aCLA antibodies as their permanent presence increase the risk of further predisposes SS pts to thrombosis. It is suggested that the cause of increased thrombembolic attacks. FVIII is the production of aPL to VIIIF, which make it insensitive to the degradation by protein C. UPS Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1l,h Int mvicOdOtngs cnAdpdidAiboces PosterPsst"cn 602 STROKE IN DOWN SYNDROME: AN ASSOCIATION WITH POSITRON EMISSION TOMOGRAPHY ABNORMALITIES IN ANTIPHOSPHOLIPID ANTIBODIES LUPUS PATIENTS WITH COGNITIVE DYSFUNCTION AND APL P.A. Gatenby'2, R.R. Tuck3, C.J. Andrews3, R. ONeil4 D. Hernosillo-Romo, S.L. Holliday, J. Xiong, J. Lancaster, R.L. Brey 'Clinical Imnmunology Department 2ANU Medical School, UTHSCSA, San Antonio, TX, USA 3Neurology Department, 'Medical Imaging Department, A pilot brain MRI/ FDG-PET study was performed to evaluate for The Canberra Hospital, Canberra, ACT, Australia fimctional and anatomic to brain abnormalities in Lupus patients with and Two females are presented with Down syndrome and stroke in association without aPL. Six subjects with moderate to severe cognitive with antiphospholipid antibodies. Both were in their third decade of life. impairment/with aPL and 4 with normal cognitive functioning/without aPL were studied. Visual inspection of FDG-PET images showed One suffered a dense L hemiparesis , dysarthria and a L homonymous mild hemianopia. Ultrasound showed no flow in the R internal carotid artery, abnormalities in frontal and temporal lobes in 2/6 impaired subjects. Voxel- cerebral CT scan revealed an R frontoparietal infarct. IgG-aCL were by-voxel quantitative comparisons between groups demonstrated a meditum positive and remain so. The second case was more complex with a significantly decreased FDG uptake in multiple discrete regions, including stuttering onset initially affecting both carotid territories, but progressing to the white matter in bilateral frontal lobe and the gray matter in the right basilar artery disease. Appearances on MRA are similar to those described inferior frontal lobe. Three gray matter and 5 white matter regions were also previously as Moyamoya. Liupus anticoaguilant and antibodies to beta2GPl analyzed using MRI Ti and T2 relaxometry. Relaxation times were different were detected. Individuals with Down syndrome have an increased between the cognitively impaired and cognitively normal groups (p < 0.10) incidence of autoimmune disease. These two cases extend the scope of these in 2 brain regions. TI in posterior white matter (728 vs. 686 msec) and T2 in observations. frontal white matter (67.1 vs. 62.6 msec) were longer in the impaired group. These observations are consistent with: (1) a possible loss of myelin that results in a greater change in Ti than T2 in posterior white matter and (2) a more recent event with compensatory fornation of edema in frontal white matter. These results suggest that robust functional and anatomic abnormalities are seen in cognitively impaired Lupus patients with aPL. Functional and anatomic brain imaging studies have great potential for uncovering the mechanism of these and other neurologic manifestations of aPL. ANTICARDIOLIPIN ANTIBODIES IN ACUTE STROKE COGNITIVE FUNCTIONING IN SECONDARY APS: AGE SUBTYPES EFFECTS AND THE ROLE OF ANTI-B2GP2 K.N. Vemmos, P.G. Konstantopoulou, G.K. Tsivgoulis, S.L. Holliday', D. Hennosillo Romo2 , A. Saklad2, R.L. Brey2 C.D. Evangelopoulos, D.A. Sotos, A.J. Leventakos, M. Dalesios 'South Texas Veterans Health Care System, 2University ofTexas Health Clinical Therapeutics ofMedical School ofAthens University, Greece Science Center, San Antonio, TX, USA Background: Anticardiolipin antibodies (aCL) are a recognized marker for We compared 11 patients who met published criteria for secondary Anti- an increased risk of thrombosis including stroke. Aim of our study was to Phospholipid Syndrome (APS) and 21 patients with clinical APS features examine a possible relation of elevated aCL and stroke subtype and the and repeatedly positive aB2GP values (APS-AB2GP) with 130 patients with possible influence on outcome. Methods: We studied a series of 262 first- neither syndrome. All patients met ACR criteria for SLE. A computer- ever stroke patients admitted to our hospital within 24 hours after symptoms administered battery assessing cognitive speed/efficiency (ANAM) and onset. Patients were classified according to the etiopathogenic mechanism standard measures for depression, SLE disease damage/activity, and into stroke subtypes. Sere were obtained within 4 days of admission and medications were collected every four months for two years. Mixed Linear measured by ELISA method. Patients were followed up to eight years. Model Analyses with fixed effects for age group (<35, 36-55, >55), visit Survival was assessed by Kaplan-Meier limit method. Results: Study number, and the presence of the two APS syndromes were examined. At the patients were 168 men and 94 women with amean age 65+12.6 and 69±14.0 initial study visit, the APS group did not differ from non-APS SLE patients years respectively. The prevalence of positive aCL among stroke subtypes in age, education, Ethnicity, SLE disease damage/activity, depression, was: atherosclerosis 10/50 (20%), cardioembolism 15/59 (20.3%), lacunar prednisone dose, aspirin dose, or on any ANAM tests. The APS-aB2GP stroke 13/42 (23.6%), cryptogenic infarcts 7/29 (19.4%), miscellaneous 5/13 group had more Hispanics (p=.003) and scored lower on the ANAM (27.8%) and intracerebral hemorrhage (ICH) 12/17 (41.4%). No statistical Steinberg test (p=.04). The longitudinal analysis found significant main significanice was measured among ischemic stroke subtypes (p=0.26). effects for age group and visit number on average ANAM score (p<.001), Patients with ICH had a higher prevalence aCL compared to all ischemic suggesting robust age and practice effects. Patients with APS scored lower strokes (p=0.017). Mortality at one month was 22.5% in patients with on ANAM, both for the traditional APS group (p=.010) and more so for positive aCL compared to the patients with negative aCL 5.3% (log rank APS-AB2GP group (p<.001). An APS-aB2GP/age group interaction was p-O.OOOl). Cumulative long-term survival was as well higher in patients also found in which the middle-aged group was more impaired than the with positive aCL during a period of 8 years (log rank p=0.019). youngest or oldest groups (p<.001). The results suggest that ANAM is Conclusion: The prevalence of aCL antibodies is similar among ischemic sensitive to impaired cognitive speed/efficiency in traditionally defined APS stroke subtypes and unexpectable high in patients with ICH. Our results and that APS-aB2GP appears even more strongly associated with this indicate that aCL antibodies might be a marker of severity of impairment. The age/APS-aB2GP interaction found suggests that aB2GP- cerebrovascular accident. associated cognitive impairment may be specific to middle age in this SLE cohort. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 llhIrnt8naard Qiess onkAiomioddAdtles PoPstar ANTI-ICAM AB PARTIALLY IMPROVE NEUROLOGIC SILENT ISCHEMIA IN SLE AND APS: CLINICAL AND 603 DYSFUNCTION IN MICE WITH EXPERIMENTAL APS MORPHOLOGICAL FEATURES IN CEREBRAL MAGNETIC RESONANCE IMAGING L. Ziporen, J. Chapman, L. Rauova, R. Brey, Y. Shoenfeld M. S. P. Garcia L. Diez Medicine Chaim Sheba Medical Center, Jl. Bernardino, Mora, Novo, Raya, Porres, 'Department of Internal B', P. N. A. Gil Sackder School ofMedicine, Tel-Aviv University, Israel A. Robles, F. Garcia Iglesias, Lavilla, Sastre, Aguado La Paz University Hospital. Madrid. Spain Background: Ani-phospholipid antibodies (aPLs) are involved in endothelial cell activation and thrombus formation. Microthrombi and mild Aim: To evaluate the frequency, clinical characteristics and morphological inflammation were demonstrated in brain specimens of mice with APS and changes in cerebral magnetic resonance imaging (MRI) of silent ischemia in neurological manifestations. Objective: To cvaluatc the anti-inflammatory SLE and APS patients. Patients and Methods: One hundred and twenty eight therapeutic effect of anti-ICAM antibodies on the clinical and neurological MRI studies were performed. MRI scans were analysed for the presence of state of mice with APS. Methods: Three groups of BALB/c mice (n=15 cortical infarcts, white matter changes [periventricular hyperintensity (PVH; each), received a single immunization of b2GPI, or of BSA (10mg) in 0-6), deep white matter hyperintensity (WMH; 0-24), basal ganglia controls (n=15), emulsified in CFA. 2 weeks later, mice received 5 weekly hyperintensity (BGH; 0-30), infratentorial foci of hyperintensity (1TF; 0-30) i.p injections of 500mg of anti-ICAM abs, or an irrelevant substance, as and atrophy (Pasquier scale; 0-39) and calcifications. Silent ischemia was control. Serological and clinical parameters of APS were tested. We defined by the presence of white matter hyperintensities and atrophy on assessed the neurological status of the mice by Rota-rod treadmill, T-maze MRI studies without neurological manifestations of stroke nor TIA. Results: and neurological reflexes. Results and Conclusions: b2GPI-immunized mice Thirty nine patients (30,5%) had silent ischemia (20 with SLE and 19 with developed high levels of aCL and anti-b2GPI abs (OD405=0.2 and 1.8, APS). Silent ischemia was more prevalent in males (52% vs 24%, p< 0.05). respectively), compared to the control mice (0.1 and 0.2) and Patients with silent ischemia were older (pe0,001), with a mean (SD) age of thrombocytopenia (no.of plt=1035X103/mm3, and 735X103/mm3, 54 (17)y. White matter hyperintensity changes and cerebral atrophy scores respectively). APS mice displayed incoordinaton on the Rota-Rod by were higher in patients with silent ischemia (PVH p<0.05, WMH p<0.001, stayiing shorter onl the rotating bar (Mean+SD=59scc, and 39scc, ITF p<0.001 and Pasquier p<0.001). We found a significant association respectively, p<0.01), performed worse the T-maze learning task (p=0.01, between silent ischemia and arterial and venous thrombosis (p<0,001; repeated measures ANOVA), indicating cognitive impairment and showed a p<0,029). No relation was found between the titres and isotypes of tendency for hyperactivity in a stair-case system (Mean no. of steps=- 1.4, antiphospholipid antibodies and MRI changes. Conclusions: Silent ischemia compared to 9.6 of controls). Anti-ICAM treatment of b2GPI-immunized is a common feature in patients with SLE and APS. These findings should mice resulted in significant improvement in motor coordination (59sec, be consider to establish preventive therapy. compared to 39sec in non-treated mice), in hyperactivity (9.2 steps, compared to 11.4 steps), but did not change their cognitive abilities. We assume that ICAM-mediated processes account for some but not all neurological defects in APS. CEREBRAL MAGNETIC RESONANCE IMAGING (mRI) IN CEREBRAL VENOUS THROMBOSIS AND APS - REPORT OF A PATIENTS WITH SLE AND APS CASE J.I. Bernardino, M. Mora, S. Novo, P. Garcia Raya, L. Diez Porres, A.C.S. Grilo, F. C. Soromcnho, M. V. Riscado A. Robles, F. Garcia Iglesias, P. Lavilla, N. Sastre, A. Gil Aguado Hospital de Curry Cabral, Lisboa, Portugal Paz Madrid. La University Hospital. Spain Antiphospholipid syndrome is a disorder of a recurrent vascular thrombosis Aim: To examine morphological changes by cerebral MRI in patients with and pregnancy losses associated with elevated level of antiphospholipids SLE and APS and to establish a possible correlation with clinical parameters (aPL) antibodies. In this disease all sites in vascular tree may be subject to and antiphospholipid antibodies (aPL). Methods: 136 patients were included thrombosis. Cerebral ischemia is the most common arterial manifestation of in the study. 58 patients with SLE, 36 PAPS, 11 SAPS, 11 asymptomatic arterial compromise. In venous circulation deep and superficial vein of the aPL and 12 with other autoimmune diseases. The median age was 46 y leg are the most frequently reported sites of thrombosis, but a less usual (range 18-89) and 77% were female. MRI scans were analysed for the form is the cerebral venous sinus thrombosis. The authors describe the case presence of cortical infarets, white matter changes [periventricular of a 24-year-old woman with a history of thrombocytopenia treated / hyperintensity (PVH; 0-6), deep white matter hyperintensity (WMH; 0-24), dependent of corticosteroids, who came to the hospital complaining of basal ganglia hyperintensity (BGH; 0-30), infratentorial foci of migraine and blurred vision. In the observation she had papilar edema. We hyperintensity (ITF; 0-30), atrophy (Pasquier scale; 0-39) and calcifications. did a computted tomography scan of the brain, which was nornal, followed Results: 81% of the MRI studies were abnornal. Cortical infarcts were by a magnetic ressonance that revealed cerebral venous thrombosis of the observed in 6% and calcifications in 14%. Cerebrovascular events were lateral sinus. She was treated with intravenous heparin, followed by expressed as stroke in 16% and TIA in 19%. 39 patients without warfarin. We have done a trombophilia screen protein C, protein S, neurological symptoms had MRI changes (30.5%). There were significant antithrombin III, factor V Leiden, aPL antibodies and lupus anticoagulant, association between arterial thrombosis and PVH (p<0.001), ITF (p< 0.001), that were within normal limits. She recovered nonnal vision but maintain HBG (p<0.05) and Pasquier (p<0.001). Cognitive impairment was related to headches. Two years latter she had a stronglypositive aPL antibodies. higher values of PVH (p<0.001), WMH (p<0.05) and Pasquier (p< 0.05). No relation was found between white matter changes or atrophy and aPL except for ITF (p< 0.001). A good relation between beta2GPI and antiprothrombin antibodies, LAC and cortical infarcts was detected (p< 0.05; p< 0.001; p< 0.05). The age was the only independent variable related with abnormal MRI. Conclusion: MRI abnormalities are frequent in these patients. The age and the presence of aBeta2GPI, aPT and LAC are important risk factors. Wus Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lClh ktnicui (ngs onAtiiosplidAtibocUes PotPreim s 604 ACTIVATION OF ERK IN BRAIN ENDOTHELIAL CELLS BY INDUCTION OF EXPERIMENTAL APS WITH CNS THROMBIN AND ANTIPHOSPHOLIPID ANTIBODIES AND ITS INVOLVEMENT IN 5 DIFFERENT MOUSE STRAINS MODULATION BY THE RAS INHIBITOR, A. Katzav1, C.G. Pick2, A.D. Korczyn', Y. Litvinjuk'4, S. Shrotl, FARNESYLTHIOSALICILATE M. Blank3, Y. Shoenfeld3, P. Sirota4, J. Chapman' R. Aronovichl, D. Gurwitz', Y. Kloog2, J. Chapman' 'Department ofPhysiology and Pharmacology, Sackler Facultv Of 'Sackler Faculty ofMedicine, 2 Wise Faculty ofLife Sciences, Medicine, 2Department ofAnatomy, Sackler Faculty ofMedicine, Tel Aviv Tel-Aviv University, Tel-Aviv, Israel University, TelAviv, 3Department ofMedicine B and Research Unit Of Autoimmune Diseases, Chaim Sheba Medical Center, TelHashomer, Background: The antiphospholipid syndrome (APS) commonly affects the `Abarbanel Mental Health Center, Bat Yam, Israel central nervous system through mechanisms that may include small vessel pathology and activation of thrombin. Antiphospholipid antibodies (aPL) Objective: The antiphospholipid syndrome (APS) includes systemic and have been reported to activate endothelial cells, however the activation of central nervous system (CNS) pathology associated with antibodies to a brain vascular endothelial cells (BVEC) and the receptors and signaling complex of phospholipids and b2-glycoprotein 1 (b2-GPI). We have recently pathways involved have not been fully characterized. Objective: To examine reported the induction of APS associated with behavioral and cognitive whether aPL and thrombin activate brain vascular endothelial cells through deficits in Balb/C femnale mice that dcvcloped 4-5 months following the Ras-ERK pathway. Methods: SV40 immortalized rat BVEC (line GP8; immunization with b2-GPI. In the present study, we examined the influence donation of J. Greenwood, London, UK) were grown to confluence on of genetic factors on the ability to induce experimental APS with CNS by testing several mouse strains immunized with b2-GPI. collagen-coated 24-well plates. IgG fractions were purified from 5 APS involvement patients on a protein G column and applied to confluent BVEC for 2 h. Methods: Female mice from five strains were immunized once with b2-GPI Levels of ERK phosphorylation in the IgG exposed BVEC were measured in complete Freund's adjuvant (CFA) or with CFA alone (controls). by Western immunoblots utilizing an antibody directed towards Neurological assessment in a staircase test and in a T-maze alternation test was 4-5 month following the immunization. phosphorylated ERK. Results: Enhanced phosphorylation of ERK was performed Results: Immunization with b2-GPI resulted in elevated levels of antibodies against observed following exposure of BVEC to thrombin (0.1 Unit/ml; 5 min) or to LPS (10 ug/ml; 5 min) and this increase was blocked by the Ras inhibitor negatively charged phospholipids and b2-GPI in all five mouse strains. famesylthiosalicilate (FTS; 25 uM; applied 2 h prior to thrombin or LPS). Autoantibody levels were significantly higher in Balb/C, ICR, and C57/BI Exposure of BVEC to aPL IgG (1:100 relative to serum) from 3 patients mouse strains compared to AKR and C3H/eb mouse strains. Hyperactivity also induced ERK phosphorylation. This activation was almost completely reflected by higher number of stairs climbed in 3 minutes, was seen in the blocked by FTS, 25 mM. Conclusions: Activation of the Ras-ERK pathway Balb/C, ICR, and C3H/eb mouse strains. Anxiety reflected by more frequent is an effect of both APS IgG and thrombin. This pathway is potentially rears, was seen in the Balb/C, AKR, and C3Hleb mouse strains. Cognitive amenable to drugs such as FTS and may serve as a therapeutic target in decline in the T-maze altemation test was seen in the Balb/C, C3H/eb, and APS. C57tBl mouse strains. Conclusion: In this study we demonstrate the ability to induce APS with CNS involvement in different mouse strains. These results indicate that there is no clear correlation between autoantibody levels and neurological impairment, and suggest a complex genetic immune interaction. STROKES SECONDARY TO ANTIPHOSPHOLIPID ANTIBODY APS EXACERBATES COGNITIVE IMPAIRMENT IN AN APP SYNDROME IN SLE MOUSE MODEL OFALZHEIMER'S DISEASE M.R. Sivakumar A. Katzav', C.G. Pick2, A.D. Korczyn', M. Blank3, Y. Shoenfeld3, J. Chapman', Apollo Hospitals, Chennai, Tamilnadu, India 'Department ofPhysiology and Pharmacology, Sackler Faculty of Objective: To evaluate the prevalence of Strokes in Systemic Lupus Medicine, 2Department ofAnatomy, Sackler Faculty ofMedicine, Tel Aviv Erythematosus(SLE) from India. Background: SLE is a severe autoimmune University, TelAviv, 3Department ofMedicine B and Research Unit of disease which affects multiple organs. In 1999, The American College of Autoimmune Diseases, Chaim Sheba Medical Center, Tel Hashomer, Israel Rheumatology (ACR), developed case definitions for 19 NPSLE syndromes. Cerebrovascular disease is often associated with the Immune and inflammatory mechanisms have been consistently implicated in Antiphospholipid Syndrome (APS). Design/Methods: 551 consecutive the pathogenesis of neurodegenerative diseases. Conversely, many immune patients from January 1985 till June 2001, who satisfied more than 4 of the mediated diseases of the central nervous system lead eventually to 1982 ACR revised criteria for the diagnosis of SLE, were evaluated. neurodegeneration. Clinically, genes influencing neurodegenerative Neuroimaging with MRI of Brain and Spinal cord, MR Angiogram, 4 vessel diseases, such as APOE, also influence the course of neuroimmunological Digital Substraction Angiography, EEG and Cerebrospinal Fluid analysis diseases such as multiple sclerosis. We have investigated whether immune were done. Antibodies to ANA, ds DNA, Cardiolipin (IgG, IgA and IgM), challenges can modify the course of animal models of neurodegenerative diseases. The animal model chosen is a mouse strain carrying a pathogenic Beta 2 Glycoprotein I, Anti-Ul RNP, Anti-Sm were performed. Lupus Anticoagulant (LAC) [screening and confirmatory], RPR test and mutation in the bAPP gene. The immune challenge used was induction of Complement CH50, C3 and C4 were estimated. Patients with Stroke were antiphospholipid antibodies by immunization with b2-glycoprotein 1. This evaluated in detail. Results: There were 502 women and 49 men with a inununization has previously been found to induce behavioral and cognitive mean age of 35(range: 19-68) Cerebrovascular disease were present in 72 dysfunction in a number of normal mouse strains, but not in C57BL, the patients (13%). Arterial ischemic stroke were seen in 47(8.53 %), venous background for the transgenic mice used in the present study. The mice were infarctions in 8 (1.45%), recurrent ischemic strokes in 12 (2.18%) and immunized at the age of 4-5 months and 4.5 months later were tested for Intracerebral hemorrhage in 5 (0.91%). The following antibodies were hyperactivity and anxiety on a staircase apparatus and for cognitive function present: Anticardiolipin antibodies-31 (5.62%) [IgG-21 and IgM-10], Anti in a swim T-maze. There were significant differences in behavior between beta2 Glycoprotein I -in 4 (0.73%), LAC-16 (2.90%) and false positive the APP transgenic mice compared to the controls in both tests. Induction of antiphospholipid antibodies impaired performance in the cognitive test only RPR- 34 (6.17%). Couclusions: Cerebrovascular disease was an important manifestation and was associated with severe, active disease and the in the APP transgenic mice and had no significant effect in the wild type presence of anticardiolipin antibodies. Early recognition and initiation of controls. The immunization had no significant effect in the behavioral assay. anticoagulant therapy is life saving. These results indicate the potential role ofimmune mediated mechanisms in the pathogenesis of neurodegenerative processes and point to the potential use ofimmunomodulatory therapies in such diseases. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 IrllXhtndOr*Sss anhAitopdAIo bodes Psrscm 605 THROMBOPHILIA AS A PREDICTOR OF RECURRENT CLINICAL SIGNIFICANCE OF ACTIVATED FACTOR XIII IN MISCARRIAGE PRIMARY AlS M.O. Sugiura, Y. linuinia, K. Katano, Y. Ozaki, K. Suzumnori P.R.J. Amesl, A. Margarita2, L. Ialusaccone3, V. Brancaccio' Nagoya City University Medical School, Nagoya, Japan 'Department ofHaematology, Countess of Chester Hospital, Chester, 2Department ofAngiology, Lupus Research Unit, UK, 3Coagulation Units, To investigate whether decrease of protein C (PC), protein S (PS), Carderelli Hospital, Naples, Italy antithrombin III (ATIII), factor X1I (FXII) and factor XIII (FXIII) have predictive value for subsequent miscarriages. A total of 536 cases with a Factor XIII is involved in the cross-linking offibrinogen and stabilisation of history of two or more first trimester miscarriages were examined. Of the the fibrin clot. We aimed to investigate its significance in primary 536, 112 cases started ASA at 4 weeks' gestation because they were antiphospholipid syndrome (PAPS). Activated FXIII (FXIIIa, amydolitic antiphospholipid antibodies (aPL) positive or had a previous history of assay) was measured in 36 PAPS patients (M12, F24, mean age 38±16 failure with paternal lymphocyte immunization therapy. PC, PS, ATIII, FXII years) and in 28 non thrombotic carriers of idiopathic antiphospholipid and FXIII were examined at exactly 4 weeks' gestation. The subsequent antibodies (aPL) (M4, F24, mean age 36±13 years). It was also measured in pregnancy outcome for the 424 cases treated with no ASA were compared 15 non APS thrombotics (M6, F9, mean age 36&13 years) and in 20 normal between cases with abnormal and normal values for each parameters. We subjects (1OMF, 38±16 years) who served as positive and negative control also analyzed the effect of ASA on subsequent live birth in 112 patients groups. FXIIIa was highest in PAPS (132±33%) than in aPL (118±33%), separately with respect to various parameters and aPL. Data for cases caused non PAPS thrombotics (113±31%) and normal controls (106±22%)(p<0.01). by abnormal embryonal karyotype were excluded from the analysis. There In PAPS, FXIIIa was non significantly higher in patients with arterial than were no differences in subsequent miscarriage rates between abnormal and venous thrombosis (144±40% vs 127±29%) but strongly correlated to the normal PC, PS, ATIII and FXIII. The rate with abnorrnal FXII is number of thrombotic events (r-0.6, p=<0.0001). In 25 PAPS patients signiificantly higher than that with normal FXII. A higher miscarriage rate intima media thickness (IMT) of three segments of carotid arteries was found in patients receiving no medication than in those administered (common, bifurcation, internal) was measured by high resolution aspirin with decreased FXI (p=0.03, Odds ratio=28) or with aPL (p=0.0l, sonography. In this group, FXIIa was correlated to anticardiolipin antibody Odds ratio=4.8). Factor XII decrease but not PC, PS, ATIII or FXIII predicts titre (r=0.47, p=O.01), plasma fibrinogen (r=0.5, p=0.02) and most subsequent miscarriage in paticents with a history of first triimiester recurrent importantly to IMT of common carotid (r-'0.46, p=0.0l) and carotid miscarriages. Low dose aspirin has potential for prevention of miscarriage bifurcation (r=0.59, p=0.002). Enhanced activation ofFXII in patients with caused not only by aPL but also factor XII decrease. PAPS may contribute the development of thrombosis and atherosclerosis in PAPS. FACTOR V LEIDEN, FII G20210A AND MTHFR C677T AND THE COMPARISON OF THREE DIFFERENT VACUTAINER RISK OF VENOUS THROMBOEMBOLISM, ARTERIAL COLLECTION SYSTEMS FOR THE DETERMINATION OF THE THROMBOSIS AND RECURRENT MISCARRIAGE IN APS DILUTE RUSSELLS VIPER VENOM TIME F. Pieroni, M.V.C. Freitas, M.A. Zago, A.G. Araujo, M.H. Tavella, W. F. Warner, A. Bowyer, M. Makris, M. Akil, D. Pastorelli, P. Louzada-Jr, R.F. Franco C. Brookfield, E. Hampton, S. Kitchen School ofMedicine, Ribeirao Preto University ofSao Paulo, Brazil Royal Hallamshire Hospital, Sheffield, UK The contribution of genetic thrombophilia to tihe occurrence of venous We have compared three types ofBD Vacutainer evacuated blood collection thromboembolism (VTE), arterial thrombosis (AT) and recurrent tubes: siliconised glass containing either 0.105 M buffered sodium citrate miscaniage (RM) in APS is still under investigation. The aim of our study (Glass-Citrate) or 0.109 M buffered sodium citrate, theophylline, adenosine, was to determine whether genetic factors for thrombophilia influence the dipyridamole Glass-CTAD); and plastic BD Vacutainer Plus Citrate tubes risk of thrombosis and RM in APS Brazilian patients. The frequencies of containing 0.109 M buffered sodium citrate (Plus-Citrate) for dilute factor V Leiden (FVL), factor II (FII) G20210A and methylene- Russell's viper venom time (DRVVT) testing. Samples were collected in tetrahydrofolate redutase (MTHFR) C677T polymorphisms were random order from 20 normal subjects and 11 patients with previously determined in three different groups of unrelated patients with APS: [G1] abnonnal DRVVTs. All samples were centrifuged twice at 2000 g for 15 APS-VTE (37 patients, mean age 36ys); [G2] APS-AT (20 patients, mean mins. and stored at 70C prior to testing. DRVVTs were performned manually age 35ys); [G3] APS-RM (22 patients, mean age 35ys). As control group, on each sample using Diagnostic Reagents materials (Oxford, UK), and we evaluated 387 healthy blood donors. Odds ratios (OR) for APS-VTE subsequent corrections using freeze-thaw fractured platelets (correction related to heterozygous FVL was 2.4 and for heterozygous FIIG2021OA, the ratio). In normal subjects the mean DRVVT ratios (with SDs in brackets) OR was 6.7. Overall OR for MTHFR C677T was 0.9. OR for APS and were: Glass-Citrate 1.01 (0.04); Plus-Citrate 1.05 (0.04); Glass-CTAD 1.04 arterial thrombosis linked to heterozygous FVL was 2.2, for FII G20210A (0.04). Although the differences were small, results in Glass-Citrate were the OR was zero, and OR associated with MTHFR C677T were: overall OR: statistically lower (ANOVA, p Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 iamh htangard OQgess onictqhiqidAtibDdes Post Pmsftsetk 606 THROMBOSIS IN LUPUS PATIENTS. ANTIPHOSPHOLIPID PROTEIN C INHIBITOR, ISOLATED TOGETHER WITH ANTIBODIES, ANTI - INHIBITOR PROTEIN ANTIBODIES OF BETA2GLYCOPROTEIN 1, REACTED WITH APS SERA BLOOD COAGULATION, AND NON INMUNE RISK FACTORS B. Bozic', S. Cucnikl, I. Krizaj2, T. Kvederl STUDY 'University Medical Centre, Department ofRheumatology, M. Mauri, J. Ordi-Ros, F. Paredes, J. Villarreal, E. Balada, M. Vilardell 2National Institute JosefStefan, Department ofMolecular Biology, Hospital Vall D'Hebron, Barcelona, Spain Ljubljana, Slovenia Objective: To evaluate the immune and non immune risk factors associated Beta2-glycoprotein I is the major target molecule for the so-called to thrombosis in patients with SLE. Methods: 239 patients with SLE (203 anticardiolipin antibodies. The isolation of the antigen is believed to be the females, 36 males) were evaluated. Non immune risk factors for arterial crucial phase, due to a possible cleavage of the fifth domain, mediated by (AT) and venous (VT) thrombosis were assessed. Immune risk factors were plasmin or by perchloric acid. Our work was oriented in the evaluation of canied out by independent ELISA methods for IgG and IgM antibodies the isolation procedure of the beta2GPI with the special emphasis on the against aCL, aPE, beta2GPI, FIL PC, PS, ATIII and FXII. LA was tested by possible presence of a/ different forms of beta2GPI, b/ immune complexes, coagulation tests. Homocysteine levels were assessed by an ELISA. A c/ some other co-isolated proteins. The isolations were perfonned by pearson Chi-Square was applied as the statistical analysis. Results: Sixty perchloric acid precipitation at, 18 and 50 minutes, followed by heparin SLE patients (25%) presented one or more thrombotic events. 55% affinity and cationic exchange chromatography. The properties of isolated presented a VT and 45% an AT event. Seventeen patients presented a proteins were tested by rocket electrophoresis, ELISA, SDS-PAGE, rethrombosis. The mean number per patient of non-immune risk factors for immunoblotting and N-terminal sequencing. Three distinct fiactions all AT was 2.7 and for VT 2.39.The non-immune risk factors associated with containing beta2GPI, were observed. The isolation procedure with 3-minute arterial thrombosis (p<0.005) were the time of disease duration, precipitation gave the highest yield of beta2GPI, but contaminated with hypertension, use of steroids, and hyperhomocysteinemia. The non immune immunoglobulins. The isolation procedure with 18-minute precipitation risk factors for venous thrombosis were male sex, disease activity, gave the best yield of unnicked beta2GPI, which was used for the pregnancy - puerperium, and steroids' use. The mean number per patient of preparation of the affinity column. The isolation procedure with 50-minute immune risk factors for AT was 3.6 and for VT 3.3.The significant immune precipitation gave the highest yield of proteins in the third peak, where a risk factors for arterial and venous thrombosis were LA, IgG aCL, and anti- protein C inhibitor was identified beside beta2GPI. It reacted with sera from beta2GPI IgG antibodies. No statiscal association was found neither with the patients with primary APS. The clinical importance of antibodies against other antibodies tested nor with IgM isotypes. Conclusions: Thrombosis is a protein C inhibitor is not known yet. But a/ antibodies against protein C common complication in patients with SLE Our results suggest that the inhibitor could simulate the APS reactivity in tested sera if protein C mechanism of thrombosis in SLE patients is multifactorial when the inhibitor is present as impurity or b/ protein C inhibitor could be the novel presence of both non-immune and immune risk factors interact. However, target antigen for the autoimmune response. antiphospholipid-protein antibodies are the major factors implicated. HETEROGENEITIES OF LUPUS ANTICOAGULANT ANTIBODIES INSENSITIVITY OF THROMBIN GENERATION TO ACTIVATED (LA): DIFFERENT LIGAND-SPECIFICITIES DETERMINE A PROTEIN C IN SELECTED PATIENTS WITH CLINICAL FEATURES OF APS ANTIPHOSPHOLIPID ANTIBODIES M. Yamazakil, M. Kaneda', T. Yoshida2, Y. Ontachil, T. Mizutanil, T. Ito', V. Regnault1, S. Beguin2, E. De Maistre3, D. Wahl"4, H.C. Hemker2, H. Asakural, S. Nakao' T. Lecompte"3 'Kanazawa Graduate University School ofMedicine, 2Kanazawa University 'EA 3452 - Faculte de Medecine de l'UHP - Vandoeuvre-Les-Nancy, Hospital, Japan France, 2Synapse B. V., University ofMaastricht, The Netherlands, We previously demonstrated that 4 subtypes of anti-prothrombin antibodies 3Hematologie Biologique, 4Medecine Interne et Medecine Generale II, CHU (aPT) and anti-beta2-GPI reflect differences in clinical features of APS. de Nancy, France Patients with LA bound only to human prothrombin (hFI1) in the presence An association between acquired resistance to activated protein C (APC) of PL and Ca ions (aPT #1) as well as anti-beta2-GPI antibody (ab2-GPI) and antiphospholipid antibodies is widely postulated but difficult to developed both arterial and venous thrombosis at a significant higher evidence. Thus we decided to investigate this phenomenon using an incidence than other aPTs. LA bound only to hFIl in the absence of PL and automatically registered thrombin generation curve (the thromb(in)ogram). Ca ions (aPT #2) was related to venous thrombosis but not to arterial Thrombograms were measured as previously described (Thromb Haemost, thrombosis. In contrast, LA bound to both human and bovine prothrombin 2000, 84: 747) with citrated plasma in the presence of patients' platelets or (bFII) (aPT #3 and #4) was not related to any thrombosis. To clarify DOPS/DOPE/DOPC vesicles. Four selected patients LA-positive on the mechanisms responsible for different hemostatic effects ofthe 5 subtypes of basis of the accepted criteria of the ISTH were compared to two normals LA, we determined effects of each LA antibody on hemostatic functions of that were at the limits of the interindividual variation of thrombin endothelial cells, monocytes, and platelets. Both aPT #1 and ab2-GPI generation. With the patients' plasma, the burst of thrombin generation inhibited PC activation, and augmented both TF production and PAI-1 started later and reached a lower peak than with normal plasma. The peak release significantly from endothelial cells. Moreover, aPT #1 significantly and the area under the curve were very sensitive to addition of APC in enhanced TF production on monocytes and platelet activation and ab2-GPI normal plasma but resistant to it in the patients' plasma. APC resistance was significantly activated platelet compared with normal IgG. APT #2 inhibited observed with all patients' samples in the presence of phospholipids from PC activation on endothelial cells. APT #3 and #4 had no effects on their proper platelets but not necessarily with added phospholipids. It mulst hemostatic functions of endothelial cells, monocytes, or platelets. These be pointed out that the plasma of our selected patients could be completely findings suggest that differences in hemostatic effects among subtypes of resistant to APC, whereas a subject homozygous for the V Leiden LA antibodies may account for heterogeneity of clinical features of APS. polymorphism remains 40 % sensitive to APC in comparable experiments. Detenrnination of ligand-specificity of LA is useful for predicting clinical In conclusion, (1) measuring thrombin generation can evidence the course of APS. coexistence of an anticoagulant effect (prolonged initiation phase) and of a procoagulant effect (acquired APC resistance); (2) this resistance may be so intense as to blunt any additive effect ofa possibly associated genetic defect, such as the common V Leiden polymorphism. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1amtletimiceMOrVglss i o l dAltoce-s obster tmseUloms ANTIPHOSPHOLIPID ANTIBODIES AND RHEUMATOID COFACTOR INDEPENDENT ANTICARDIOLIPIN ANTIBODIES IN 607 ARTHRITIS: PREVALENCE, CLINICAL SIGNIFICANCE IN A PATIENTS WITH LYMPHOPROLIFERATIVE DISEASES COHORT OF 200 CONSECUTIVE PATIENTS S.B. Andrejevic', M.N. Bukilica2, B.M. Bonaci-Nikolic' P. Sarzi-Puttinil, E. Rossi2, F. Atzenil, G. Santalenal, G. Randisil, 'Institute ofAllergy and Immunology, University Clinical Center, M. Carrabba' 2Institute ofRheumatology, University Clinical Center, Belgrade, Serbia, 'Rheumatology Unit, 2Transfusion Centre, L. Sacco University Hospital, Yugoslavia Milan, Italy Clinical features observed in the presence of anti-phospholipid (aPL) Background: To determine the prevalence, clinical significance of aPL and antibodies includes thromboses and recurrent fetal losses are usually not LAC in a cohort of 200 consecutive patients with Rheumatoid Arthritis present during lymphoproliferative diseases (LPD). The present study was (RA). Thromboembolic events are frequently observed in subjects positive aimed at characterizing aCL antibodies found during LPD by differentiating for aPL and/or LAC expecially if affected by autoimmune diseases; we serum cofactor-dependent and independent aCL antibodies in an ELISA therefore assess if the presence of aPL or LAC was associated with any performed with either bovine serum or gelatin respectively. Also, sera was clinical or biological manifestations of RA. Methods A group of 200 tested for the presence of anti-beta2GPI antibodies by commercial ELISA. consecutive patients (156 women and 44 men; mean age 48±12 years), Group of 16 LPD patients with previously confirmed lupus anticoagulant fulfillinig criteria for diagnosis of RA (1987 ACR criteria) were investigated (LA) activity was compared with group of patients with primary in a cross-sectional design. Main endpoints were: a) the occurrence of aPL antiphospholipid syndrome (PAPS). In the LPD group high prevalence of positivity b) the clinical and/or serological correlation with the presence of serum cofactor independent aCL were detected in the presence of gelatin. these autoantibodies. Results: aPL was found in 23 patients; IgG in 18 (15 Presence of bovine serum which contain beta2 GPI decreased significantly had low titer, 3 high titer) and IgM only in 6 patients (5 low titer, and only I the detection ofaCL in sera ofLPD patients. Presence of IgM anti-beta2GPI with a high titer). Only 4 patients had LAC. Only 2 patients with both aPL antibodies were detected by ELISA in sera at 3/16 LPD patients. and LAC had recurrent venous thrombosis and miscarriages. No correlation Conversely, in the sera of 34 PAPS patients detection of aCL was markedly was observed between the severity of the articular disease and the presence reduced in the presence of gelatin, and 27/34 PAPS patients had high of aPL. Moreover, no correlation was observed between the presence of concentrations of anti-beta2GPI antibodies. In conclusion: during LPD as extra-articular manifestations and the presence of aPL. Patient with RA and infectious diseases aCL were generally found to be cofactor independent aPL presented a higher prevalence of hypergammaglobulinemia and and directed against cardiolipin alone. antinuclear antibodies at low titer. Conclusions: aPL prevalence in RA patients is low (11.5% of the patients), with a close correlation with hyperganimaglobulinemia. The observation of antiphospholipid syndrome in presence of RA is unusual with only 2 patients of our cohort group that manifested this association. PERSISTENTLY HIGH IGG ACL RELATED TO THROMBOTIC HIGH INCIDENCE OF ARTERIAL THROMBOSIS DUE TO FEATURES IN BEHCET'S DISEASE, TAKAYASU'S ARTERITIS ANTIPHOSPHOLIPID ANTIBODIES IN PATIENTS WITH EVANS' AND WEGENER'S GRANULOMATOSIS SYNDROME; SIGNIFICANCE OF DETECTING LUPUS RIA. Levy', M.N. Elisa', A. Bozza2, B.E.G. Bica2, R.A. Asherson3 ANTICOAGULANT AS A PREDICTOR M. T. M. lUniversidade do Estado do Rio de Janeiro, 2Universidade Federal do Yamazakil, Yoshida2, Kaneda', Y. Ontachi', T. Mizutani', T. Ito', H. S. Nakao' Rio de Janeiro, Brazil, 3Johannesburg, South Africa Asakura', 'Kanazawa Gradutate School Most publications of aPL in primary necrotizing vasculitides are case University ofMedicine,Kanazawa, reports or small series and aPL is not always accompanied of clinical 2Kanazawa University Hospital, Kanazawa, Japan features of anti-phospholipid syndrome (APS). Thrombotic features in Evans' syndrome (EV) is an immune thrombocytopenia complicated by Behcet's disease (BD), Takayasu's arteritis (TA) and Wegener's autoimmune hemolytic anemia (AIHA). Thrombotic complications granulomatosis (WG) are well recognized but have been attributed to occasionally occur in patients with EV, particularly when platelet counts several reasons. Some have suggested that the aPL found in these patients increase after inmnunosuppressive therapy, although the exact incidence of are not related to clinical features of APS and have no clinical relevance. such complications in unknown. To clarify the mechanisms of thromboses We investigated IgG and IgM aCL by standardized method and beta 2 GP-I in patients with EV, we studied 14 EV patients (2 males, 12 females, 4 to 79 dependencies by an adapted ELISA method in 13 such patients. Of the six yo., median 25 yo.) for the presence of antiphospholipid antibodies (APAs) patients with BD, APS classification according to Sapporo's criteria could including anticardiolipin antibody (aCL), aCLJbeta2-GPI antibody (ab2- be fulfilled in, and clinical improvement was observed with warfarin in all, GPI), lupus anticoagulant (LA), and biological false positive (BFP) as well the sixth patient had recurrent migranes and thrombocytopenia that as for thrombotic complications. Twelve of the 14 patients (86%) had at improved with clopidogrel and hydroxychloroquine therapy. Of the 5 least one of the above APAs. Nine of the 12 patients with APA developed patients with TA, the only one with at least 2 moderate or high aCL titers thrombosis in the artery (6 patients) and in the vein (4 patients) while no had a stroke history and was on warfarin and ASA therapy. Of the 2 patients thrombosis occurred in 2 paticnts without any APAs. Among APAs with WG studied, both had moderate or high GPL in more than 2 occasions, detected, the positive LA test correlated most with occurrence of one patient is a 16 years old female with recurrent leg ulcers that improved thrombosis; all that patients who developed thrombosis had shown positive after infliximab therapy was started and the other had previous stroke and LA tests and 9/11 (82 %) patients with LA developed thrombosis. These deep veini lhrombosis and is on warfarin. The role of aPL in the findings indicate that APAs are frequently detected in EV-patients and that pathogenesis of the vasculopathy found in primary vasculitides has to be this probably accounts for the high incidence of thrombosis in them. The considered and if correctly approached, may improve survival. positive LA tests in the most reliable predictor for thrombosis in EV- patients. When LA is revealed in EV-patients responding to immunosuppressive therapy, administration of antithrombotic agents is recommended. bpsPL Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 10ahItaiaiui Omgess an AiopnhoidAniboles PoFteRsetan 608 LUPUS ANTICOAGULANT, ANTICARDIOLIPIN ANTIBODIES PREVALENCE OF CONGENITAL THROMBOPHILIA DEFECTS AND ANTIBETA2GLYCOPROTEIN 1 ANTIBODIES IN PATIENTS IN PATIENTS WITH APS WITH THALASSEMIA G. Papaioannoul, M. Speletas', S. Vasilikiotil, E. Katodritoul, Z. Mondher', C.M. Heithem2, L. Lilia', A.M. Slim2, S. Maryam', A. Pavlitou-Tsiontsi2 M.F. Ben Dridi2, M. Sondes' 'Haematology Department, 21mmunologyLaboratory, Papageorgiou 'Immunology Department, 2Pediatric Department, La Rabta, Tunis, Tunisia General Hospital, Thessaloniki, Greece Objectives: Because of the high prevalence of thrombosis in thalasemia we Interactions between genetic and acquired factors could contribute to decided to investigate the prevalence of antiphospholipid antibodies in a thrombosis development. This study set out to detennine the prevalence of prospective study of 22 children with tlhalassemia. Methods: IgG, Igm-, IgA congenital thrombophilia defects (FVLeiden, 20210A->G PT- anticardiolipin and IgG antibeta2glycoprotein I antibodies were tested by an polymporphism, a-nd MTHFR-677C->T as well as protein C, protein S and ELISA method and Lupus anticoagulant was detected by coagulation tests. antithrombin-III deficiencies) in 61 patients with antiphospholipid syndrome Results: 4 patients had antiphospholipid antibodies and none had lupus (APS), to evaluate whether these genetic alterations are risk factors for anticoagulant. Among these 4 patients, 2 had IgG anticardiolipin and thrombosis in APS. As control group we considered the prevalence of these antibeta2glycoprotein 1 antibodies associated to cerebral stroke and the 2 alterations that has been estimated in our area in previous published studies. others children had only IgA anticardiolipin antibodies. FVLeiden was found in 15 patients (24.6% compared to 3% of controls, Conclusion: antiphospholipid antibodies does not seem to be frequent in p<0.05), while 9 patients were carriers of the PT-20210A allele (14.7% thalassemia despite the thrombotic events. However there was no compared to 2% of controls, p<0.05). Protein C deficiency was established relationship between high levels of these autoantibodies and this disease. in 3 patients (4.9%), protein S deficiency in I patient (1.6%), and antithrombin-III deficiency in 1 patient (1.6%), compared to 0.1% of controls (p<0.05). However, no significant variation was found between the patient group and the controls, regarding the prevalence of homozygotes and/or heterozygotes for the mutated MTHFR-677T allele (24 of 61 patients, 39.3% versus 40% of controls). Interestingly, 5 patients displayed both FVLeiden and MTHFR-677C->T alteration, and 3 patients exhibited both PT-20210G->A polymporphism and the MTHFR-677C->T alteration. Eventually, 20 of APS patients (32.7%) did not display any well-defined genetic defect that predispose to thrombosis. Conclusively, it is obvious that the FVLeiden, and the PT-20210A->G polymporphism, as well as the protein C, protein S and antithrombin-III deficiencies are important risk factors that could increase the risk ofthrombosis in patients with APS. THE BENEFICIAL EFFECT OF ANTIMALARIALS IN THE GENETIC FORMS OF THROMBOPHILIA IN THE STRUCTURE TREATMENT OF IDIOPATHIC THROMBOCYTOPENIC OF GESTOSIS PURPURA A.D. Makatsaria, S.V. Baymuradova, V.0. Bitsadze, O.S. Alyautdina, E. Toubil, A. Kessell, D. Atias', B. Brener2 N.V. Dolgushina, D.T. Salickram 'Bnai-Zion Medical Center, 2Rambam Medical Center, Haifa, Israel Moscow Medical Academy, Russia Background: Many patients with refractory idiopathic thrombocytopenic 197 pregnant women aged between 21 and 45 years were examined for the purpura(ITP),require long corticosteroid therapy,followed frequently by presence of genetic and combined forms of thrombophilia. 50 women were splenectomy. It is therefore important to keep looking for more efficient healthy (I group) and 147 pregnants had gcstosis (II group). In 98 women regimens. Objective: Antimalarials were reported widely to be both anti- PCR method was used retrospectively. MTHFR mutation was revealed in 54 inflammatory and anti-thrombotic. In addition, these drugs were shown to women (55%) (homozygous fonn in 24 (24%) and heterozygous form in 30 down-regulate platelet activation markers. We therefore asked if they could women (31%)), FV/Leiden mutation - in 15 women (15%) (4 had improve thrombocytopenia in ITP patients and be added to the list of homozygous form (4%) and 11 had heterozygous form (11%)), combined potential treatments in this disease. Patients: Antimalarials were given to genetic forms of thrombophilia - in 15 women (15%); genetic forms three ITP patients in whom prednisolone (P, 15-20mg/d) and in one together with APS in 76 women (77%). In genieral, genietic defects of splenectomy also, were insufficient to increase thrombocytes above the hemostasis were detected in 69 women of the II group (70%/o). In this group range of 60-80 k/ul. Patients were followed for 8-12 months. Results: pregnancy was complicated with early and severe gestosis, miscarridge in Hydroxychloroquine (HCQ) in one and the combination of HCQ and the term of 22-32 weeks of gestation due to severe gestosis in 24 women Quinacrine (Qn) in another two, improved and kept thromhocytes in the (25%), fetal growth retardation in 2 women (2%), eclampsia in 2 women range of 100-120 k/ul and allowed the decrease of P dosage to 2.5-5 mgld (2%) and stillbirth in 28 women (28%). 49 women were examined during the period of follow-up. Conclusion: Antimalarials are shown to be prospectively: MTHFR mutation was revealed in 25 women (52%) beneficial in improving thrombocytopenia and should be considered among (homozygous forn in 10 (21%) and heterozygous fomi in 15 (31%) of other regimens in the treatment of ITP. The possibility that they have women), FV/Leiden mutation - in 5 women (11%) (2 had homozygous form modulatory effects on platelet activation and may attenuate anti-platelet (5%) and 3 had heterozygous form (6%)), PtG20210A mutation in 3 antibody production should be investigated. It is of interest to investigate women (6%), combined genetic forms of thrombophilia - in 8 women whether a combination of HCQ and Qn is more beneficial than HCQ alone (18%), genetic forms together with APS in 37 women (75%), APS only in in ITP. 7 women (7%). In general, genetic forms of thrombophilia were detected in 33 women (67%). In the I group heterozygous form of MTHFR mutation was revealed in 3 women (7%) and heterozygous form of FV/Leiden mutation - in 1 woman (3%). Small doses of aspirin were administered to all pregnants with genetic forms of thrombophilia beginning from the conceptional cycle. Folic acid and vitamins of B group were also prescribed from early terrns of pregnancy in the presence of MTHFR mutation. D- dimer detection, FV/Leiden mutation and PtG2021OA mutation were the indication for LMWH (Fraxiparin) use. bWm Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 10thlrtanicid Owgss mPFon iicipdhd1imod P&nPresatioM RELATIONSHIP BETWEEN P2-GLYCOPROTEIN I SER316SER BETA2-GLYCOPROTEIN-I VARIANT WITH 609 POLYMORPHISMS AND ITS PLASMA CONCENTRATION REDUCED BINDING TO PHOSPHOLIPID ASSOCIATED WITH ANTI-BETA2-GLYCOPROTEIN I ANTIBODIES IN A PATIENT F. Lin', C. Feighery2, E. Casey3, J. Jackson' WITH PREGNANCY COMPLICATIONS 'Def,artment ofBiological Sciences, Dublin Institute of Technology, R.S. Camilleri, M.J. Nash, R. Leisner, I.J. Mackie, S.J. Machin, H. Cohen Department ofImmunology, 3Department ofRheumatology, St James' Hospital, Dublin, Ireland Haematology Department, University College London, UK 02-glycoprotein I (02GPI) is a phospholipid-binding protein thought to be Beta2-glycoprotein I (beta2GPI) is an important target antigen of involved in the regulation of the coagulation cascade. To date, four point antiphospholipid antibodies (aPL) and serves as a cofactor in the binding of mutations have been well characterized in the,B2GPI gene at positions 88 aPL to lipid surfaces. Previous studies have shown that individuals with a (Ser/Asn), 247 (Val/Leu), 306 (Cys/Gly), and 316 (Trp/Ser). 02GPI polymorphism at codon 316 of the beta2GPI gene (resulting in a tryptophan polymorphisms apparently affect plasma ,B2GPI levels, as shown by the to serine substitution) have reduced binding of beta2GPI to phospholipid surfaces. It has also been that this may be observation that polymorphisms at the codons 306 and 316 may have a reported polymorphism protective significant lowering cffcct in a dose-dependent fashion (Ruiu G et al. 1997; against the production of aPL as it may prevent expression of the epitope Kamboh MI et al. and Mehdi H et al. 1999). In this study, we measured that promotes aPL production. Our patient (36 years old) presented with a past obstetric history of a stillbirth at 28 weeks associated with hypertension P2GPI levels (using a sandwich ELISA) and analyzed the four P2GPI chain reaction followed by allele-specific and severe intrauterine growti restriction and an early miscarriage. She was polymorphisms (using polyrmerase subsequently found to be persistently positive for both IgG anticardiolipin restriction enzyme digestion using Tsp509I, RsaI, iVszl, and BstBI (New 157 healthy Irish individuals. antibodies (10-20 GPLU, normal<5 GPLU) and anti-beta2GPI antibodies England Biolabs Inc, Berverly, MA, USA) in (28-40%, normal<3%). She has since successfully delivered three healthy While no statistical difference in 32GPI levels were observed among the babies when managed antenatally. Genotyping of this patient has revealed genotypes of the mutations at position 88 (P=0.8564) and 247 (P=0.3061), she is homozygous for the codon 316 Trp to Ser beta2GPI polymorphism. extremely significant differences in P2GPI concentrations were found Studies examining the binding capability of the patients beta2GPI to among the genotypes of the mutations at position 306 and 316 (P<0.000l cardiolipin reveal markedly reduced binding in comparison to normal for both). For the mutations at positions 306 and 316, the presence of the beta2GP1. It is of particular interest that this patient possesses a Ser316Ser miutated allele in the genome had a significant lowering effect on the plasma beta2GPl variant, which lacks lipid binding but still has a significant titre of j32GPI levels. The potential significance of these findings with regard to anti-beta2GPI antibody activity that is manifesting as multiple pregnancy thrombophilia remain to be elucidated. complications. A possible explanation is that patemal beta2GPl expressed on trophoblastic tissue may be acting as both a stimulant to, and a target of, the matemal immune response, resulting in pregnancy loss. APS IN THE UVA CLINICAL DATA REPOSITORY (CDR) A CASE OF PRIMARY APS CONVERTED IN SLE AFTER 10 1996-2001 YEARS G.A. McCarty'2, T.E. Cason', K.W. Scully2 G. Filsel, E. Vittori, C. Oliveri, F. Puppo, F. Indiveri 'Division ofRheumatology and Immunology, 2Department ofHealth Department ofInternal Medicine, University ofGenoa, Italy Evaluation Sciences, UVA Health Systems, Charlottesville VA, USA Patient C.A., a 45 year old male, came for the first time to our observation in Objective: APS is diagnosed in patients (pts) if a proven thrombosis occurs 1990 due to recurrent episodes of deep venous thrombosis and seizures. w/ pos. anticardiolipin(aCL) and/or lupus anticoag- ulant(LAC)by Sapporo Laboratory analyses demonstrated circulating anti-phospholipid antibodies Cnrtena. From 7/00-5101, 244 pts. w/ APS were seen in Rheum Clinic. (IgG and IgM), reduced platelet count (73000/mmc), prolonged activated Using presence in the Clinic as an outcome variable, we ascertained the partial thromboplastin time (45 sec). Anti-nuclear antibodies were negative completeness of cachment of APS pts.there relative to the UVA CDR, a and complement was not consumed. We diagnosed the primary anti- system-wide legacy database. Methods: CDR 6/96-12/01 was queried by phospholipid syndrome and treatment with acenocumarol and aspirin was SQL for unique case identification of all pts w/ >1 pos. administered. The patient, yearly monitored, did not present recurrence of aCTJaPTT/DRVVVT test & a proven thrombosis & a primary/secondary thrombosis. In February 2002 the patient came again to our observation ICD9/CPT code of SLE and/or APS, with at least 1 outpt (OPV) or showing edema of the legs and numbness associated with elevated ESR (90 inpt(OPV) visit of age >Sy. Demographics, IPV, OPV, Service (SVC), Total mm/h), macrohaematuria, proteinuria (> 20 gr/day), reduced proteinemia Charges (TOTCHG), Total Cost (TOTCST), physician charge (PHYSCHG), (3.5 gr%/o), increased creatinemia (1.8 mg%), positivity of Coombs test, physician cost (PHYSCST), Payor (PAYOR), and financial screen level presence of anti-nuclear antibodies (> 1:160, homogeneous pattern), and (FSLI-7 =!00% selfpay) were determined. ACR and Sapporo Criteria were highly consumed C3 and C4 complement factors. The echocardiography used for diagnoses (Dxs) Results: 176 unique visits were made by 143 pts; showed the presence of a small pericardial effusion. Renal biopsy 91.6% F, 73.4%W, median age 39.0y. 27.9%> 50y, 11.9% < 21y. Dxs 3 demonstrated a proliferative (class V) and membranous (class IV) were: SLE 31.8%, Other Dxs 3 1.8%, APS 19.9%, Misc 8.5%, RA 4.0%, NA glomerulonephritis with sub-epithelial and sub-endothelial deposition of 4%. SVCs were Rheum 51.5%, Int Med 17.6%, NA 6.3%, Peds 3.4%, immunocomplexes. The patient developed crises of malignant arterial Neuro 2.8%, Heme/OBGyn 0.6%. Median TOTCHG was $566 hypertension (220/130 mmHg) causing reversible posterior ($2-$330,589, median TOTCST $399 ($37-$229,217) and median leucoencephalopathy. On the basis ofclinical, serological and bioptic results PHYSCHG was $214(0-$143,020. Multiple visits, age, sex, IPV, OPV, yar, we made diagnosis of SLE for which we started treatment with high dose and Dx was not significantly associated w/ presence in Rheum Clinic methylprednisolone (1 gr i.v. boli for 3 consecutive days) and cycles of nonwhite race and FSL 7 as a proxy for poor SES was significantly plasmapheresis followed after 24 hours by cyclophosphamide boli (1 gr i.v.) associated (P=0.0042 and P=0.0007 respectively). Only 33.6% of all pts repeated every 3 weeks. This case supports the concept that the primary identified had yet been seen in Rheum Clinic-2/3 were not yet identified as anti-phospholipid syndrome can switch to SLE even after long term from APS despite meeting Sapporo Criteria. APS is associated with high care diagnosis. utilization in visits and high hospital charges due to recurrent CVA, PTE, DVT, stent thrombosis, and inadequate Rx. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 10G IktemauiOaless onAtpioudliddAribocies P iseUrn 610 THE ASSOCIATION OF THE ANTIPHOSPHOLIPID ANTIBODY ANTICARDIOLIPIN ANTIBODIES IN GREEK PATIENTS WITH RESPONSE WITH DRB1/DQB1 CODING AND QBP PROMOTER CHRONIC HEPATITIS C AND B (HBEAG NEGATIVE) VIRUS POLYMORPHISM INFECTION D. Logar', B. Vidan-Jeras2, A. Ambrozic', V. Dolzan3, M. Hojnik' A. Pavlitoul, G. Germanidis2, A. Giannakoul, J. Xanthakis2, A. Gantzarou2, 'Clinical Centre, 2Tissue Typing Centre, Blood Transfusion Centre, V. Galanopoulou 2, R. Zilidou', A. Fleval, E. Pagkalos' 3Institule ofBiochenmistry, Faculty ofMedicine, University ofLjubijana, 'Department ofImmunology, '1st Department ofInternal Medicine, Slovenia Papageorgiou General Hospital, Thessaloniki, Greece Objective: The antiphospholipid syndrome consists of arterial or venous Aim of the study: To determine the prevalence of anticardiolipin (aCL) and thrombosis and/or recurrent fetal loss in the presence of anticardiolipin anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in Greek patients with antibodies (aCL) and/or lupus anticoagulants. The aim of our study was, to chronic hepatitis C and chronic hepatitis B (HlBeAg negative) virus ascertain if the polymorphism ofHLA-DR, DQA aisd DQB alleles and QAP infection. Patients and Methods: Fifty one (51) consecutive patients with and QBP promoters influences the production of aCL and anti-beta2GPI in biopsy proven chronic hepatitis C and HCV RNA positive in serum were SLE. Methods: We analysed a group of 65 consecutive unrelated Slovenian studied. In 8 patients the histological diagnosis was cirrhosis (15,7%). Forty SLE patients (all female, mean agc 36.3 ycars, mcan follow-up 93 months). one (41) patients are actually on treatment. Twenty four (24) consecutive The control group consisted of 74 unrelated healthy adults. aCL and anti- patients with biopsy proven chronic hepatitis B (HBeAg negative) and HBV were by in-house beta2GPl (IgG, 1gM and IgA isotypes) determined ELISA. DNA positive in serum were also studied. Ten (10) patients (41,7%) were controls were typed for DRBI, DQBI, QAP and The patients and QBP cirrhotic and 20 are actually on treatnent. IgG and IgM aCL and anti- alleles by PCR-SSO, using the 12t IHW pnrmers, probes and protocols. The beta2GPI antibodies in senum samples before and at 6 months during subtyping of DQB I alleles as well as DQAI typing were carried out with treatment, were measured by ELISA. A wide spectrum of autoantibodies selected Dynal SSP primers. Allelic and deduced haplotypic frequenieis in were also tested. Results: In chronic hepatitis C patients, IgG-aCL patients and controls were compared using Fishee's exact test. Results: 32 of antibodies were positive in 1/51 (2%). IgM anti-beta2GPI antibodies were were positive for IgG and/or aCL while 16 of 65 SLE patients (49%) 1gM positive in another patient 1/51 (2%). ANA 1/160 in 3/51 (6%), 65 (25%) were positive for anti-beta2GPI. Significantly increased ASMA 1/80 in 13/51 (25%), RF 100 IU/mL in 6/51 (12%) and anti-TPO frequencies of the DRBI*07, DQB1*0202, DQAI*0102 haplotype were 1 00 IU/mL in 3/51 (6%) were also detected. In chronic hepatitis B patients, found in the anti-beta2GPI negative as well as in the aCL negative group of IgM-aCL antibodies were positive in 1/24 (4%). ANA 1/160 in 1/24 (4%), patients compared with controls (P=0.0057 and P=0.001 I respectively). On the other hand, the DQBI *0301 allele and it's promoter QBP3.1 were ASMA 1/80 in 11/24 (46%), RF 100 IU/mL in 10/24 (41,7%) were also detected. No has changed during treatment. significantly underrepresented in the anti-beta2GPI negative as well as in antibody reactivity The positive the aCL negative patients compared with controls (P=0.001 and P=0.06 detection of aCL and anti-beta2GPI antibodies was not correlated with any respectively) Conclusions: The DRB 1*07, DQB 1*0202, DQAl*0102 clinical manifestation. Conclusion: The prevalence of aCL and anti- haplotype may have a protective role against aCL and anti-beta2GPI beta2GPI antibodies in Greek patients with well characterized chronic immune response. The DQB 1*0301 allele and its promoter QBIP3.1 did not hepatitis C and B is not significant and does not change during treatment. appear to have a significant role in directing aCL and anti-beta2GPI Their presence is not correlated with any clinical manifestation. autoimmune response in our SLE patients. THE ASSOCIATION OF HLA DR2 AND THE SUSCEPTIBILITY OF SLE aPL INDUCED BY IMMUNIZATION WITH THE PHOSPHOLIPID- AND PRIMARY APS IN INDONESIA BINDING SITE OFB2GLYCOPROTEIN I REGOGNIZE K. Handonol, H. Kalim2 CONFORMATIONAL EPITOPES ON A BACTERIAL PEPTIDE 'Department of Clinical Pathology, 2Department ofInternal Medicine, Faculty of S.S. Pierangelil, M. Blank2, R.G. Espinola', Y. Shoenfeld', E.N. Harris', Medicine, Brawyaya University, Malang, Indonesia A. Gharavi' Compared with the Caucasian SLE, Indonesian patients have more severe diseas, 'Morehouse School ofMedicine, Atlanta, GA USA, 2Chaim Sheba Medical activity, younger disease onset and lower life expectancy. Such poor prognosis ha. Center, TelAviv, Israel been found in Oriental Chinese patients too. These differences are thought to b4 We previously reported the production of monoclonal antiphospholipid with the differenc of genetic; background in Caucasian and Indonesia o associated (aPL) antibody in mice after immunization with GDKV (a peptide that Oriental Chinese. The aim of the study was to determine the association between HLI represents the phospholipid binding site on the Vth domain of B2GPI) clas 11 with the risk of SLE and primary APS. Cases to be studied were 91 unrelate( (GDKV-MoAb). This antibody was pathogenic in vivo and had similar patients with SLE and 23 patients with primary APS, age 26.92 ± 8.79 years, female: 94.51% from several University Hospital in Indonesia. As controls, 126 unrelatem binding characteristics to autoimmune aPL. Recently, a peptide (CATLRVYKGG) (named peptide A) identified in bacterial antigens that healthy persons matched in age, sex and race. Subject from these are shared the sami HLA haplotype. All of the samples were assayed for autoantibody expression and HLI shares sequence similarities with region ITI of B2GPI (PNAS (USA) 1999; class II genes. Autoantibodies (anti-ds DNA, anti Sm, anti-Ro/SSA, anti-La/SSB, anti 96:5164) was shown to induce pathogenic aPL antibodies in mice RNP, ACA IgG, ACA IgM) were determined by ELISA procedure. HLA class I: suggesting, molecular mimicry between bacterial antigens and self-proteins. typing (HLA-DRB, -DQA, -DQB, -DPB) was performed by PCR SSO dot blot ant Sera from 17 patients with APS and GDKV-MoAb reacted strongly with reverse dot blot. The data were analyzed by Chisquere test or Fisher exact with Yate: peptide A by ELISA suggesting possible cross-reactivity of conformational correction. The level of significance was set at 0.05. Estimated relative risk (OR) wa. epitopes presented by A and those recognized by aPL. To address this calculated by Wolfs formula, with Haldane's modification. The result of the stud) question, we carried out competion/inhibition studies using GDKV-MoAb. showed that there was a positive association between susceptibility to SLE with HLA GDKV-MoAb was treated with CL liposomes (I mg/ml), or with CL/B2GPI DRBI*15011-DRB5-0101, DQAI-0102 and DPBI*0202 (susceptible alleles). On the suspension (CL:lmg/mlB2GPI:100 microg/ml) or with CL/peptide A other hand, HLA-DRB1*1202-DRB 3*0301, DQAI*0601, DQB*0301 an( suspension (CL:1 mg/ml, peptide A: 100 microg/ml) or with B2GPI solution DPBI *2801 were found lo decrease the risk of SLE (protective alleles). Further more (100 microg/ml) or with peptide A solution (100 microg/ml) for hour at was (HLA-DRB1* 15011) tended to be it found that patients with the sucepitible allele room temperature and overnight at 4 C. Subsequently mixtures were younger, more severe clinical manifestations and had more specific autoantibod) had centrifuged and the supernatants were tested by ELISA for hinding to CL, to expressions with with protective allele or another allele. Subjeck. compared the patients peptide A, to B2GPI and to GDKV. Binding of GDKV-MoAb to CL was had higher risk o: with HLA-DRBI*15011-DRB5*0101, DQAI*0102, DQB1*0501 inhibited by 38% by CL alone, by 61% by CL/B2GPI liposomes and by anti-dsDNA, anti Ro/SSA, anti-RNP, ACA-IgG and ACA-IgM antibodies expression 74% by liposomes made of CL/peptide A. No inhibition of binding to CL In the contrary, subject with HLA-DRBI*1202-DRB3*0301, DQAI*0601 was observed when GDKV-induced aPL was treated with B2GPI alone or DQB1*0301 had lower risk of the autoantibodies expression. The result in patient: peptide A alone. Binding of GDKV-MoAb to B2GPI was inhibited 28% by with primary APS showed that aPL expression (aCL-IgM) was positively associate: CL liposomes, 60% by CL/B2GPI liposomes and 20% by B2GP1 alone. The with HLA-DRB1*15011 and DRB5*0102. Conversely, no association was founc between aPL-IgG and HLA-DRBI gene. HLA-DRB1*1202 and DRB3*0301 genes binding of GDKV-MoAb to peptide A was inhibited by 40% by CL alone, were negatively associated with aCL-IgG and IgM. It was concluded that the genetii 35% by CL/B2GPI, 46% by CL/peptide A but no inhibition was observed susceptibility to SLE and primary APS in Indonesian patients were similar,ic HLA when antibody was treated with peptide A, or B2GPI alone. Binding of DR2 (DRB1*1501 1-DRB5*0101). These alleles were different to what had been founr GDKV-MoAb to GDKV was inhibited by CL (60%), by CL/B2GPI (88%), in Caucasians but was similar to what had been reported in Oriental Chinese. Thes by CL/A (45%) but no inhibition occurred wheh the antibody was treated findings suggested that the worse prognosis of SLE iq Indonesian patients wa. with A or B2GPI alone. These studies suggest that a conformational epitope probably due to the genetic background. It appears that the role of HLA gene not onl is recognized by GDKV MoAb, presented by CL/B2GPI complex, in the occurrence of SLE but also in the disease severity. CL/peptide A and to a less extent by CL liposomes alone but not by peptide A or B2GPI alone. Keywords: Human leucocyte antigen, SLE, APS, Genetic susceptibility Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lhiIrtminacM (sxmss n ipdAtes Pistacm ANTIPHOSPHOLIPID ANTIBODIES ASSOCIATED WITH USE OF LOW-MOLECULAR WEIGHT HEPARIN IN PREGNANT 611 SEVERE MALARIA INFECTION WOMEN WITH APS M. Ehrenfeld, M. Bar-Natan, Y. Sidi, E. Schwartz A.D. Makatsaria, V.0. Bitsadze, N.V. Dolgushina, A.T. Safarov Chaim Sheba Medical Center and Tel-Aviv University, Tel-Hashomer, Moscow Medical Academy, Moscow, Russia Israel We have an experience of LMWH (Fraxiparin) use in pregnants with Malaria, the most important human parasitic disease, affects more than 400 different disorders (recurrent fetal loss, herpes infection, recurrent million people in the world, and leads to 1-3-million deaths each year. thrombosis and artificial heart valve), where antiphospholipid antibody Among returning travelers with imported malaria approximately 10% of the syndrome (APS) was the main cause of pathology and complications. affected individuals develop severe, often fatal, falciparum malaria with Administration of LMWHs to patients with APS is pathogenically correct, cerebral, renal, pulmonary and coagulation system involvement. It is not because these dregs influence the same links of hemostasis, that are fuilly understood why these complications develop in only some of the damaged in APS, namely the supression of anticoagulante properties of the patients. We describe two patients, who did not take prophylaxis while blood: violation of protein C activation, AT-M, TFPI and prostacycline travelling in Africa, and developed severe falciparum malaria upon release. The results of our study showed that early and continued returning, with ARDS in one of them, and pulmonary embolism and splenic prescription of fraxiparin (nadroparin) in patients with APS is effective in infarction, in the second. Both were found to have high levels of prevention of fetal loss and thrombotic complications, contributes to normal anticardiolipin antibodies (ACL), 19.6 MPL U/ml (N 1-10) in the first, and placental development and decreases the risk of premature delivery. 100 GPL U/ml (N 10-18), and 59 MPL U/ml in the second. High levels of Efficiency of long-term use of LMMWH was proved by clinical (absence of circulating anticoagulant (CAC) aPTT 2.9 (N 0.9-1.45), and DRVVT 1.8 (N thromboembolic obstetrical complications) and laboratory criteria 0.9-1.3), were also found in this patient. Both patients recovered completely, (thrombophilia markers disappearance, normalization of platelet function). though IgM-ACL still remain high at 6 months follow-up. Our 2 patients, Thus, Fraxiparine is highly effective and harmless for use in obstetrical are to the best of our knowledge, the first to be reported with falciparum practice. The dominating role of combined forms of thrombophilia malaria and APS. We suggest that the infection might have unmasked the (inherited and acquired) in pathogenesis of obstetrical complications makes APS, thus leading to the complicated course of the malarial infection, by LMWH the drug of choice in treatment ofthis group of pregnants. adding a thrombotic tendency to the thromboembolic complications associated with falciparum malaria, thus APL may play a significant role in the pathogenesis of this complication of malaria, in at least some of the patients. APS AND PREGNANCY: A SEVEN YEAR EXPERIENCE OCCURENCE OF SOME PLACENTAL PATHOLOGIC MANIFESTATIONS IN MATERNAL SLE, PRIMARY APS AND A. Maina, V. Donvito, L. Balbi HEALTHY MOTHER CONTROLS Servizio di Medicina Interna, OIRM, S. Anna, Torino, Italy C. Dostall, J. Stejskal2, K. Andelova3, J. Madar3, J. Kovarik3, Setting: A large tertiary care ob-gyn hospital (7,500 deliveries a year). A. Sucharova4, D. Tegzoval, V. Vlasakova5 Background: APS is a common cause of recurrent fetal loss and 'Institute ofRheumatology, 1Institute ofPathology, 3Institutefor the Care of complications of pregnancy such as severe preclampsia, IUGR, thrombosis, Mother and Child, Prague, Obstetric Clinic, Charles University, abruption placenta, prematurity. Aim of the study: To revise the effects of Ilnternal Unit, Regional Hospital, Ceske Budejovice, Charles University, medical and obstetrical care in a consecutive series of 72 patients who had a Prague, Czech Republic pregnancy after a confinned diagnosis of APS and 1 or more uneventful pregnancy. Methods: Patients were taken in charge after the previous Objective: To identify the presence of gross changes and light microscopy pregnancy failure or referred by other specialists before the index features as well as immunofluorescence findings in placentas from SLE pregnancy. Clinical and laboratory assessment included: detaliled history, and/or APS mothers when compared with healthy mother controls. physical examination, clinical chemistry, autoimmunity in order to select the Methods: JR STE and/or APS mothers (8 with SLE, 5 with SLE and patients and give them appropriate therapy. Patients were followed till a few secondary APS and 5 with primary APS) and 16 healthy control mothers months after delivery. Results: Good overall results of the assistance were monitored during pregnancy and delivery. Placentas from all followed program (>90% live births)if compared with historic controls, mothers were examined. Gross inspection was focused on fetus/placenta notwithstanding high percentage of Cesarean Sections (>70%). Secondary weight rate and the presence and extent of infarction. Light microscopy APS showed an increase of late clinical complications compared to primary examined age of placenta and presence of inflammatory, vascular and A.PS. LAC positive patients seem more at risk of treatment failure. degenerative changes. Immunofluorescence perforned in unfixed frozen sections followed the presence, extent and localization of IgG, IgM, IgA, C3, Clq and fibrinogen. Results: Significant pathological findings in the placenta of systemically treated SLE and/or APS mothers were rarely observed. Inmunofluorescence examination discloses mostly nonspecific quantitative differences in comparison with healthy controls but that of IgG (p < 0.05). Significantly, the IgG staining of nuclei, observed in four SLE mothers may be similar as described in lupus nephritis. Conclusion: Placental pathological changes were observed in SLE and APS in those mothers only whose pregnancy was complicated by exacerbation of the disease (nefritis, hypertension) and followed by loss of the fetus (observed in tlhree mothers). No similar changes were evidenced in other diseased mothers and in healthy controls. Supported by research project ofInstitute ofRheumatology No 00000023728. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 loth Irtei ana Ongess an kAiomiqdAribocles PoseUi1ma 612 THE ESTIMATION OF B AND B-1 LYMPHOCYTES AND EFFECT OF ANTIPHOSPHOLIPID ANTIBODY SUBTYPES ON ANTICARDIOLIPIN ANTIBODIES IN PREGNANT WOMEN WITH PREGNANCY OUTCOME PRE-ECLAMPSIA. M. Backos, R. Rai, L. Regan D.A. Darmochwal-Kolarz, B. Leszczynska-Gorzelak, J. Rolinski, Department ofReproductive Science and Medicine, ICSM, J. Oleszczuk St. Mary's Hospital, London, UK University School ofMedicine, Lublin, Poland Objective: To determine the prospective pregnancy outcome of women with Background: The aim of the study was to investigate the population of B CD a history of recurrent miscarriage (RM) associated with aPL, according to 19+ cells and autoreactive B-1 CD19+5+ lymphocytes in peripheral blood aPL types. Subjects and Methods: Pregnancy outcome of 218 women and the presence of anticardiolipin antibodies in the serum of patients with (median age 33 years; range 21- 45) with a history of RM (median 4; range pre-eclampsia and healthy pregnant women. Material and methods: 32 3-9) in association with persistently positive aPL were studied. The study patients with pre-eclampsia and 20 healthy pregnant women were included population were divided into 3 groups according to aPL subclass: 26 (12%) in the study. The expressions of surface molecules were estimated using were lupus anticoagulant (LA) positive, 87 (40%) IgG anticardiolipin (aCL) two-color flow cytometric method. The total concentrations of class IgG, positive and 105 (48%) IgM aCL positive. All women were treated with IgM and IgA anticardiolipin antibodies were measured with the use of semi- aspirin (75mg daily) and heparin (Clexane 20mg daily) during pregnancy. quantitative ELISA immunoenzymathic method. Results: The percentage of Results: The live birth rate was similar in the three groups [LA 50%; IgG B CD 19+ cells did not differ in the group of patients with pre-eclampsia in aCL 67%; IgM aCL 64%]. Pre-eclampsia complicated 21% of LA comparison with healthy, pregnant women. The percentage of BL CD19+5+ pregnancies, 10% of IgG aCL pregnancies and 0% ofIgM aCL pregnancies lymphocytes, which are responsible for the production of autoantibodies, did (p = 0.003). Placental abruption occurred in 7% of LA pregnancies, 3% of not exceed 1.5 % in both studied groups. The negative value of IgG aCL pregnancies and 0% of IgM aCL pregnancies. Preterm delivery anticardiolipin antibodies was present in 71.9 % patients with pre-eclampsia rate in LA group (36%) was similar to that in IgG aCL group (14%, and in 75.0 % of healthy women, the borderline value of ACA had 15.6 % p = NS), but significantly higher than that in IgM aCL group (7%, p = 0.01). of patients from the study group and 15.0 % of healthy pregnant women. Of the 14 live born infants to mothers with LA, one resulted in a neonatal The positive value of ACA had 12.5 % of patients with pre-eclamnpsia and death due to prematurity and 5 (36%) were small for gestational age. In 10.0 % from the control group Conclusions: The results obtained suggest contrast, only 7% (p = 0.01) of infants in the IgG group and 1% (p = 0.0004) that anticardiolipin antibodies are not the leading cause of pre-eclampsia. of infants in the IgM group were small for gestational age. However, it seems possible that thay may play a pathogenic role in some Conclusion: Treated pregnancies associated with IgM aCL are at far less cases ofpre-eclampsia. risk of obstetric complications than those associated with LA or IgG aCL. FETAL LOSS SYNDROME IN WOMEN WITH HERPES AND APS PRECLAMPSIA AS THE FIRST MANIFESTATION OF APS N.V. Dolgushina, V.0. Bitsadze, A.D. Makatsaria N.R. Jesus', R.A. Levy"2, E.M.N. Albuquerque', L.C.M.S. Porto" 2 Moscow Medical Academy, Russia JUniversidade do Estado do Rio de Janeiro, 2Labs Patologia Clinica, Rio de Janeiro, Brazil Basing on our experience herpes virus infection is the cause of antiphospholipid antibody syndrome (APS) in 47,4% of cases. Initiation of Preeclampsia is widely reported in the primary and secondary forns of this process is connected with molecular mimicry phenomenon and antiphospholipid syndrome (APS). But little is known on the frequency and production of cytokines in the place of injury. The cytokines increase the implications of preeclampsia as the opening feature of primary APS. The apoptosis of endotheliocytes with exposure of anionic phospholipids on their identification of these patients may improve their long-term morbidity and membranes and further production of autoimmune antibodies (anti- mortality rate. To study the frequency of anticardiolipin (aCL) in a cohort of phosphatidylserine antibodies). Antiphospholipid antibodies (APA) and previously healthy patients that developed preeclampsia of a multiracial herpes virus itself cause the endothelium damage with the further background, we tested by standardized aCL ELISA serum samples of thrombophylic state development. The pregnants with APS and herpes have twenty-eight patients that developed preeclampsia and as controls 30 a double risk for miscarridge and different obstetrical complications samples from nornal pregnancies, obtained at the same tinme period. The (hestosis, fetal growth retardation, placental insufficiency). The cause of age, number of gestations and race characteristics were similar between the miscarridge is implantation damage in early stages of gestation and 2 groups. Of the 28 patients with preeclampsia, three (10.7%) had low IgG thrombotic vasculopathy of placental spiral arteries in the later terms of aCL (15-35 GPL), five (17.8%) had moderate or high IgG aCL (>35GPL), pregnancy. In these women herpetic infection is taking a severe course due while in the control group (n=30), we found two samples (6.6%) with low, to optimal conditions for its replication and transplacental transmission and none with moderate or high titer. None of the samples tested were IgM (stagnanty-thrombotic changes in placenta). That's why the success of aCL positive. We recommend that the patients with low titers continue to be management of these patients depends on timeliness and continuity of tested every 4 to 6 months, and those with persistently moderate or high therapy. The drugs of choice are low molecular weight heparins. We have titers, once identified, should be managed at least with anti-platelet an experience of fraxiparine administration in several hundreds of pregnants aggregating agents and additional risk factors must be avoided. Prospective with genital herpes and APS with 100% of successful deliveries with multicenter collaborative studies on the aCL role in predicting small for healthy newborns without any signs of infection. We also have a positive gestational age infants and preeclampsia, as well as the role ofpositive lupus experience of intravenous immunoglobulin prescription in this group of anticoagulant tests and uterine artery Doppler studies at 22-24 weeks of patients. the mechanism of its action is connected with the direct viral gestation must be generated. suppression and regress of autoimmune process due to fraction of antiidiotypic antibodies contained in the medication. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1chInIfa8flSCE g anlAtqSpdAtIoces PBS8tQa ANTIPHOPHOLIPID ASSOCIATED THROMBOCYTOPENIA: GESTATIONAL AND NEONATAL OUTCOME IN SLE AND 613 AS A RISK FOR THROMBOSIS AND PREGNANCY MORBIDITY ANTIPHOSPOLIPID SYNDROME (APS), IN SLE WITH ANTIPHOSPHOLIPIDS ANTIBODIES (APL) S. Furukawa', T. Atsumil, M. Ieko2, Y. Sakai', S. Jodo', Y. Amasaki', K. Ichikawa', T. Koike' D. Faden', A. Lojacono', L. Barbetti', S. Zattil, M. Spunghi', M. Mottal, G. Balestrieri', R. Gorla2, A. Doria2, F. Vescovil, A. Tincani' 'Department ofMedicineII, Hokkaido University Graduate School of Medicine, Sapporo, 2Department ofMedicine, Health Sciences University of tBrescia University, Brescia, 2Rheumatology Department, University of Hokkaido, Toubetsu, Japan Padova, Italy Background: Thrombocytopenia is one of the classic manifestations Objective: To compare gestational and neonatal complications in three associatcd with antipospholipid antibodies (aPL). In Sapporo Criteria, groups of patients: SLE (group 1), SLE and APS (group 2), SLE and APL however, thrombocytopenia is not included as a clinical feature to define (anticardiolipin antibodies) (group 3). Patients and methods: from 1983 to antipospholipid syndrome (APS). In this study, we explored the significance 2001, 147 pregnancies in 102 patients with mean age 30 years (range 18-43) of thrombocytopenia in patients positive for aPL in variety of autoimmune were followed by the obstetrical-rheumatological team. Group 1: 93 diseases. Patients and Methods: This study comprises 342 patients with pregnancies (3 set of twins) (69 patients). Group 2: 18 pregnancies (16 autoimmune diseases (299 female; 187 systemic lupus erythematosus, 40 patients). Group 3: 26 pregnancies (24 patients). Rcsults: primary APS, 115 others). Lupus anticoagulant, anticardiolipin antibody and Group I Group 2 Group3 phosphatidylserine-dependent antiprothrombin antibody were tested. The Intrauterine fetal deaths 5 (5,3%) 1(5,5%) 1(3,9%) presence of those aPL was compared with clinical manifestations of APS, Miscarriages 10(10,7%) 1(5,5%) 3(11,5%) defined by Sapporo Criteria, and thrombocytopenia (less than Newbom 78(84%) 16(88,9%) 22(84,6%) 100,000/micro litter). Results: Thrombocytopenia was found in 64 (19%) Mean gestational age(w) 37,4 36,1 37,5 and 175 (51%) had at least one of aPL. Clinical manifestations of APS were Mean weight at birth (g) 2727 2550 3049 found in 106 (31%). Excluding these 106, a prevalence of thrombocytopenia Range (g) 2380-4020 1760-3220 14104250 was significantly higher in aPL positive patients than aPL negative (Odds Preterm premature rupture Ratio 2.7 [1.3-5.7], p=0.0148). In a second set of analysis among the of membranes 13(16,6"A 1(6,2%) 0 patients with thrombocytopenia, 31/51(61%) aPL positive patients had Preterm delivery (< 37w) 23(29,41'/0) 8(50%) 4(18%) clinical manifestations of APS. In contrast, none of 13 patients without aPL Preeclampsia 7(9%) 4(25%) 2(9%) had APS manifestations (p=0.0003). Conclusion: In non-APS autoimmune Small for gestational age 6(7,6%) 0 0 diseases, significant correlation beween aPL and thrombocytopenia was Thrombocytopenia found, thus aPL was indepandently related with thrombocytopenia. The and iatrogenic Hypoadrenia 0 1(6,2%) 0 presence of aPL in patients with thrombocytopenia increased the risk for Neonatal infections 2(2,6%) 0 0 having clinical manifestaitons of APS, therefore such patients may be Conclusions: there aren't statistically significant differences between the descriminated from aPL negative patients with thrombocytopenia. groups; probably an appropriate manegement in group 2 and 3 may reduced possible adverse pregnancy outcome related to antiphospholipids antibodies. There is higher incidence of preeclampsia as well as preterm delivery in group, but it doesn't reach statistical significance. ANTIPHOSPHOLIPID ANTIBODIES PREVENT EXTRAVILLOUS PREGNANCY LOSS IN SLE, APS AND RELATED CONDITIONS TROPHOBLAST DIFFERENTIATION A. Gil Aguado, M. Janez, P. Lavilla, D. Pascual Salcedo, M. Cuesta, S. Quenby', S. Monmtfield', L. Chamley2, R. Farquharson3, G. Vince' J.1. Bemardino, F. Garcia Iglesias, M. Mora, A. Robles, F. Gaya On BehalfofThe APS Study Cooperative Group 'University ofLiver?Tool, UK, 2National Womens Hospital, Auckland, New Zealand, Liverpool Women's Hospital, Liverpool, UK La Paz University Hospital, Madrid, Spain Objectives: Positive test results for antiphospholipid antibodies (Apl) are Objective: To analyze pregnancy loss, the correlation with antiphospholipid associated with adverse pregnancy outcomes, especially recurrent antibodies (aPL) and the efficacy of AAS in the prevention of abortions/fetal miscarriage in both the first and second trimester, intrauterine growth loss in women with SLE, APS and related diseases. Methods: 122 women restriction, and pre-eclamnpsia. As Apl are also associated with thromobsis, with a total of 316 pregnancies were evaluated. There were: 55 SLE the pathogenesis of this condition in pregnancy was thought to be by a patients, 35 primary APS (PAPS), 10 with secondary APS (SAPS) and 22 thrombotic mechanism. In this work we are investigating the more recent with related conditions. The efficacy of the treatmient with aspirin (100 hypothesis that Apl have a detrimental effect on human trophoblasts. In this mg/daily) was controlled in 24 pregnancies. Results: A total of 93 study we have investigated the effect of Apl on extravillous tropohoblast miscarriages (30%) was observed, most of them (58%) occurred before the (EVT). These EVT are the cells that invade the matemal decidua and spiral 10th week of gestation. The prevalenlce was higher in PAPS (69%) alid arteries. Hence abnormalities in their ability to invade would account for the SAPS (60%) than in SLE (35%) or related diseases (23%). In the total series large range of obstetric pathology seen with Apl. a correlation was found with high titers of aCL-IgG/IgM, aBeta2GPI-IgG Methods: Extravillous trophoblast was isolated from term placentas from and aPTPS-lgG/IgM. No correlation was found between the time of women who had normal pregnancies. A laboratory model for EVT was presentation of losses and the presence or different isotypes of aPL. In SLE designed. In vivo EVT differentiate into giant multinucleated cells within patients the presence of aPL (aCL-IgG/lgM, aBeta2GPI-IgG and aPTPS- the maternal decidua. A series of time course experiments found that IgG/IgM) increased the risk of abortions/fetal loss. In 24 pregnancies, from significant clumping of freshly isolated extravillous trophoblast into giant 21 women with a previous history of recurrent abortions, the treatment with multinuclear cells occurs after 48hours in culture. EVT were then cultured AAS during the preconceptional and gestational periods was associated with with and without two monoclonal antibodies to [32 glycoprotein 1. a significant decrease in miscarriages rate (78,5% without therapy versus 8,3% with aspirin; p<0.01); none of the 2 pregnacy losses observed in the Results: The anti [2-glycoprotein 1 antibody inhibited the giant treated group occurred beyond the 8th week. Conclusions: Antiphospholipid multinuclear cell formation by 20% after 24 hours, 41% after 36 hours and antibodies are a high risk factor for miscarriages in pregnant women with 50% after 48 hours of culture. APS or SLE. Treatment with low dose aspirin during pregnancy should be Conclusions: 1 antibodies can inhibit EVT in the 12-glycoprotein clumping considered in these patients. laboratory. Thus Apl may cause obstetric complications by directly preventing EVT invasion of the matemal decidua. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 10 Itmanai O( on ArthiiopioddAribocies PosterPsetmiai 614 INTRAUTERINE GROWTH RETARDATION (IGR) AND ANTICARDIOLIPIN AUTOANTIBODIES IN WOMEN SUFFERING PREMATURE BIRTH (PB) IN APS, SLE, AND RELATED PREGNANCY DISORDERS DISEASES J. Madarl, K. Sulal, A. Chaloupkova2, D. Novakova3, Z. Ulcova-Gallova4, A. Gil Aguado, M. Janez, P. Lavilla, D. Pascual Salcedo, M. Cuesta, R. Kinsky' F. Garcia Iglesias, J.I. Bemardino, L. Diez Porres, A. Robles, F. Gaya 'Institutefor the Care ofMother and Child, 21nstitute ofMolecular Genetics, On BehalfofThe APS Study Cooperative Group Czech Academy ofSciences, 3Charles University, 3rd Faculty ofMedicine, La Paz University Hospital, Madrid, Spain Prague. 4Charles University, Faculty ofMedicine in Pilsen, Czech Republic Objective: To analyze the prevalence of IGR and PB, the correlation with Object: To determine selected immune markers in women suffering antiphospholipid antibodies (aPL) and the efficacy ofaspirin in women with pregnancy disorders (miscaniage, cctopic prcgnancy). Paticnts and APS, SLE and related conditions. Methods: 122 patients with a total of 316 Methods: The study comprises four groups of patients experienced, within pregnancies were evaluated. There were 55 patients with SLE, 35 with the last 3 years: (1) delivery of healthy child and no pathological pregnancy primary APS (PAPS), 10 with secondary APS (SAPS) and 22 with other (control group, n=35); (2) the last of two or more subsequent first trimester related conditions. The efficacy of aspirin in the prevention of these miscarriages (early aborts, n=135); (3) miscarriage in weeks 20 - 26 (late obstetrical abnonnalities was evaluated in 21 pregnancies. Results: In 197 aborts, n=38); (4) extrauterine pregnancy (GEU, n=45). Blood samples were pregnancies evaluated a total of 21 IGR (10,7%) was observed. The tested for titers of IgG and IgM ACLA, total immunoglobulins, and prevalence of IGR was higher in SLE women (29%) than in PAPS (17%), circulating immunocomplexes using standard methods. The level of anti- SAPS (13%) or related diseases (9%). The presence of APL or the titters of trophoblast cell-mediated immunity (AT-CMI) was measured by the different isotypes did not influence in the frequency of IGR for the total migration inhibition test of blood leukocytes (Dimitrov et al, J.Jmmunol. series or for SLE patients. PB was found in 14 pregnancies (9%), all ofthem Methods 154,147,1992). Results: The subsequent differences have been in women with aPL. The frequency of PB was: 22,5% in SLE+APS, and found, as compared with control group: In the group (2) - early aborts - the 18,4% in PAPS and SAPS. A strongly correlation was found between the mean titers of both IgG- and IgM-ACLA were significantly higher; prevalence of PB with LAC, aCL-IgG, aBeta2GPI-IgG/IgM, aPT-IgM and percentagc of AT-CMI-positive patients was significantly higher; othcr aPSPT-IgG/IgM. In 21 pregnancies, from 19 patients with recurrent markers were not significantly different. In the group (3) - late aborts - the abortions, the gestational treatment with aspirin (100 mg/daily) induced a ACLA titers were also elevated, even more markedly than in the group (2); significantly decrease in PB rate (4,8%). Aspirin therapy failed in diminish the percentage of AT-CMl-positive patients, as well as the other markers the rate of IGR (29%). Conclusions: Antiphospholipid antibodies are an were not significantly different. For the group (4) - GEU - the elevation of important risk factor for premature birth but have no influence on the percentage of AT-CMI-positive patients was typical. Conclusions: Elevated incidence of IGR. Treatnent with aspirin should be recommended for the titers of anticardiolipin autoantibodies have been found namely in the group prevention ofpremature birth in these patients. of late miscarriages. For early pregnancy losses the increase of antitrophoblast immunity is more typical. Acknowledgement: This study was supported by IGA grant No. 6463-3 from the Czech Ministry ofHealth. ENOXAPARIN ADAPTED TO FERTILITY PROGRAM TO PREVENT PRECLINICAL, EMBRYONIC AND FETAL LOSS IN WOMEN WITH RPL AND THROMBOPHILIA A. Sarto, M. Martinez, A. Falco, A. Marazzi, A. Paganini, V. Smaltino, C. Capmany, C. Quintans, S. Pasqualini Halitus Instituto Medico, Buenos Aires, Argentina APS AND PREGNANCY. A RETROSPECTIVE STUDY We evaluated the gestational outcome in 162 women with RPL (2 or more J.O. Latino, G.R. Bierfass, O.G. Carballo, D.S. Femandez Romero, preclinical, embryonic or fetal loss) who presented thrombophilic pattern, in M.V. Paolini, M.E. Prada, U. Scher, M. Pesaresi, A.M. Di Losiardo treatment with enoxaparin. Patients had at least one of the following Immunology Unit, Hospital Dr. C.G. Durand, Buenos Aires, Argentina thrombophilic markers: hypofibrinolysis, antiphospholipid antibodies, abnormal APCR, protein S, C or antithrombin III deficiency, Our aim was to determine pregnancy outcome and perinatal results in a hyperhomocysteinemia, FV Leiden, Phrotrombin 20210, MTHFRC677T. series of APS patients. 188 patients were followed up from 1988 to 2001. Before diagnosis of thrombophilia they had a total of491 gestations, 86% of Ninety two (49 percent) patients fulfilled 4 or more criteria of the ACR them ended in pregnancy loss (preclinical 128, embryonic 245, fetal 49). classification for Systemic Erythematosus Lupus (SLE), 24 (12 percent) After diagnosis of thrombophilia, 180 subsequent pregnancies were treated fulfilled criteria for APS from Sapporo Convention in 1998. 14 (58 percent) with enoxaparin. Assisted reproductive techniques were applied in 45 cases of them were primary APS and the others secondary APS (8 SLE, 1 to achieved pregnancy. In 87 pregnancies of those women who had at least Rheumatoid Arthritis and I Scleroderma). The follow up protocol consisted one preclinic pregnancy loss, Enoxaparin (20 or 40 mg/day)was started in an obstetric control, immunological and general laboratory tests every 21 Patients were treated with low dose Subcutaneous previous to conception according to the fertility program, continuing with days. Aspirin (AAS), Heparin (H) ], Glucocorticosteroids (GC) and Intravenous Gammaglobulin gestational regime. Ninety-three pregnancies of those women who had only embryonic or fetal loss started therapy with enoxaparin after a biochemical (IVIG) at different combinations. In this series we had 58 percent successful pregnancy diagnosis. During gestations the Enoxaparin dose was adjusted pregnancies with delivery of a viable infant. Pregnancy failure occurred in with Anti Xa test (range: 0.3 at 0.6 u/ml). One hundred and fifty two 42 percent of the patients without taking into account the gestational age, ended in similar to the reported literature. The more frequent complications were (84.5%) pregnancies live birth of the treated pregnancies vs. 69 of early membrane disruption and intrauterine retardation. 491 (14.5%) of the previous pregnancies untreated (p < 0.001). Preclinical growth Concluding the outcome of pregnancies did not seem to be related to more aggressive pregnancy loss rate was 3.3% vs 26%, embryonic 10.6% vs 50% and fetal loss rate was 1.6% vs 10% in treated and untreated gestations. Enoxaparin treatments adapted to fertility program is effective to prevent preclinical , embryonic and fetal loss in women with RPL and thrombophilia. RESULTS: Gestational outcomc in untreated and treatcd prtgnancies with Enoxaparin in wonmen with RPL and thrombophilia. Untreated pregnancies Treated Pregpancies p- valuc N pregnanacies 491 180 Pregnancy lasn 86% (422) 15.5 (28) <0.001 Prtchnical 26% (128) 3.3% (6) <0.001 Embryonic 50 (245) 10.6% (19) <0.001 Feul 10 (49) 1.6% (3) <0.001 live bite 14% (69) 84.5% (152) <0.001 8 twins Intuterinle growdh 14.4 (10) 3.8% (6) 0.005 retardation Preecaiampsia 13% (9) 1.3% (2) <0.001 UPSf Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 Ilhirten(ardQ hsson fi dIm odes PMStiQ HELLP SYNDROME IN THE ANTIPHOSPHOLIPID SYNDROME PERIPHERAL BLOOD NATURAL KILLER CELL NUMBER IN 615 (APS) WOMEN WITH RECURRENT MISCARRIAGE AND APS N. Ticulicl, D. LcThi Huong', B. Wechsler', D. Vauthicr-Brouzcs2, B. Cork, S. El-Gaddal, M. Backos, R. Rai, S. Jivraj, L. Regan 0. Aumaitre3, Z. Amoural, J.C. Piettel Department ofReproductive Science and Medicine, ICSMat St AMary's, 'Department ofInternal Medicine, ~~~~~~~~~~2Department of Obstetrics, NorfolkPlace, London, UK Pitie-Salpetriere Hospital, Paris, 3Department oflnternal Medicine, Introduction and Objective: Antiphospholipid syndrome (APS) affects 15% France Clermont-Ferrand, of women with recurrent miscarriage (RM). Although there has been an We report on 8 women with APS who presented with HELLP syndrome. increase in the understanding of the underlying mechanisms involved in APS was prinary in 4 cases and it secondary to SLE in 4. Mean age was 30 APS-related pregnancy loss, the non-thrombotic mechanisms remain to be years (range: 24-34). hree women were nulliparous. The HELLP syndrome determined. Natural killer (NK) cells have been postulated to be involved in occurred during the second trimester in 3 cases, during the third trimester in the implantation and placentation processes. The aim of this study was to 3 cases and the day after delivery in 2 cases. The main signs included: determine the relationship between antiphospholpid antibodies (aPL) profile hemolysis (n=4), elevated transamninases (n=8), thrombocytopenia (n=8), and peripheral blood NK cell number. Subjects & Methods: Peripheral hypertension (n=6), daily proteinuria >= 0.5 g (n-7), DIC (n=2), abdominial blood samples were obtainod from 64 non-pregnant women with of a history pain (n=5) and seizures (n=2). In 2 cases of SLE, diagnosis of APS was RM (3 or more consecutive pregnancy losses), of which 29 were aPL done before pregnancy due to an arterial (n=l) or a venous (n=l) positive and 35 were aPL negative. The investigators were blinded to the thrombosis. HELLP syndrome revealed the APS in the other 6 women, i.e. 2 aPL status. Fluorescent activated cell analysis was used to determine the women with SLE and previous asytnptomatic aPL, and 4 women with percentage of NK cells positive for CD16 and CD56. The day of the primary APS. In 3 cases, there was no therapy during pregnancy. In 4 cases menstrual cycle that the blood was taken was calculated from the date of the of SLE, 5 to 30mg/d prednisone was prescribed in addition to last menstrual period. Results: Overall the number ofNK cells was higher in hydroxychloroquine (n=2), aspirin (n=4) and heparin (n=3) because of the secretory phase of the menstrual cycle compared to the proliferative history of vascular thrombosis (n=2) or of adverse pregnancy outcome phase, with a significant increase during the mid-secretory phase. The (n-l). In a case of undiagnosed APS, aspirin was prescribed after 2 number of peripheral NK cell was significantly higher in women with aPL successive fetal deaths. Pregnancy ended in fetal death (n=2), premature positive compared to those with aPL negative. Conclusion: The study (n=4) and full-term deliveries (n=2). Four of the 6 liveboms were growth findings suggest that there is an association between circulating aPL and an retarded. Cesarian section was indicated in 7 of 8 deliveries. Two of 4 increased peripheral NK cells number. It is possible that in women with women with the APS had had another normal pregnancy while treated with APS, one possible mechanism of early pregnancy loss may be the abnormal heparin plus aspirin. APS may be revelead by HELLP syndrome. activation of cellular immunity. DOPPLER ULTRASOUND EXAMINATION IS THE BEST TEST AUTOANTIBODIES IN REPRODUCTIVE FAILURE- NOVEL FOR MONITORING SLE AND THE APS PREGNANCIES ANIMAL MODELS AND CLINICAL IMPLICATIONS D. Le Thi Huong', B. Wechsler', D. Vauthier-Brouzes?, P. Duhaut', Y. Sherer, S. Tartakover-Matalon, M. Blank, Y. Shoenfeld N. Costedoat', G. Lefebvre2, 0. Bletry', J. C. Piette' 'Department ofMedicine B and Center ofAutoimmune Diseases, 'Department ofInternal Medicine, 2Department ofObstetrics, Chaim Sheba Medical Center, Tel-Hashomer, Israel Pitie-Salpetriere Hospital, Parts, France The association of autoantibodies with reproductive failure is evident Statement of purpose: To examine the respective predictive value of clinical especially in autoimmune diseases such as the antiphospholipid syndrome examination, laboratory tests and Doppler-ultrasound examination for and systemic lupus erythematosus, in which reproductive failure includes monitoring SLE and APS pregnancies. Patients and Methods: Prospective infertility, intrauterine growth retardation and recurrent abortion. However, study of consecutive pregnancies in women with SLE and/or APS followed in addition to the clear association between antiphospholipid antibodies and in a single French tertiary referral centre. Since 1993, 116 (SLE without these manifestations there are other autoantibodies that can also induce these APS n=78, primary APS n-37, secondary APS n=28) pregnancies were effects. Recent studies indicate that anti-laminin-l autoantibodies can induce followed in 84 women. 16 ended at or before the 20th week gestatioss and/or both decreased conception rates (52% in laminin-l immunized mice versus before having the 2nd trimester Doppler ultrasound examination at 20-22 73% in controls) and highcr fctal rcsorption ratcs (24% in laminin- I weeks. We analyzed the data of 100 pregnancies who have had complete immunized mice versus 12% in controls) in an active immunization animal data including monthly clinical examination, laboratory tests and 2nd model. These autoantibodies are also found in increased frequency in trimester Doppler ultrasound examination followed by the 3rd trimester women having recurrent abortions. Anti-thyroglobulin autoantibody examination when the pregnancy evolved beyond 30-32 weeks. Adverse immunization protocol also induced reproductive failure in naive mice. pregnancy outcome was defined as fetal or neonatal death; or pregniancy These autoantibodies are relatively frequent in humans, and are associated complicated by pre-eclampsia, growth retardation or premature delivery witl various aspects of reproductive failure. Active isnnunization of mice before 34 weeks due to placental insufficiency. Results: By multivariate with thyroglobulin resulted in the production of anti-thyroglobulin analysis, the only predictor of adverse pregnancy outcome was anormal antibodies, increased fetal resorption rate, and decreased fetal and placental uterine artery on 2nd trimester examination (p=0.0006) with an odds ratio of weights compared with CFA-injected mice. Anti-thyroglobulin antibodies 12 (95% cojifidenw interval; 2.93-49.5). Conclusions: The results of the 2nd could also be eluted from the placentae of the immunized mice, but not from trimester fetal Doppler ultrasound are the best way for monitoring SLE and control mice. Demonstration of a pathogenic effect of these autoantibodies APS pregnancies. on reproduction, along with their clinical associations suggest search for them in women having reproductive failure and possibly also justify immunomodulation targeted to these antibodies. Wu8 Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1Ihktic dWgessmcniioqMfiddAtibocies PotPnrset*jrg 616 THE EFFECT OF ANTI PHOSPHATIDYL SERINE MONOCLONAL THROMBOPHILIA IN RECURRENT PREGNANCY LOSS ANTIBODY ON RAT EMBRYOS AND PLACENTA IN EX-VIVO CULTURE HJ.A. Carp, M. Dulitzky, A. Inbal S. Tartakover Department ofObstetrics and Gynecology, Insitute ofThrombosis and Matalonl', Y. Shoenfeld', M. Blank', P. von Landenberg', Chaim S. Yacoby2, A. OMoy2 Hemostasis, Sheba Medical Center, Tel Hashomer, Israel To assess the 'Laboratory of Teratology, Department ofAnatomy and Cell Objective: prevalence of hereditary thrombophilias in Biology, recurrent the live birth rate Hadassah-Hebrew University School ofMedicine, Jerusalem, for pregnancy loss, subsequent and effect of 2Center treatment. Methods: Patients with 3 or more Autoimmune Diseases, Department ofMedicine B, Chaim pregnancy losses were assessed Sheba Medical for APCR, Factor V Leiden, MTFHR, Factor II mutation (G20210A), Center, Tel-Hashomer, Sackler Faculty ofMedicine, Tel-Aviv University, Proteins C and Israel S and antithrombin III defficiencies. A cohort of patients with thrombophilias were treated with enoxaprin 40 mg or assigned to a The effect of two anti phosphatidyl serine (PS) monoclonal antibodies control group. Results: 38% of 198 patients had hereditary thrombophilias. (mAbs) on placenta and embryos growth, and on apoptosis events in the The prevalence of FVL, Factor II (G20210A), and MTHFR was similar to placenta was studied in ex-vivo embryo culture. Methods: Rat embryos. 5 the 82 parous women. The 75 thrombophilic patients had 33 previous live days old, in their yolk sacs were cultured for 28 hours in the presence of births without treatment and 278 pregnancy losses (mean 3.76±1.01). There anti-PS mAbs or non relevant control antibody. After 28 hours ofculture the were 47 subsequent pregnancies. 22 were treated with enoxaparin, 16 embryos and their yolk sacs were examined under a dissecting microscope (72.8%) reached live births compared to 9 (36%) of25 in untreated patients. to evaluate development and existence of anomalies. Apoptosis in the The live birth rate was 34.7% (17 of 49) in non thrombophilic patients placenta was detected on paraffin sections by ApopTag peroxidase labeling (similar to that of the untreated thrombophilic patients (RR - 1.04 Cl 0.54- of fragmented DNA Results: yolk sacs that were exposed to anti PS 2.01). However, the live birth rate was significantly increased by treatment antibodics grcw statistically significant less in comllparisonI to control yolk with enoxaparin, (RR = 2.35 Cl 1.12-4.93). Four miscarriages were sacs (25 % inhibition of growth (p<0.05). There were more apoptotic events karyotypically abnormal, (Trisomy 13, 16, 18 and 45XO). Two patients with in poly nucleated giant cells (GC) of the ectoplacental colne that were blighted ova who had failed to hold a pregnancy, required paternal leucocyte exposed to every one of the anti PS antibodies (20 % and 13 % cells of GC immunization or immunoglobulin to achieve a viable pregnancy with a fetal population, p<0.05) then in placentae that were exposed to non relevant heartbeat. Enoxaprin was used afterwards to prevent growth retardation and control IgG (7 % of GC population). Ex-vivo embryos that were exposed to subsequent thromboses. Conclusions: Thrombophilias are not more anti PS antibodies for 28 hours were morphologically identical to control prevalent in women with recurrent miscarriage, nor do they worsen the embryos. Conclusions: Anti PS antibodies effect directly placental growth, prognosis. However treatment with enoxaparin raises the live birth rate. and enhances apoptotic events in ectoplacental cone giant cells. ENDOTHELIAL MICROPARTICLES IN RECURRENT EARLY ASSOCIATION OF ANTICARDIOLIPIN ANTIBODY AND C677T PREGNANCY LOSS MTHFR MUTATION IN RECURRENT SPONTANEOUS H. J. A. Carp, R. Dardik, 0. Salomon, R. Eskaraev, A. Inbal ABORTING WOMEN: A NEW PATH IN THROMBOPHILIA? E. M. 'Institute ofThrombosis and Hemostasis and Department ofObstetrics and Couto, R. Barini, Samo, J3M. Annicnhino-Bizzacchi Gynecology, Chaim Sheba Medical Center, Tel-Hashomer, Israel Universidade Estadual de Campinas, Sao Paulo, Brazil Objective: To examine the prevalence of circulating microparticles in Problem: To evaluate the association between thrombogenic factors and women with recurrent early pregnancy loss. Circulating microparticles are recurrent spontaneous abortion (RSA). Method of Study: A case-control markers of cell activation associated with various prothrombotic states. As study included 88 women with RSA and 88 fertile women, matched by age hypoxia due to uteroplacental thrombosis is considered to be one of the and race. Anticardiolipin antibody (ACA), lupus anticoagulant, protein C, S causes of pregnancy loss, the presence of circulating microparticles may be and antithrombin III deficiencies were evaluated by ELISA, dRVVT, associated with pregnancy loss, in addition or as part of other procoagulant coagulometric and chromogenic methods. DNA was amplified by PCR to states such as aPL or hereditary thrombophilias. Methods: Fifty one women study the mutations Leiden factor V, G20210A in prothrombin gene and with recurrent first trimestcr pregnancy losses (3 or more consecutive C677T in methylene tetrahydrofolate reductase (MTHFR) gene. Results: miscarriages) were enrolled in the study and compared to 53 parous ACA was detected in 11 women with RSA and in one fertile woman [OR (control) women. Microparticles were measured by flow cytometry using 12,4 (CI 95%,5 to 98,5)]. Heterozygous C677T was detected in 59 women fluorescent anti-CD51IiCD31 antibodies. Results: Microparticle levels above with RSA and in 35 fertile women (OR 3,1 (CI 95% 1,7 to 5,7)]. The the upper limit of normal (45,000/mI) were detected in 9 out of 51 women concomitant presence of ACA and heterozygous C677T was found in 8 with recurrent miscarriages (17.6%). No control woman had increased women with RSA and none fertile women (p < 0,01). Conclusions: ACA microparticle levels (mean±SD: 30,000±15,000/ml). The mean±SD and heterozygous C677T in MTHFR gene are statistically associated with microparticle levels of the 9 women with elevated microparticles was RSA. The association of these two conditions is a newfound in 71,500+14,000/ml (range: 55,000-88,000); p < 0.001 compared to controls. thrombogenic factors and RSA. The titer of microparticles in this group did not correlate to either the number of pregnancy losses, Conclusions: A certain proportion of women with early pregnancy loss have elevated endothelial microparticles suggesting that endothelial damage or activation might be involved in the pathogenesis ofpregnancy loss. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1WIlrrSmnic Ozss cnbhftipdAdoe os~rPtFsat*cm 617 ROLE OF BETA 2 GLYCOPROTEIN1 IN AUTOANTIBODY CATASTROPHIC ANTIPHOSPHOLIPID SYNDROME (CAPS) OR MEDIATED PREGNANCY LOSS ANTIPHOSPHOLIPID SYNDROME (APS) WITH A CATASTROPHIC EVENT? Z. Mondher, L. Lilia, A. Houria, S. Maryam, M. Sondes D. Erkas, Y. Yazici, M.D. Lockshin Immunology Department, La Rabta, Tunis, Tunisia 'Hospitalfor Special Surgery, Weill Medical College ofCornell University, beta 2 glycoprotein I has been identified as a cofactor for the binding of New York, USA some antiphospholipid antibodies to anionic phospholipid and has been multiple demonstrated to possess anticoagulant properties in vitro. the present sudy Background: Catastrophic APS is defined as life-threatening of time but the details are role of beta 2 glycoprotein 1 in autoantibody vascular occlusions presenting over a short period attempted to deternine the of pregnancy loss. We have investigated a latex immunoassay for not standardized. Objective: To test the boundaries of the definition mediated severe measurement of beta 2 glycoprotein 1 and an ELISA for the detection of CAPS by presenting our experience with six APS patients who had clinical manifestations. Case Presentations: Case 1: 51 yo anticardiolipin and anti beta 2 glycoprotein I antibodies in 152 women with life-threatening unexplained repeated abortion (G1) and 100 healthy blood donors (G2). Chi male patient with SLE and secondary APS (deep venous thrombosis [DVT] embolus while on had renal square were used for statistical analysis Results : There is a statistical followed by DVT/pulmonary [PE] warfarin) test yo between women with unexplained repeated abortion and healthy thrombotic microangiopathy and myocardial infarction (MI). Case 2: 28 differences weeks blood donors for the presence of aCL and AB2GP1. 18 patients from Gl female patient with transient ischemic attacks (3 episodes, last one 6 less than 50% of normal beta 2 glycoprotein 1 levels against two in G2. prior) had stroke and thrombosis in the right and left atrium, and superior had presented among these 18 GI patients 7 had high level of either aCL or AB2GPI vena cava. Case 3: 30 yo male patient with primary APS (stroke) aCL AB2GP1 IgG IgM IgA IgG IgMG0 with bilateral adrenal hemorrhage. Case 4: 49 yo female patient with RA 14 6 3 7 20G2 3 3 1 0 0 and secondary APS (DVT followed by DVT/PE while on warfarin) had Conclusion: beta 2 glycoprotein 1 exert several inhibitory effects in hepatic infarcts and MI. Case 5: 62 yo male patient with renal thrombotic intrinsic coagulation pathway and in platelet aggregation. These funtions microangiopathy (on warfarin) had stroke, MI, worsening renal function and could explain the thromboses and the obstetrical complications observed in thrombocytopenia. Case 6: 69 yo male patient with prior myocardial diffuse alveolar hemorrhage followed by the presence of antiphospholipid antibodies infarction presented with subelavian vein thrombosis (I week after pulmonary hemorrhage). All patients had medium or high titer aCL and/or positive LAC test. Possible triggers, treatment regimens and outcomes of these patients will also be presented. Conclusion: These life-threatening APS events can be classified as CAPS by some, but not by all. A standardized approach to the diagnosis of CAPS (i.e., the type of event and the magnitude of the organ involvement) may help us better understand and manage this syndrome. Thus, we will present our recommendations for the definition of CAPS based on our experience in conjunction with the literature. ATYPICAL HELLP IS CRYING FOR HELP IN PREGNANT SLE AND APS: SOMETIMES A CATASTROPHIC COMBINATION WOMEN WITH ANTIPHOSPHOLIPID SYNDROME C. Noronha', A. Galrinho2, A. Panarral, M. Sousa', M.J. Barros, R. Pauznerl, M. Dolitzki2, H. Carp2, H. Mayan'. Z. Farfel', A. Many3 M. Vaz Riscado', 'Department ofMedicine E, 2Department ofObstetrics and Gynecology, 'Department ofMedicine , Vascular Intervention Unit, 3Department ofHematology, Chaim Sheba Medical Center, Tel Hashomer, Curry Cabral Hospital, Lisbon, Portugal Sackler School ofMedicine, Tel-Aviv University, Tel-Aviv, Israel The authors present the case of a 19 year-old Caucasian girl referred in May Antiphospholipid antibody syndrome (APS) is associated with adverse 1999, due to Systemic Lupus Erythematosus (SLE) based on erythematous outcome including recurrent fetal loss, intra-uterine growth skin lesions, fotossensitivity, alopecia, livedo reticularis, polyarthralgias, pregnancy leucopenia and positive ANA and anti-DNAds, with a three year evolution. restriction (IUGR) and maternal complications including thrombotic events also and early pre-eclampsia. HELLP syndrome (Hemolysis, Elevated Liver Anticardiolipin (IgG) and anti-b2GPI (IgG), in medium titers, were was documented. There was a good clinical enzymes Low Platelets) represents a unique form .in the spectrum of pre- detected. No renal involvement (40 mg), hydroxichloroquine, NSAIDs and eclampsia/eclampsia. The most conunon reason for the elevated liver response to Prednisolone syndrome, is hepatocellular damage. However, both acetylsalicilic acid, allowing gradual tapering of prednisolone to 5 mg /d. In enzymes in HELLP with left femoro-popliteal phlebothrombosis, were The incidence of November 2000, she presented hemorrhagic and thrombotic complications described. syndrome (SAPS); improvement series of patients with HELLP is very low. configuring a secondary antiphospholipid hepatic infarcts studied in large oral anticoagulation and corticosteroid We describe 4 patients with APS that presented with atypical HELLP was seen following high-intensity The echocardiogram perforned then revealed mild mitral syndrome and liver infarcts. These events occurred at the early stages of the increment. and verrucous vegetations, interpreted initially as Liebmann- pregnancy: the earliest at the 7th week of gestation, 2 at the 17th weeks, and insufficiency During the next 4 months, rapidly progressive hemato- at the 25th week of gestation. The event required termination of the Sacks endocarditis. the 4th (to nephrotic range -7,3 g/24h) and edema were noted. Active death occurred in the 4th case. All the proteinuria pregnancy in, and intra-uterine fetal prolipherative glomerulonephritis was diagnosed and damage. Patients with APS are diffuse patients recovered without any residual immunossupression with pulses of Methylprednisolone and IV very atypical HELLP syndrome, with a significant prone to develop early Cyclophosphamide was begun. Concomitantly, there were signs of heart risk for hepatic infarct. An accurate early diagnosis is important for a proper failure and marked echocardiographic deteroration - severe mitral management. Moreover, in any case of hepatic infarct during pregnancy This case should be looked for. inisufficienlcy and mild tricuspid insuffiienciiy. illustrates the anticardiolipin antibodies and Lupus anticoagulant difficulty in management of SLE with SAPS due to multiple organ involvement and the need for multidisciplinary approach. Particularly in this case, despite favorable evolution, several factors were considered (timing of cardiac valve surgery, discontinuation of immunossupression and risks of anticoagulation) and various complications occurred (acute renal insufficiency requiring emergency dyalisis and post-operative cardiac tamponade). Moreover, the authors emphasize the physiopathological role ofAPS in cardiac valvular lesions. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 Cl1h ktamiand gonssonAfiospddAMiboces Postr tns 618 TWO NEW CASES OF CATASTROPHIC APS IN PEDIATRIC PROSPECTIVE STUDY OF CARDIAC ABNORMALITIES BY PATIENTS TRANSESOPHAGEAL ECHOCARDIOGRAPHY (TEE) IN A R. Roldan', V. Perez-Guijol, M. Anton2, C. Castrol, E. Collantes' COHORT OF 56 PATIENTS AFFECTED BY PRIMARY APS 'Rheumatology Department, 2Pediatric Nephrology Unit, Reina Sofia M. Turiell, P. Sarzi-Puttini2, S. Muzzupappa', E. Rossi3, F. Atzeni2 Hospital, Cordoba, Spain 'Department of Cardiology, Istituto Ortopedico Galeazzi, University of Introduction: Systemic lupus erythematosus (SLE) is an autoimmune Milan, 'Rheumatology Unit, 3Blood Transfusion Center, L. Sacco University inflammatory disease. Although constitutional symptoms, arthritis and Hospital, Milan, Italy cutaneous disease are the most common manifestations, disease activity may Introduction: PAPS is identified by the presence of antiphospholipid produce renal, cardiopulmonary and neuropsychiatric involvement. antibodies (aPL) and is related to recurrent thrombosis (or fetal loss). The Secondary antiphospholipid antibody syndrome associated to SLE may lead purpose of this 5-year prospective study was to describe, in patients with to catastrophic syndrome (catastrophic APS) which determine a fatal final PAPS, the changes over time in the prevalence of cardiac abnormalities by outcome. In these situations nonstandard adjunctive therapies such as means of TEE. Materials and methods: A group of56 patients affected by (8 plasmapheresis play an important role. Clinical report: We report two SLE men,48 women; mean age 37±10 years; range 20-68) was included in this affected pediatric patients ( 11 and 8 years old) with catastrophic APS, that study. All subjects underwent TEE at baseline and every 1-2 years. recieved plasmapheresis in addition to standard regimen of corticosteroids, Abnormal mitral thickening assessed by TEE was defined as a thickness of cyclophosphamide and anticoagulation. Six daily membrane plasmapheresis more than 3 mm. The severity of valvular stenosis was assessed by sessions with a 5 plasmatic volumen exchange were performed and calculating valvular area by using the continuity equation. The presence of intravenous immunoglobulin ( g/Kg/d for two consecutive days) was severe spontaneous echocontrast in left atrium and/or in appendage, administered. This treatment significantly reduced disease activity intracardiac thrombi and non-bacterial valvular masses were considered as (measured by clinical outcome, and immunologic parameters). None of the potential embolic sources. Results: TEE showed initial cardiac abnormalities children suffered a bacterial or viral infection. Discussion: We describe the (mitral valve thickening and/or embolic sources) in 34 (60.7 %). Mitral clinical and serological features of two new cases of pediatric patients with valve thickening was the most common abnormality (88.2 %). Our data catastrophic APS. In the most recent series (1), comprising a total of 80 demonstrated embolic sources in 14 (41.2 %) patients (9 spontaneous patients, only 4 of them was pediatric patients (< 16 year old).Our results echocontrast and 5 Libman-Sacks endocarditis). TEE studies repeated in 50 suggest that plasmapheresis remains a therapeutic option for the treatment of patients (89.0 %) after 5-year follow-up period, revealed cardiac life-theatening SLE and catastrophic APS. 1- Ronald A.Asherson, Ricard involvement unchanged in 42 cases (84.0 %), whereas new lesions appeared Cervera, Jean-Charles Piette et al. Catastrophic Antiphospholipid Syndrome. in the remaining 8 (16.0 %) (2 cases in low-IgG aCL positive titer and 6 Clues to the pathogenesis from a series of 80 patients. Medicine 80: 355-77, cases in high-IgG aCL positive titer group). Discussion. The TEE 2001. prospective study revealed that patients with IgG aCL titer >40 U showed a higher prevalence of new cardiac abnormalities. PREVALENCE OF ANTI-THYROGLOBULIN ANTIBODIES IN APS THROMBOTIC MICROANGIOPATHIC HAEMOLYTIC ANEMIA (TM] IN APS F. Kvapill, Y. Shererl, S. TartakoverMatalon', Y. Levy', M. Blank', J. Rovensky2, L. Cehecauer2, J. Lukac2, Y. Shoenfeld3 S. Bucciarellil, G. Espinosa', R Cervera', J. Font', R.A. Asherson2, M. 'Centerfor Autoimmune Diseases, Department ofMedicine B, Chaim Sheba Ingelmol Medical Center, Tel-Hashomer, The Sackler Faculty ofMedicine, Tel-Aviv iHospital Clinic, Barcelona, Spain. 2University ofCape Town, South Africa University, Israel, 2National Institutefor Rheumatology, Piestani, Slovakia Clinical and immunologic characteristics of 25 patients with TMHA and Objective: We determined the occurrence of anti-thyroglobulin antibodies (computer-assisted review of the literature 1983-2001) are described. All pa and their association with various clinical manifestations in a large cohort of presented with a negative Coombs test and thrombocytopenia. Twenty-two ( women with primary antiphospholipid syndrome (APS). Methods: 176 sera patients were female. Mean age at presentation of TMHA was 35.9 years. Fou of women with primary APS and known clinical data were tested for the (56%) patients had primary APS, and 11 (44%) had defined systemic presence of anti-thyroglobulin antibodies. As a control served 117 sera from erythematosus (SLE). In 16 (64%) patients TMHA appeared in the conte: healthy blood donors. Results: The prevalence of anti-thyroglobulin catastrophic APS. In 15 (60%) patients, TMHA was the first clinical manifestati antibodies (10.8%) in our cohort was higher than prevalence (1%) in the APS. Nineteen (76%) patients had renal involvement, central nervous s3 normal population (p<0.003). The levels of anti-thyroglobulin antibodies in involvement was evident in 19 (76%), skin involvement in 12 (48%), cardiac the APS group were signiificantly highler than in the control population (36%), and pulmonary involvement in 4 (16%) patients. Steroids were used in 84 (p Wu Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lWikmTgiciMOisnomnimfqdpdAtI es Poisetam IMMUNOLOGICAL FINDINGS IN PATIENTS WITH ASEPTIC NECROSIS IN PATIENTS WITH PRIMARY APS 619 PULMONARY MANIFESTATIONS IN APS M.G. Tektonidou', K. Malagari', P. Vlachoyiannopoulos'. H.D. Andreeval, P.K. Petroval, V.D. Andreev', V.I. Trifonov2 H. Moutsopoulos' 'Center of Clinical Immunology, 2DepartmentfLung Diseases, 'Department ofPathophysiology, 2Secoisd Department ofRadiology, University School ofMedicine, Pleven, Bulgaria Medical School, National University ofAthens, Greece Several groups of pulmonary complications have been documented in Objective: To evaluate the prevalence of aseptic necrosis (AN) by magnetic patients with antiphospholipid antibodies (aPL), both in the context of resonance imaging (MRI) in patients with primary antiphospholipid primarv and secondary APS. We aimed to describe the clinical syndrome (PAPS), in the absence of corticosteroid use. Methods: Thirty manifestations, immunological and laboratory findings in patients with patients with PAPS who never received corticosteroids (23 women) were positive aPL and episodes of pulmonary involvement. Methods. 21 patients examined by MRI of the femoral head, in Ti and T2 sequences. MRI is the with evidence for APS were described and retrospectively reviewed for most accurate method for AN detection. The most common site ofAN is the presenting symptoms and signs of APS, together with laboratory, femoral head. Established MRI criteria were used for AN diagnosis. immunological findings and clinical outcome. The following immunological Exclusion criteria in the study were trauma, alcoholism, sickle cell disease, methods were used - serological (total aCL, b2-GPI, ANA, RF, dsDNA); pancreatitis, Gaucher disease. Results: In 3 out of the 30 PAPS patients (10 flowcytomctric analysis (blood, pleural fluid (PF), bronichoalveolar lavage percent), MRI revealcd advanced AN (doublc linc sign in T2W sequence) of fluid (BALF) with a broad spectrum of MoAbs - CD45/CD14, CD3i'CD19, both hips. None of the patients was symptomatic. One patient became CD4/CD8, CD16, CD62L/CD11b, CD25, DR, TCR). Results: The clinical symptomatic three months after the examination. Early stage AN (band diagnosis was present with symptoms raging from pleuritis / pleural effusion pattem) was seen in 3 asymptomatic patients (10 percent) (unilateral in two, (n=8), rapidly changing pulmonary infiltrates (n=3), atypical pneumonia bilateral in one). Repeat MRI at six months revealed no changes. Totally, 6 (n=3), and pulmonary fibrosis (n=4), pleuropneumonia (n-3). The patients had AN vs 24 patients without (non AN). The mean age (years) at association with other clinical manifestations of APS was found. The the time of examination in the first group was 30.71plus/minus 8.06 (95 peripheral blood lymphopeny was associated with lymphocytosis in PF due percent CI 23.26,38.17) and in the second group 41.59plus/minus 11.61 (95 to elevated T-helpers (CD4+/CD25+1DR+) or with increased number of percent CI 35.2,47.56) (p=0.035). The mean disease duration (years) in AN activated neutrophils (CD62L-/CDllb++/CD16+) in BALF. Conclusion. group was 4.57 plus/minus 4.67 (95 percent CI 0.238,8.91) and in the non Although less frequently appreciated, pulmonary involvement in APS may AN group 6.82plus/minus 4.19 (95 percent CI 4.69,8.95) (p=-0.26). Bone concurrently attack the lung compartments. The innovate flowcytometric marrow edema (unilateral) was demonstrated in 2 out of 30 patients (6,7 analysis support the diagnosis of pulmonary involvement in APS and may percent). Conclusion: AN can be detected by MRI in 20 percent of patients be used for monitoring the effect of the treatment. It is crucial for with PAPS. Younger patients tend to develop AN more frequently than pneumologists, pathologists and immunologists to work together when older. Clinicians should be aware of this possible clinical manifestation of attempting to identify lung involvement associated with APS. the PAPS because early diagnosis can lead to early treatment. IMMUNOLOGICAL DIAGNOSIS OF SYSTEMIC VASCULITIDES. ASSOCIATION BETWEEN THROMBOCYTOPENIA AND THE SIGNIFICANCE OF THE DETERMINATION OF ANTIBODIES TO ANIONIC PHOSPHOLIPIDS AND BETA2 ANTICARDIOLIPIN ANTIBODIES. GLYCOPROTEIN I IN SLE K.V. Nikolov', M.P. Baleva2, B.B. Varbanova3 M. Vadacca', A. Amoroso2, D. Caccavo3, F. Del Porto2, G.M. Fer2, S. GallUZZo2, P. Garzia2, A.P. Mitterhofes?, A. Rigon', A. Afeltra' 'Department ofDermatovenerology, Alexander University Hospital, Sofia, 'ofClintoryofCiicalImmunology, Centre ofAllergology, University 'University Campus Bio Medico, "University La Sapienza, Rome, 3 Alexander Hospital, Sofia, 3Medical University, Varna, Bulgaria University ofBari, Italy The aim of our study was to evaluate the significance of some Objective: The objective of this study was to evaluate the association immunological parameters for the diagnosis of systemic vasculitides. We between antibodies to diffcrcnt negatively chargod phospholipids and anti investigated the levels of ANA (indirect immunofluorescence on HEp-2 beta2 glycoprotein I (anti beta2 GPI) with thrombocytopenia in SLE. cells), ANCA (indirect immunofluorescence on human granulocytes), Patients and Methods: Sera were obtained from 87 patients (77 females and anticardiolipin antibodies (ACL) and rheumatoid factor (RF) isotype 10 males, median age 39 years, range 17-59; mean disease duration 8.9 (ELISA), DNA, Sm, RNP, Ro, La antibodies (counterelectrophoresis), years) affected by SLE. IgG and IgM anticardiolipin antibodies (aCL), anti- immunoglobulin, C3 and C4 complement fraction levels (radial phosphatidic acid (aPA), anti-phosphatidylinositol (aPI), anti- immunodifusion) in patients with systemic vasculitides and connective phosphatidylserine (aPS), anti-beta2-GPI were evaluated by ELISA using tissue diseases: 24 with Henoch-Schonlein purpura, 6 with Bechcet, 22 with commercially available kits (ORGenTec-Diagnostika GmbH, Mainz, urticaria vasculitis, 30 with systemic lupus erythematosus (SLE), 4 with Germany). The statistical significance of associations was evaluated by Churg-Strauss vasculitis, 4 with Wegener's granulomatosis, 60 with Fisher's exact test. Results: Thombocytopenia (platelet count less than Raynaud phenomenon, 2 with Horton vasculitis, 48 with juvenile 100,000 per microliters) was present in 18 (21%) patients. The prevalence of rheumatoid arthritis. Positive ANA were found more frequently and in different autoantibodies was: IgG -aCL 53%, -aPA 37%, -aPI 32%, -aPS higher titers in patients with SLE. cANCA were common in high titers in 38%, anti beta2-GPI 24%; IgM -aCL 15%, -aPA 17%, -aPI 18%, -aPS 14%, granulomatous vasculitides, pANCA were present in the sera of patients antibeta2-GPI 16%. Thrombocytopenia was significantly associated with with other vasculitides. The values of IgG and IgM ACL were increased IgG -aPA (p 0.0008), -aPI (p 0.02), -aPS (p 0.03), irrespective of antibody mainly in patients with SLE and usually were connected with clinical data titer. When considering IgG aCL positivity at any antibody titer, no for antiphospholipid syndrome. correlation was found with thrombocytopenia, whereas significant correlation was found between thrombocytopenia and IgG aCL at medium- high titer (p 0.046). No correlation was found between thrombocytopenia and all TgM antibodies evaluated. Conclusions- The strong association between thrombocytopenia and IgG - aPA suggests that these autoantibodies could represent a predictive serological marker for thrombocytopenia in SLE. UPS Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 tiui nrOOngess fankhtildqidAibocUes Pam Piseot 620 INTRARENAL BLOOD FLOW IN PATIENTS WITH APS RENAL INVOLVEMENT IN PRIMARY AND SECONDARY APS NEPHROPATHY IN PRIMARY AND SECONDARY APS M. Vianellil, I. Cavazzanal, P. Napodano2, A. Tincanil, R.A. Sinico2, N.A. Samokishina, V.V. Kushnir, E.A. Shahnova, N.L. Kozlovskaya, M. Cianquinil, G. Balestrieril N.A. Meteleva 'Clinical Immunology Unit, Spedali Civili di Brescia, 2Nephrology Unit, Moscow MedicalAcademy, Russia S. Carlo Hospital, Milan, Italy The aim ofthis study was to evaluate intrarenal blood flow parameters in pts Background: Renal involvement during APS has been frequently described with PAPS and SLE. We studied 28 pts, age 23- 50 (F-23, M-5): 8 pts with as thrombotic occlusions at any level of vascular tree; nevertheless PAPS (gr.1), 14 pts with SLE with APS (gr.2) and 6 lupus nephritis (1EN) pts non-thrombotic features have been reported in few cases (Griffiths, 2000). without APS (gr.3). The intrarenal blood flow was investigated by Duplex Aim of the Study: To evaluate the prevalence and histologic features of Doppler ultrasonography method. We measured systolic (Vs) and diastolic renal involvement in patients (pts) affected by PAPS and secondary APS. (Vd) velocities and resistance Index (RI) in segmental, interlobar and Pts and methods: six cases out of 115 pts with PAPS and 17 cases out of 43 arcuate arteries. Results: see Table 1. pts affected by systemic lupus erythematosus with APS have been selected because of abnormal urinanalysis (microhematuria, proteinuria more than Table 1. The intrarenal blood tlow velocities in PAPS and SLE pts.(X+/-sd) 300 mg/day) or elevated serum creatinine (>1.5 mg/dl). Fifteen pts performed a percutaneous renal biopsy. Results: Microhematuria and Gr.pts V segmental (cnls) V interlobar(cm/s) V arcuatc (ctrs) proteinuria were recorded in 5 pts with PAPS and in 12 pts with secondary Vs Vd Vs Vd Vs Vd = APS, nephrosic syndrome was detected in one patient with PAPS and in 2 Gr. 1 70,+/-6,4 29+1-4,2 38+/-8,4 16+/-5,1 8+/-11,3 3+/.3,9 cases with secondary APS. Three pts had chronic renal insufficiency. Four Gr. 2 93+/-26,3 38+1-8,4 51+/-13,4 22+/-6,0 15+/-s1/5,9 7+/-7,5 thrombotic microangiopathy (2 PAPS and 2 secondary APS), 4 membranous Gr. 3 98+/-19,5 40+/-7,0 52+/-11,0 23+/-4,7 32+/-7,6 15+!-2,2 GN (2 PAPS), 5 prolipherative GN (one PAPS) and 2 necrotizing GN were detected. Conclusions: Renal features were detected in 5.2% and 39.5% of The pts of the gr. I had lower parameters of blood flow in comparison with primary and secondary APS, resectively. Three pts with PAPS had the 3d The had lower of blood pts group (p<0,05). PAPS pts parameters glomerulonephritis (prolipherative and membranous) without any histologic in and flow segmental interlobar arteries than those with SLE and APS features of thrombosis: all of theme were anti-native DNA, anti-ENA were (p PREVALENCE AND CLINICAL SIGNIFICANCE OF RENAL INVOLVEMENT IN PRIMARY ANTIPHOSHOLIPID ANTICARDIOLIPIN, ANTI-DS-DNA AND ANTINUCLEAR SYNDROME: REPORT OF TWO CASES ANTIBODIES IN PATIENTS WITH SLE WITH AND WITHOUT S. Rednicl, I. Andrei', D. Vladutiu2, M. Petrescu', N.R. Rednic4, RENAL INVOLVEMENT H.D. Bolosiul M.K. Nikolova', I.D. Vlahov2, A.B. Ilievl, B.N. Kiperoval, R.J. Djerassil 'Department ofRheumatology, 2Department of ?sfephrology, 3Department of 'Clinic ofNephrology, 2University Alexander Hospital, Faculty ofMedicine, Pathology, 'Department ofInternal Medicine IV, University ofMedicine Sofia, Bulgaria and Pharmacy, ClujNapoca, Romania Objective: to investigate the prevalence and clinical significance of Background: The renal involvement attributable to antiphospholipid anticardiolipin (ACL), anti-ds-DNA and antinuclear (ANA) antibodies in syndrome (APS) may take several forms, most of which may be associated patients with systemic lupus erythematosus (SLE) with and without lupus with renal hypertension. Case studies: We describe two young patients with nephritis (LN). Materials and Methods: we investigated the levels of IgG primary APS who presented with severe and accelerated hypertension. No and IgM ACL (enzyme immunoassay), ds-DNA (counter evidence of systemic disease was observed and the physical examination immunoelectrophoresis) and ANA (indirect immunofluorescence on HEp-2) revealed only livedo reticularis. The anticardiolipin (ACL) antibodies titers in the sera of 87 SLE patients (12 male and 75 female, mean age 43.11, were repeatedly extremely high. The diagnostic of primary APS was range 11-77) divided in two groups: group I with biopsy-proven LN (45 formulated. Renal angiography found nornal proximal renal arteries and patients) and group It(42 patients)-without renal involvement. The levels of other renal investigations were normal. Renal biopsy showed ischaemic ACL were compared with the clinical manifestations of antiphospholipid glomeruli without proliferative lesions, but with focal intimal fibrosis, syndrome (APS). RESULTS: IgG and/or IgM ACL, ds-DNA and ANA thrombosis of the intrarenal vessels in the absence of vasculitis. They were were found more frequently in group I (22/45 vs. 3/42; 34/45 vs. 21/42; treated with antihypertensive drugs, anticoagulant and antiaggregant 30/45 vs. 21/42 respectively, p<0.01 in all cases), as were clinical data for therapy. [nstead of this treatment one of the patients developed progressive A.PS (17/45 vs. 3/42, p<0.01). In group I we found no correlation between multiorgan failure with malignant hypertension, renal failure, central the presence of ACL, ds-DNA and ANA and renal histological pattem, nor nervous system manifestation (grand mal seizures, hemiparesis, amaurosis between the levels of ACL and the severity of APS manifestations. There fugax, steady deterioration of consciousness) and gastrointestinal was a significant correlation between ACL/APS and the presence of LN involvement (abdominal pain due to ischemic ulceration of the bowel), (r='0.37 and 0.45 respectively, p<0.01). In conclusion, our data demonstrate actually a catastrophic APS. Discussion: The cases suggest that higher prevalence of ACL, ds-DNA and ANA in SLE patients with renal hypertension and renal failure should be recognized as complication ofAPTS, involvement and a significant correlation between ACL/APS and the the etiology being primary intrarenal vascular disease with presence of LN. Therefore, ACL appear to be an important diagnostic and nephroangiosclerosis. It's illustrates also how primary APS presenting with prognostic marker in LN patients especially in the presence of other renal manifestations can result in a fatal form of catastrophic APS. thrombophylic factors, such as nephrotic syndrome, corticosteroid treatment, dehydratation, etc. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lntInatiii Qgss an ioswici-dhibodes PosterPrestatkm CARDIAC ABNORMALITIES IN SECONDARY AND PRIMARY TRANSESOPHAGEAL ECHOCARDIOGRAPHY VALUE TO 621 APS EVALUATE APS RELATED VALVULAR LESIONS T.M. Reshetnyakl, O.A. Fomicheva2, G.P. Kotelnicoval, E.M.N. Albuquerque', 0. Ribeiro', S.S. Xavier2, M. Castierl, E.N. Alexandrova2, T.V. Popkoval, I.E. Shirocoval, V.A. Nassonova', J.A.S. Papi2, D.S. Silveira2, M.I.D. Souto2, A.C. Fonseca2, E.M. Klumb', E.L. Nassonovi R.A. Levy] ]Institute ofRheumatology ofRAMS, 2Cardiac Research Center, 'Universidade do Estado do Rio de Janeiro, Universidade Federal do Moscow, Russia Rio de Janeiro, RJ, Brazil Aim: To describe the cardiological abnormalities in patients with systemic To study the prevalence of cardiac valvular alterations in patients with lupus erythematosus (SLE) with antiphospholipid antibodies (aPL) and in antiphospholipid syndrome (APS), we examnined by transthoracic (TT) and patients with primary APS (PAPS) with estimation these features during multiplan transesophageal echocardiography (TE-ECHO) 61 patients with follow-up. Patients and methods: There were 195 pts followed up during APS according to Wilsons criteria. Twenty-three patients had primary and nine years in this study. All patients were divided into three groups: the I-st 38 had APS secondary to SLE. Clinical manifestations and TE-ECHO Gr. included 51 pts (9M; 32F) with SLE; the Il-nd Gr. 88 pts (23M; 65F) findings of the 2 groups were compared. The same examiner, with the same with SLE and with aPL; the III-d Gr. 56 pts (ISM; 41F) with PAPS. equipment, blinded for patients diagnosis, performed all exams. In 11 (53%) Results. Myocardial infarction (MI) was detected in 9 pts (under 45 yrs of 23 primary APS patients and 26 (68%) of38 secondary, valvar alterations age): in 5 (6%) pts from the II-nd Gr. and in 5 (9%) pts from the 1II-d Gr. were found by TE-ECHO, one had altered TT-ECHO. The most commonly Occlusions of coronary artery were detected by coronary angiography with found alterations were: valvular regurgitation (47.8% in primary group and signs of atherosclerosis in vessels affected only in 2 of 9 pts with MI. The 52.6% in the secondary); valvar thickening, that occurred frequently in both frequency of different valvular lesions was thc highcst in Ill-d Gr. in 24 of groups (39% and 34.2%, respectively), valvar vegetations, that were more 56 (43%) pts versus the II-nd Gr. pts (27% p=,0l) and especially in common on the primary APS group (17.4% x 13.2%, respectively). The comparison with the I-st Gr.(4%) (p =,0002). Abnormalities of aortic valve most commonly affected valve was the mitral, with dysfunction (26% in were more often in II-d Gr.pts comparison with the I-st and lI-nd Gr. in 11 primary APS and 47.4% in secondary) and thickening (39% and 34.2%), of 56 (20%) (p =,01). The replacement of mitral valve was done in one pts followed by the aortic. Vegetations in primary APS were evenly distributed with PAPS. During 9 yTs follow-up 10 pts with cardiac abnormalities died; 4 among mitral and aortic valves; while the vegetations found in 5 secondary of them because of heart failure. Conclusion: Cardiac abnormalities are APS were all restricted to mitral valve. We did not observe ventricular associated with elevated aPL. Valvular lesions predominant in PAPS, dysfunction in none of the patients with primarv APS, while it was found in especially aortic valve involvement correlates with PAPS. 13% of the patients with secondary APS. TE-ECHO is a powerful diagnostic tool for evaluating valvular function in both primary and secondary APS. ANTIPHOSPHOLIPID ANTIBODIES AND THE CLINICAL MANIFESTATION OF THE ANTIPHOPHOLIPID SYNDROME W. Miesbach, B. Buehler, G. Asmelash, 1. Scharrer J. W Goethe-University, Frankfurt, Germany Antiphospholipid (aPL) antibodies are known to be associated with thrombotic events in the antiphospholipid syndrome (APS), which appears CARDIAC AND CEREBRAL MANIFESTATIONS OF THE APS to be the most common acquired thrombophilic defect. The labaratory W. Miesbach, F. Jung, I. Scharrer detection of aPL antibodies can be perfonned either using solid phase Anticardiolipin (aCL)-ELISA assays (IgG-aCL, IgM-aCL) or coagulation Goethe University, Frankfurt, Germany assays to detect lupus anticoagulants (LA). We describe cardiac and cerebral manifestations in 27 patients with Patients: The present paper describes the clinical features of 3 groups of antiphospholipid syndrome (APS). Patients and Methods: We studied 27 patients (n=1 00) with either a) IgG-aCL-titers >100 GPL-U/ml (n=34), patients with APS with either cardiac, cerebral or both manifestations. All b) IgM-aCL-titers >80 MPL-U/ml (n=27) and c) patients with positive lupus patients fulfilled the proposed classification criteria for the APS according anticoagulants in two different tests and negative aCL-assays (n=39) to the SSC of the ISTH. Two patients died and had an autopsy. Results: 18 (normal range of IgG-aCL: 9-11,7 GPL-U/ml; normal range of lgM-aCL: 3- of the 25 patients had coronary artery disease with either thrombotic 4,8 MPL-U/ml). All patients fullfilled the proposed classification criteria for coronary occlusions, high grade stenosis or complete vessel occlusion. In the APS according to the SSC ofthe ISTH. three patients the left main artery was involved. 10 patients had involvement a) 19/34 patients with IgG-aCL-titer >100 GPL-U/ml presented with arterial of the left anterior descending artery (LAD), 6 patients of the circumflex thrombosis, 16/34 patients with venous thrombosis, and 5/34 patients artery (RCX) and 5 patients had involvement of the right coronary artery without any thrombembolic disease. (RCA). As valvular lesions have been described in association with the APS b) 12/27 patients with IgM-aCLtiter >80 MPL-U/ml had previous arterial it is to note that in 16 cases there was mitral- or aortic valve disease. Three thrombosis; 6/27 patients were suffering from a venous thrombosis and patients underwent mitral valve or aortic valve replacement. 7 patients 11/27 patients didn't have any thrombembolic disease before. presented with a history of cerebrovascular involvement. 5 of these patients c) In over 50 % of patients with persistent positive LA and negative ACL- had cerebral infarction and one patient recurrent cerebral bleeding under assay (20/39) no thrombembolic disease could be found. 12/39 patients had oral anticoagulant therapy. 3 of the 7 patients had a prior history of secondary APS or malignancy and in 8/39 patients LA were normalized. myocardial infarction, whereas 3 patients underwent mitral or aortic valve Results: By including only patients with either high elevated IgG-aCL- and replacement. Conclusion: Based on our findings APS is more frequently IgM-aCL-titers or LA without any elevated aCL antibodies, the clinical associated with cardiac or cerebral manifestations than hitherto described. feature of these patients can clearly be distinguished. IgG-aCL seems to be Also valvular lesions can be associated with the APS. the strongest risk faktor for thrombembolic disease, so over 85 % of these patients were suffering from thrombembolic disease, whereas nearly 60 % of patients with IgM-aCL >80 MPL-U/ml were suffering from thrombembolic disease. In both groups arterial thrombosis was more frequeiat than venous thrombosis. 32,3 % ofpatients with IgG-aCL >100 GPL-U/ml as well as 35 % of patients with IgM-aCL>80 MPL-U/ml were suffering fom cerebral infarction. Otherwise 15 % of patients with IgG-aCL >100 GPL-U/ml didn't have any thrombembolic disease before. In over 40 % of the patients with IgM-aCL > 80 MPL-U/ml thrombembolic disease could not be found. Most of the patients with persistent positive LA and negative ACL-assays (51,3 %) have not complained a thrombembolic disease in the past. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 ahemadandQmgenssm tionphpdArtibocIes PofPws ts 622 APS NEPHROPATHY IN PATIENTS WITH ISOLATED ANTIPHOSPHATIDYLETHANOLAMINE ANTIBODIES AND ANTIPHOSPHOLIPID ANTIBODIES VALVULOPATHIES IN PATIENTS WITH SLE E. D. V. Daugas', Nochy2, Caudwell3, C. Jacquot4 E. Balada, J. Ordi-Ros, M. Mauri, F. Paredes, J. Villarreal, M. Labrador, 'Service de Nephrologie B, Hopital Tenon, Paris, 2Services d'Anatomo- M. Vilardell Pathologie et de 4Nephrologie, HEGP, Paris, 3Service de Nephrologie, Hospital Vall dHebron, Barcelona, Spain Hopital Louise Michel, Evry, France Objective: To evaluate and correlate the presence of antiphospha- APS associated nephropathy (APSN) is a vascular nephropathy associating tidylethanolamine antibodies (aPE) and valvulopathies in patients with acute thombotic lesions (thrombotic microangiopathy), chronic vascular systemic lupus erythematosus (SLE). Methods: A total of 51 patients who lesions (fibrous intimal hyperplasia or organiized thrombi of small arterics) suffered from cardiac valve disease were included in this study. All of them and/or downstream focal cortical atrophy. APSN occurs in primary APS and fulfilled the revised criteria of the American College of Rheumatology in secondary APS in lupus where it worsens the renal prognosis. In lupus, (ACR) for the classification of SLE. They were tested for aPE and anti- APSN may be diagnosed isolated, without extrarenal manifestation of the cardiolipin antibodies (aCl) by two independent ELISAs. Lupus APS. The aim of this study was: should APSN be suspected in patients with anticoagulant (LA) was assessed by coagulometric methods. Fifty healthy antiphospholipid antibodies but not APS in non lupus patient? We studied blood donors' plasmas were included as controls. Results: Statistically retrospectively 18 patients having isolated antiphospholipid antibodies - significant differences werc observed between IgG aPE-positive and without APS- (sustained LA or aCI >=15 GPLU in 8 and 11 patients negative patients (p=0.002). Thirty-nine (76.47%) patients had mitral valve respectively), who had a renal biopsy. Patients with primary APS or lupus involvement, 12 (23.52%) had aortic valve involvement, and 6 (11.76%) and patients with end stage renal disease were excluded. Of these 18 had both mitral and aortic valve involvement. Mitral and/or aortic valve patients: 2 had fibrous endarteritis without specific glomerular changes regurgitation were the common manifestations. Five of the patients required (nephrosclerosis), 1 had acute tubular necrosis, I had an acute immuno- valve surgery. No patient presented with stenosis or valve thrombosis. IgG allergic insterstitial nephritis; the 14 others had glomerular lesions aPE were equally found in tho.se patients with mitral valve involvement and including: 6 focal and segmental glomerulonephritis (GN), 3 in those with aortic valve involvement (66.66%). Five out of the 6 patients 2 membranoproliferative GN, mesangial IgA GN, 1 minimal change GN, 1 with both mitral and aortic valve involvement had IgG aPE antibodies. membranous GN, 1 endocapillary proliferative GN (post-infectious). Statistically significant differences were also found when considering IgG Tbrombotic microangiopathy (TMA), the less specific lesion of APSN, was aPE as the sole aPL antibodies (p=0.028). Correlation with valvulopathies present in only one patient with IgA GN and 30 GPLU of anticardiolipin remained even at aPE cut-offs of 0.600 (8 s.d.) (p=0.019). Conclusions: aPE antibodies without microangiopathic hemolytic anemia. No patient had correlated with valvulopathies. Since valvulopathies are found in some lesion of chronic APSN. We conclude that APSN is a very rare event in patients with antiphospholipid syndrome (APS), we conclude that the patients with isolated antiphospholipid antibodies. Thus, in non lupus evaluation of aPE may allow us to detect some more secondary APS patients, whithout APS, antiphospholipid antibodies are poorly predictive of patients in the SLE population. APSN. CHRONIC THROMBOEMBOLIC PULMONARY HYPERTENSION CENTRAL SEROUS RETINOPATHY IN ASSOCIATION WITH IN THE ANTIPHOSPHOLIPID ANTIBODY SYNDROME: A CASE ANTIPHOSPHOLIPID ANTIBODIES REPORT K. Ta', J. Coppeto2, A.M. Conti-Kelly2, T. Greco3 K.T. Calamia, M.E. Menefee, J.M.S. Pascual 'Yale Primary Care Program, Yale University School ofMedicine, Mayo Clinic, Jacksonville, FL, USA Waterbury and New Haven, CT, 2St. Mary's Hospital, Waterbury, CT, A 65 year-old female was evaluated for dyspnea and thrombocytopenia. 3Yale University School of.7Medicine, St. Mary's Hospital, New Haven and Three years earlier she presented with deep venous thrombosis with Waterbury, CT, USA pulmonary embolism and warfarin was initiated. One and one half years Purposc: Ischcmia of choroidal lobules resultinlg in choroidal later dyspnea and hypoxia following hysterectomy led to a chest CT scan hyperperneability has been hypothesized as the initial event in central that showed evidence of chronic clot. An inferior vena cave filter was serous retinopathy. This study reports a potential association of central placed. Thrombocytopenia developed, unresponsive to prednisone. She was serous retinopathy with antiphospholipid antibodies (aPL), which may play able to return to usual activities, but exertional dyspnea progressed over 6 a role in choroidal ischemia. Methods: Review of case studies from a patient months prior to evaluation. The PT was 27.1 with an INR of 2.5. The cohort prospectively evaluated for aPL. Results: All four patients identified platelet count was 42,000/ml. Anticardiolipin antibodies were positive. with acute and chronic central serous retinopathy confirmed by fluorescein Pulmonary function testing revealed a DLCO at 63% of predicted. Oxygen angiography were found to have high titers of aPL (anticardiolipin, saturation was 91% at rest and 81% on exertion. An echocardiogram antiphosphatidylserine, and anti-beta2 glycoproteinl antibodies). Two revealed marked right ventricular enlargement with hypokinesis. There was patients had concommitant diagnosis of antiphospholipid syndrome (APS). severe tricuspid regurgitation with an estimated RV systolic pressure of 114 Another patient had a family member with APS. All patients were treated mm Hg. Pulmonary angiography showed smooth narrowing of the with aspirin and/or warfarin, and remain stable. Conclusion: Elevated titers pulmonary arteries with occlusions of multiple branches. The patient was of antiphospholipid antibodies may be seen in patients with central serous concemed about the risks of thromboendarterectomy, especially in the retinopathy. Our findings suggest a role for antiphospholipid testing in setting of thrombocytopenia, and elected against surgery. Anticoagulation patients with central serous retinopathy. Prospective studies are warranted. was maintained but she died of respiratory failure several weeks later. This case suggests that, in presence of anticardiolopin antibodies, progressive hemodynamic and structural derangement of the lung circulation may occur after pulmonary thromboembolism, despite adequate anticoagulation and IVC filter placement. Associated thrombocytopenia was not protective, and might represent persistent thrombotic activity. Nonetheless, because of the poor prognosis of patients with chronic pulmonary thromboembolic syndrome, thromboendarectomy should be considered. bpm Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 IflhIntinai Oxgess ton iospipd docles PostrPmset*6m aPL IN PATIENTS SUFFERING FROM AUTOIMMUNE PLASMAPHERESIS USING IN COMPLEX TREATMENT OF 623 HEPATITIS AND PRIMARY BILIARY CIRRHOSIS PREGNANT WOMEN HAVING APS E. Toubi, A. Kessel, K. Hallas, E. Zuckerniai T.N. Demina, E.B. Yakovleva, S.V. Cheremnych Bnai-Zion Medical Center, Haifa, Israel State Medical University, Donetsk Ukraine Background: Previous studies documented the presence and persistence of Aim ofthe study was substantiation of including and working out ofoptimal anticardiolipin antibodies (aCL) overtime, in patients chronically infected methods of plasmapheresis in complex therapy of antiphospho- with HCV. However, no such data is available regarding aCL and lipidsyndrome during a pregnancy. Materials and Methods: 70 pregnant autoimmune liver diseases. Objective: We studied the dynamics of aCL and women with antiphospholipidsyndrome underwent complex treatment at anti beta2 glycoprotein I (a b2 GPI) overtime, in patients with autoimmune pregnancy term of 6-12 weeks. By trecatmnt of 37 pregnant women of the hepatitis (AIH) and primary biliary cirrhosis (PBC). Patients and Methods: 1st group complex therapy was completed with filter plasmapheresis; 33 Thirty patients with AIH(group I), were investigated at baseline and six pregnant women of the 2nd group (group of comparison) underwent months following immunosuppressive (IS) treatment. Group II included 35 complex therapy adopted in the Centre. 800/1000 ml of plasma was PBC patients followed over a year. Thirty age-sex matched normal removed at one procedure. Standard plasma replacement was done by individuals served as a control group. Antibodies to HEP-2, smooth muscle, albumin, reoplyglucin, immunoglobulin intravenous. Ratio of removed mitochondna, aCL, a b2 GPI, and complement levels were detennined in plasma and plasma substitutes was 1:1.4. plasmapheresis course lasted 14 all. Results: In group 1, 9/30 (30%) reacted low-moderately positive to aCL days and consisted of 3 procedures with intervals of 4-5 days. Results: Data compared to 2/30 controls and in 6/30 (20%) to a b2 GPI compared to non received showed that patients had changes in thrombocytopenia, in controls (p=0.01 and 0.02 respectively). Following treatment and in hyperaggregation, hypercoagulation in plasma and increasing of fibrinolytic parallel to a clinical-serological improvernent, aCL disappcared in 5/9 and a potential was marked. Fibrinogen concentration was 4.9/0.3 gram/litre, b2 GPI in 3/6 patients. Those in whom aCL / a b2 GPI persisted, required summary evaluation of coagulability factors were enough high. In 18.5% longer IS therapy and frequently relapsed upon treatment discontinuation. In cases moderate compensated hypercoagulation prevailed it wasn't followed group I1, 5/35 (14%) and in 4/35 (11%) were found positive to aCL and a b2 by fibrinolysis activation. In 15.5% cases were marked changes which had GPI respectively (p= N.S. in comparison to controls). Conclusions: Whereas decompensed character and appropriated 2-3 phase of disseminated aPL were found in correlation with AIH severity and turned to disappear intravascular coagulation syndrome. After plasmapheresis being done in upon improvement, they were found infrequently in PBC patients. We target group the decreasing of fibrinogen concentration from 4.9/0.3 to suggest that aCL and a b2 GPI are of different origin and have different 3.7/0.2 gram/litre, partially activated thromboplastin time fiom 52/0.2 to significance in both diseases. 41.2/0.2 sec, kaolin time from 69.4/0.2 to 44.7 sec, the increasing of thrombocyte concentration from 164.000 to 220.000 was marked. Conclusion. plasmapheresis including in complex therapy of antiphospholipidsyndrome is clinically effective and an important reserve of perinatal loss prevention. EFFICACY OF AFFINITY PURIFIED ANTI-dsDNA ANTI- FRAXIPARIN USING IN PREGNANT WOMEN HAVING APS IDIOTYPIC ANTIBODIES IN THE TREATMENT OF V.K. Chaica, T.N. Demina, S. Dghelomanova EXPERIMENTAL SLE State Medical University, Donesk, Ukraine, Y. Shoenfeld', L. Rauova'3, B. Gilburel, L. Zi?oren', F. Kvapil', I. Goldberg2, J. Kopolovic2, J. Rovensky M. Blanc' The aim of the investigation is to study the fraxiparin influence on the fetoplacental complex function in patients with antiphospholipid svndrome. 'Centerfor Autoimmune Diseases, Medicine B, Chaim Sheba Department of Methods: 90 pregnant women with antiphospholipid syndrome were Medical Center, Tel-Hashomer, The Sackler Faculty ofMedicine, Tel-Aviv examined and divided into 2 groups. The 1 group consisted of 40 pregnant University, 2Department ofPathology, Chaim Sheba Medical Center, women treated with fraxiparin according to the new method, the 2 group 50 Tel-Hashomer, Israel, "Research Institute Rheumatic of Diseases, Piestany, pregnant women treated according to the conventional therapy including Slovakia heparin. Fibrinogen, partially activated thromboplastin time, prothrombin We have employed an anti-idiotypic modulation by concentrated specific index, products of fibrin fibrinogen factor I degradation, coagulability to Li natural polyclonal anti-dsDNA anti-idiotypic antibodies from IVIG in White, antithrombin, kaolin time, esythematosis anticoagulant, treatment of experimental SLE. Methods: Specific natural polyclonal anti- antiphospholipid and anticardiolipin antibodies imunoglobulin defined. dsDNA anti-idiotypic antibodies (IVIG-aID) were affinity purified from Results: Investigating hemostasis system in the I group patients before and IVIG on anti-dsDNA-Sepharose column constructed with anti-dsDNA after using of fraxiparin quick and steady nonnalisation of idiotypes affinity purified from 55 patients with SLE. The NZBtW Fl mice hemostasnogramm was marked. In the 2 group patients after abolition of were treated with 3 weekly injections of IVIG-aID (2 mg/kg/inj) and regular heparin was marked hypercoagulation: expressed hyperfibrinogenemia, IVIG (400 mg/kg/inj) after developing anti-dsDNA antibodies at the age of raised kaolin time value and partially activated thromboplastin time. 21-22 weeks. Results: The IVIG-aID treated mice showed a decline in the Pregnancy outcome in the 1 group was as follows all infants were bom in titcr of a-dsDNA antibodies during the treatment, reaching maximum one ternwith weight 3 kilogram, Apgar scale, early neonatal period was without week after the last injection. The significant difference in the proteinuria complications. In the 2 group the pregnancy was terminated in the 1 level in IVIG-alD treated compared to control group was observed. trimester in 8 women, in the 2 trimester in, premature labor took place in 4 Imnmunohistology showed the different pattem of IgG deposition, with patients. In tenn born infants weighted 3 kilogram, Apgar scale 4. mesangial and capillary walls deposits in control and IVIG treated group Conclusion: Fraxiparin using in complex treatment ofpregnant women with and just mesangial deposits in IVIG-aID treated group. Conclusion: We antiphospholipid syndrome provided quicker and more steady nonnalisation hawe shown, that it is possible to isolate the specific anti-dsDNA-anti-ID of hemostasis system parameters in comparison with those in treatment with from regular IVIG and this preparation is in vivo 200 times more effective heparin. in suppression of humoral and clinical signs of SLE than regular IVIG. Wus Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 11hsticuS Og en dqiodAribockes Po Bs8taIG1 624 CONVENTIONAL ORAL ANTICOAGULATION (INR 2-3) DOWNREGULATION OF EXPERIMENTAL SLE AND APS BY EFFECTIVELY PREVENTS RE-THROMBOSIS IN PRIMARY TNFALFA DNA VACCINATION ANTIPHOSPHOLIPID ANTIBODY SYNDROME M. Blank', I. Krause', G. Wildbaum2, N. Karin2, Y. Shoenfeld' P.R.J. Ames', A. Ciampa2, E. Grandone3, M. Margaglione4, 'Centerfor Autoimmune Diseases, Department ofMedicine B, Chaim Sheba G. Scenna5, L. Iannaccones, V. Brancaccios Medical Center, Tel-Hashomer, Sackler Faculty ofMedicine, 'Department ofRheumatology, North Middlesex Hospital, London, UK, Tel-Aviv-University, Tel-Aviv, 2Bruce Rappaport Faculty ofMedicine, 2Coagulation Unit, SGM Hospital, Avellino, 3Atherosclerosis Unit, IRCCS Technion, Haifa, Israel Opera Padre Pio, S. Giovanni Rotondo, Foggia, 'Medical Genetics, Naked DNA encoding TNFaIf was introduced to BALB/c mice with University ofFoggia, 'Coagulation Unit, Cardarelli Hospital, Naples, Italy experimental SLE induced by 16/6 Id and to mice with experimental APS Higher than average oral anticoagulation (OA)(INR 3-4) seems required to induced by beta2GPI. Administration of naked DNA encoding TNFalfa prevent re-thrombosis in the antiphospholipid syndrome (APS). From 1989 resulted in the generation of immunological memory to its gene product. OA (warfarin) was initiated at a conventional range (INR 2-3) in 34 patients Upon induction of either SLE or APS, this memory inhibited the (12M, 22F, median age at initiation of OA 32 years, range 14-60 years) with development of high titers of anti-ssDNA anti-histones and antiphospholipid primary APS. Reasons for OA were: deep vein thrombosis n=22 (64%), and anti-beta2GPI antibodies (32%-41% reduction as compared to the ischaemic stroke n=3 (8%), myocardial infarction n=2 (6%), venous + untreated group vaccinated with control naked DNA) in experimental lupus. arterial thrombosis n=4 (11%), recurrent events (before starting OA) n= 3 Downregulation of TNFalfa in the mice with the autoimmune conditions (8%), pregnancy related thrombosis n= 6/22 (27%). The median follow-up affected the balance between Thl/Th2 cytokines by suppression of IL-4 and was 34 months equalling to 126 years/patient. There was only 1 thrombotic IL-10 expression. No significant effect on Thl related cytokines (IL-2 and recurrence (fatal ischaemic stroke) equalling to 0.8% years/patient. This was INFgama) was detected. Administration of TNFalfa DNA construct led to offset by I major bleeding event (subdural haemorrhage) (0.8% amelioration of the clinical manifestations of experimental lupus and years/patient) and 21 minor bleeding events (16.7% years/patient). In 7 experimental APS. The murine anti-TNFalfa antibodies which developed in patients OA was temporarily suspended (surgery n=2, delivery n=3, the DNA vaccinated mice and could adoptively transfer the beneficial effect thrombocytopaenia n=2) and switched to a prophylactic dose of low of the vaccine to other mice with the disease. The current study suggest a molecular weigh heparin (LMWH). Despite the latter, 5/7 (71%) of these way by which a DNA vaccine can be used for induction of protective patients re-thrombosed (ischaemic stroke n=l, deep vein thrombosis n-3, immunity in experimental SLE and APS. deep vein thrombosis + pulmonary embolism n=-1). Conventional intensity OA (INR 2-3) effectively prevents recurrent thrombosis in primary APS. At variance, LMWH in prophylactic doses fails to prevent recurrences in those primary APS patients who require temporary anticoagulant cessation. ANTIPHOSPHOLIPID ANTIBODIES (APA) IN PATIENTS WITH INDUCTION OF ORAL TOLERANCE IN EXPERIMENTAL APS LES: PREVENTION OF THROMBOSIS BY FEEDING WITH POLYCLONAL IMMUNOGLOBULINS P. Mesiano, L. Colla, L. Besso, F. Bermond, A. Messuerotti, C. Marcuccio, I. Krause, M. Blank, Y. Sherer, B. Gilburd, F. Kvapil, Y. Shoenfeld C. Canavese, P. Stratta CenterforAutoimmune Diseases, Chaim Sheba Medical Center, University ofTurin, Internal Medicine Department, Nephrology Section, Tel-Hashomer, Israel Turin, Italy We attempted to induce tolerance in an experimental APS model by oral APA are subject to a constant update. Aim of this study was to verify the administration of intravenous immunoglobulins (IVIG). Experimental APS progresses in prevention of APA-related thrombotic risk in LES. Out of 165 was induced by active immunization with beta-2GPI. Naive mice were fed patients (110 biopsed) observed from 1968 to 2001, 51 were APA positives, with IVIG, as a whole molecule, F(ab)2 or Fc fragments. Feeding was 14 sporadic and 37 persistent. At least one thrombotic event occurred in 23 performed before APS-induction, at an early or late stages of the disease. persistents, none in transients. No correlation between APA class [LAC, Significantly diminished humoral response was noted in the groups of mice ACL, anti-beta(2)GPI] and thrombosis was found. Of the 17 patients tested tolerized with WIG either as a whole molecule or with IVIG-F(ab)2 for anti-beta(2)GP 1, 6 thrombosis occurred among the 9 negatives, 3 among fragments, accompanied by significant attenuation of clinical the ones positives. A thrombotic event appeared de novo or recurred in 2 on manifestations. The maximal effect was achieved in mice tolerized before ticlopidine, in 3 on dipiridamole, in 2/16 anticoagulated (INR 2-2,5), in none disease induction. Significant improvement in serologic and clinical of the 12 on low-dose ASA. Recurrent thrombosis have been observed, in parameters was also noted in mice fed at an early stage of experimental particular in patients with Sneddon Syndrome. Patient survival free of APS, while IVIG feeding at a late stage did not produce tolerance. thrombotic event was significantly lower in the APA positive group Abrogation of T lymphocyte proliferation to beta-2GPI was detected in the (p=0.001). In our experience: 1) the predictability for APA rclatcd mice fed with IVIG prior to beta-2GPI immunization, mediated by TGF- thrombosis risk is still linked with a non typical clinical presentation ofLES beta and IL-10 secretion. The effect of IVIG-feeding was nonspecific, since or with a biochemical to characterise the activity of similar inhibition of T-cell proliferation was observed in WIG-fed mice sporadic finding; 2) Ovalbumin (OVA). The tolerance induced by IVIG-feeding APA towards ever changing epitops, the lab should offer a package of the immunized with most for thrombotic genetics risk factors could be could be adoptively transferred to syngeneic mice by CD8+ cells. These updated tests; 3) looking cells were found to secrete high levels of TGF-beta and IL-10. Similar helpful; 4) it is important to search for APA in the follow-up ofpatients with levels in a first low-dose ASA out cautious and effectives if inhibitory effect was observed for anti-OVA OVA-immunized negative test; 5) turned In summary, oral tolerance has a non-specific began at the very start of the clinical presentation of LES, keeping on mice. IVIG-induced moderate patients with past thrombotic event. immunomodulatory effect in experimental APS, mediated by TGF-beta and anticoagulation (INR 2-2,5) IL-0-secreting CD8+ cells. Our results point to a possible application of WIG in the induction oforal tolerance against various autoimmune diseases. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1U1kl1niQn Qigs onfA ridpdAltkdes Past rFshicm 625 ANTIPHOSPHOLIPID ANTIBODIES- CLINICAL CORRELATION APOPTOSIS OFT CELLS IN WOMEN WITH A HISTORY OF AND STATISTICS OF THE DEPARTMENT (1984-2002) THROMBOPHILLA AND RECURRENT SPONTANEOUS ABORTION C.M.C.A. Tonel,J M Matoso Fereira,T. Pereira, L. Araujo, J. Lourenco, C. Pina, A. Ribeiro, M. Alves, F. Esteves, L. Morais, M. Noronha M.M. Jerzak1w2, M. Kasprzycka3, M. Semczuk2, A. Gorski13 Hospital de Reynaldo Dos Santos, Vila Franca De Xira, Portugal 'Department ofReproductive Immunology, Ludwik Hirszfeld Institute of Immunology and Experiemental Therapy, Polish Academy ofSciences, The authors present 17 patients with diagnostic criteria for Antiphospholipid Wroclaw, 2Department ofObstetrics and Pathology ofPregnancy, Syndrome (Sapporo Criteria). The majority of the group (10 patients) has University School ofMedicine,.Lublin, 3Department ofClinical Primary Antiphospholipid Syndrome; the remainder has Systemic Lupus Immunology, University School ofMedicine, Warsaw, Poland Erythematosus (6 patients), while I patient has Rheumatoid Arthritis. There are still 25 patients in study, who have clinical or laboratory features of the Introduction: T cells apoptosis has been proposed as a mechanism for syndrome, but not both. Thrombotic events (arterial and venous) are the maintaining immune privilege. Transient expression of extracellular matrix commonest clinical features among the Antiphospholipid Syndrome group. (ECM) proteins reflects trophoblast differentiation. Therefore, the purpose Anticardiolipin antibody (Ig G) was the main laboratory finding. Under anti- of the study was to determnine the role of T cell apoptosis in ECM coagulant therapy, all patients remain assymptomatic, and 6 of them had 8 environment in women with a history of thrombophilia and recurrent successful pregnancies (on therapy with low molecular weight heparine plus spontaneous abortion (RSA). Materials and Methods: Thirty nonpregnant women ten women aspirin). Currently, one patient with high levels of anticardiolipin, with the history of RSA and nonnal healthy with the pregnancy outcome were antiphospholipids and b2-glycoprotein l(Ig G) but no clinical symptoms is previous successful studied. We studied apoptosis in the late 1st trimester of pregnancy, under therapy with low molecular of peripheral blood T cells after culture with mAb OKT-3 alone and with weight heparine plus aspirin. mAb OKT-3 and the following ECM: fibronectin or collagen IV. We used Cell Death Detection ELISA- a photometric enzyme immunoassay for the quantitative in vitro determination of cytoplasmic histone-associated DNA fragments after induced cell death. In addition, apoptotic peripheral blood T cells were identified by annexin V-PE staining protocol using flow cytometry. Results: The highest values of enrichment factor: mU of the sample (dying/dead cells) per mU of the corresponding control (viable cells) were observed after peripheral T cells culture with fibronectin or collagen IV in samples of women with the history of RSA. However, percentage of apoptotic T cells stained by annexin V was significantly lower in RSA women compared to control (P<0.05). The existence of thrombophilia does not influence these results. Conclusions: Preliminary data suggest that apoptosis of activated cytotoxic T cells might be an interesting possible explanation of successful pregnancy outcome. This work was supported by grant nr 4 P05E 005 18 (KBN). THE EFFECT OF SERA FROM WOMEN WITH SLE OR APS AND RECURRENT ABORTIONS ON HUMAN PLACENTAL EXPLANTS IN CULTURE CLINICAL AND RESEARCH DATABASE FOR SLE AND APS WITH SAPPORO AND ACR CRITERIA LINKED TO POINT-OF A. Ornoy', S. Yacobi', Z. Blumenfeld2, R. K. Miller' SERVICE ENTRY 'Laboratory of Teratology, Department ofAnatomy and Cell Biology, G.A. McCarty"3, T.E. Cason', J. Meade2, M. Zang2 Hebrew University Hadassah Medical School, Jerusalem, 2Department of Obstetrics and Gynecology, Rambam Medical Center,Haifa, Israel, 'Division ofRheumatology and Immunology, 2Department ofMedicine, 3Rochester School ofMedicine, Rochester, New York, USA 3Department ofHealth Evaluation Sciences, UVA, Charlottesville, VA, USA Systemic Lupus Erythematosus (SLE) with or without evidence of Objective: A web-based relational database prototype for an electronic antiphospholipid antibodies (aPA) and antiphospolipid syndrome (APS) is medical record applicable to rheumatic diseases but optimized for SLE and associated with a high rate of recurrent pregnancy loss (RPL). The placenta to both APS has been developed with point-of-service entry forms linked is thought to be the site of pathological damage in many of these abortions. accepted clinical criteria (Sapporo, ACR, SLICC, SLEDAI), outcome Placental explants were shown to be affected by several substances (i.e. measures, and clinical documnentation requirements for both new & return cadmium) added to the chemically defined culture medium of F-12 DMEM and pt. evaluation as part of a Master's practicum in medical informatics and 10% FCS. In order to study the possible effect of sera from women with health resources management. Methods: Existing DBs were elementalized SLE/APS on the placental explants we first cultured 5.5-7.5 weeks old (ARAMIS,Arion, ACR Standardized Forms, SLICC, SLEDAI, BILAG) and human placental explants on serum from normal non-pregnant women and were reviewed with goal structure of currcnt forms in use at UVA established that human placental explants grew better in normal human secretaries, researchers,nurses). identification from all end users(clinicians, serum for 96 hours thani in the chemically defined medium, Enhanced Sybase!Oracle interfaces with existing informatics initiatives & platforms, growth and higher secretion rates of human chorionic gonadotropin (hCG) Codd's rules for entity-relationship databases normalized to 3rd form, data and estradiol (E2) were manifested with no change in progesterone (PGN) reduction were assessed. Results: validity, SQL/Access reporting, and data secretion, Placental explants were then cultured using human sera from non- concensus from mo; response End user input time for building varied 3d4 pregnant women with SLE/APS prior to or after treatment. The usual 23 E-R tables addressed pattern was RNs>secretanes>MDs>researchers, treatment was by heparin, glucocorticosteroids and low dose aspirin. work Subj/Obj/Assessment/Plan elements of clinical incl. Secretion rates of hCG, of E2 and PGN by placental explants using sera measures, Treatment/Procedure/Attending Notes, SLE/APS SF36/MHAQ from treated and untreated women with SLE/APS was similar to that of a one-page (2-sided) web based elements in a two-page(2-sided) New Pt and control. Further, serum from untreated women with SLE/APS produced a requirements were met. Data validity or hard copy form. HCFA and CMS significant decrease in the proliferation rate of the trophoblastic cells and an was verified in 5/20 randomly chosen pts (R.0.99) by 2 MD- data entry increase of apoptosis. Successful treatnent significanfly reduced the 24-52% vs. paper pairs; handwriting volume &time reduction varied from apopotic rate and increased cell proliferation, but the cell proliferation rate physician fonns, and DB queries resulted in valid output. Patient and was still lower than noted in controls. We conclude that sera from women clinical acceptance was not compromized. Conclusion: A point of service with SLE/APS may directly damage the developing placenta reducing and research DB optimized for SLE and APS is achievable. proliferation and enhancing apoptosis. The placental explants in culture are devoid of blood circulation, that is thought to be impaired in APS. This points to the possibility that the trophoblastic damage resulted from a direct effect of the antiphospholipid antibodies. Successfull.treatment diminishes that damage. Supported by Grant No. 032 5160 from the Israeli ministry of Health and by grant 037-4264 from the Israeli American Binational Fund. Wus Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 Oltiktamiavd Qm onAridqidAIibocies PFsrmseo 626 ANTI-B2GPI ANTIBODIES AS THE SOLE ANTIBODIES: ABOUT A SIMPLE RELATIONSHIP BETWEEN TWO APTT REAGENTS 11 PATIENTS AS A CONFIRMATORY TEST IN THE DIAGNOSIS OF LUPUS ANTICOAGULANT (LA) IN ORAL ANTICOAGULATED (OA) C. Desauwl, E. Hachulla', M. Lambert', S. Dubucquoi2, U. Pasturell, PATIENTS A.L. Fauchais', V. Queyrel', P.Y. Hatron', B. Devulderl L.C. Gennari, A.N. Blanco, S.H. Grosso, P. Cassais, M.A. Lazzari 'Service de Medecine Interne A, Hospital Huriez, CHRU. Lille, 2Laboratoire dImmunologie, Faculte de Medecine de Lille, France Departamento de Hemostasia y Trombosis, Instituto de Investigaciones Hematologicas 'Mariano R Castex, Academia Nacional de Medicina, Since the description by Cabral et al. in 1996 of 6 primary antiphospholipid- Buenos Airev, Argentina cofactor syndromes with anti-Beta-2-GPI isolated, these last ones were not enclosed in the criteria of Sapporo. We studied the characteristics of our It is widely known the importance of using adequate criteria for LA; mainly presenting patients with anti-Beta-2-GPI alone. Results: 2451 dosages of in OA patients because of the defect induced by the anticoagulants. Among anti-Beta-2-GPI were prescribed in our service between November 1997 and confirmatory tests, the use of two aPTT reagents, one with high and the December 2000. This antibodies (without anticardiolipin and Lupus other with low sensitivity to LA can be mentioned. Our aim was to find out Anticoagulant) was observed at least oncc in 67 patients. Eleven paticnts (10 the best relationship (R) between reagents with high (A) and low (B) women) presented anti-Beta-2-GPI on 2 or more occasions at least 8 weeks sensitivity that better discriminates among LA+ and LA- and whether apart. The mean age at disease onset was 38 years +/- 11 with a follow-up of different cut-off are needed according to the patient status, OA+ or OA-. 48 months+/-35. Five of them presented at least one clinical criteria of APS aPTTA (PTT-LA, Stago) and aPTTB (aPTT L. Grifolds) were analyzed in such as defined to Sapporo without associated pathology. Their 206 patients: 104LA-/OA-; 40LA-/OA+; 25LA+/OA-; 37LA+/OA+. characteristics were described in the table: Different R were calculated and evaluated as an evidence of the Pt. Sex Age Clin. Manifestations Isotye Follow phospholipid dependency of LA: the delta between both aPTT, divided by either aPTIB or aPTTA (Rl=aPTTA-aPITB/aPTTB; I F 41 LR,RY I 30 R2=aPlTA- aPTTB/aPTTA) and the R between normalized aPTT values 2 F 33 FLx3,RY I 27 3 F 52 SVT, LC IgM 57 (aPTTr=aPlTpatient/aPTTnormal) (Rrl=(aPTTrA-aPTTrB)iaPTTrB; 4 F 30 FLx4, SVTx2, RY, TIA IgG 105 Rr2=(aPTTrA-aPTlrB)/aP'ITrA). Considering LA-/OA+ cut off (higher or equal: mean+2SD), sensitivity (S) and specificity (SP) for LA+/OA+, results 5 F 27 RAT IM 41 LR: Livedo Reticularis, RY: Raynauds phenomenon, FL: fetal loss, SVT: superficial vein were RI(0.66): S=92%, SP=98%; R2(0.41): S-86%, SP-98%; RrI(0.33): thrombosis, LC: lacunar cerebral infarcts, TIA: transient ischemic attack, RAT: radial artery S=73%, SP=95%; Rr2(0.31): S=68%, SP=95%; while applying LA-/OA- thrombosis cut off for LA+/OA-, results were R1(0.48): S=68%, SP=99%; R2(0.34): The 6 other patients presented systemic diseases. Conclusion: We describe 5 S=64%, SP=98%; Rrl(0.12): S=72%, SP=98%; Rr2(0.11): S=72%, new cases of APS with anti-Beta-2-GPI isolated. These antibodies were SP=98%. Despite the best criteria for LA+/OA+ is that calculated from OA+ asymptomatic in 4 patients with connective tissue disease. The recent controls, it is possible to apply a single cut off based on OA- controls. In proposition of minor laboratory criteria for APS which included anti-Beta-2- OA- a better discrimination was obtained with Rrn or Rr2. We consider it GPI will be a progress for studying the APS. would be useful to employ Rrl or Rr2 as a single criterion for both, OA+ and OA- patients. CONFIRMATORY dRWT ON MIX SAMPLES IMPROVED CLINICAL SIGNIFICANCE OF ANTICARDIOLIPIN AND ANTI- SENSITIVITY AND SPECIFICITY FOR LUPUS ANTICOAGULANT beta2-GLYCOPROTEIN I ANTIBODIES (LA) IN PATIENTS UNDER ORAL ANTICOAGULATION (OA) G. Obermoser', W. Bittcrlich', F. Kunm2, N. Sepp' A,N. Blanco, L.P. Gennari, S.H. Grosso, P. Cassais, M.A. Lazzari 'Department ofDermatology, 2Department ofinternal Medicine, Departamento de Hemostasia y Trombosis, Instituto de Investigaciones University ofInnsbruck, Austria Henmatol6gicas 'Mariano R. Castex Academia Nacional de Medicina, PURPOSE: Anticardiolipin and anti-beta2-glycoprotein 1 antibodies are Buenos Aires, Argentina characteristic laboratory features of the antiphospholipid syndrome. We The diagnosis of LA in patients under OA might be difficult because of the investigated their prevalence and assessed their correlation with clinical coagulation defect induced by anticoagulants. Considering the importance of manifestations of the antiphospholipid syndrome. In particular, the the dRVVT, we evaluated the usefulness of applying different criteria for diagnostic values of anticardiolipin and anti-beta2-glycoprotein I antibodies screening and confirmatory tests. Thirty seven OA patients (P) were and lupus anticoagulant were compared. METHODS: 3600 consecutive sera analyzed: 13 LA+ based on PTTLA results and 24 LA- (controls). APTT were tested for IgG and IgM anticardiolipin antibodies between January (PTTLA, Stago), dRVVT (Sigma), their corresponding mixing tests (P:N) 1999 and June 2001. All anticardiolipin positive samples or if specifically and the phospholipid neutralization (PLN) of both P and P:N samples were requested were further analysed for IgG anti-beta2-glycoprotein I antibodies performed. The following dRVVT criteria were analyzed (cut off >mean (in total 1238 assays). Retrospectively, the data of anticardiolipin or anti- controls+2SD). For mixing tests A=P:N/N>1.21, B=P:N/N)-(P/N)>1.09 and beta2-glycoprotein I antibody positive patients were correlated with clinical C=[(P:N/N)-l]/(P/N)>0.lI were applied. While, for confirmatory tests, we symptoms of the antiphospholipid syndrome and other laboratory considered PLN of both P (D=(P/N)-(P/N)PL>0.17; E={[(P/N)- parameters by review of medical records. RESULTS: 147 anticardiolipin (P/N)PLJ/(P/N)}xl00>I.02), and P:N (F=(P:N/N)-(P:N/N)PL>0.06; antibody positive patients were identified, 35 (23.8%K) of them were also G={[(P:N/N)-(P:N/N)PL]/(P:N/N)}xiOO>5.7). The analysis, excluding 3/13 positive for anti-beta2-glycoprotein I antibodies. In 9 of 1238 (0.73%) tested LA+ with prolonged dRVVT corrected by nornal plasma, showed a better sera only anti-beta2-glycoprotein I antibodies but not anticardiolipin sensitivity/specificity with A (90%/o/100%) and C (80%/100%) for inixing antibodies were detected. The presence of both autoantibodies was strongly studies; or F (100%/100%) and G (100%/100%) for the PLN of P:N. Less associated with clinical symptoms of the antiphospholipid syndrome sensitivity was observed when PLN of P criteria were considered (D: (p=0.007) compared to p-0.008 for lupus anticoagulant. Similarly, the 60%/o/96%; E: 50%/96%). Applying D and E criteria, 1/24 was a false specificity and positive prodictive value of anticardiolipin antibodies were positive result; perhaps, due to the increase of phospholipid concentration in increased by a positive anti-beta2-glycoprotein I antibody test (7.4% to 83% a deficient plasma, like OA sarnples; while more false negative results were and 32% to 48% respectively) compared to 73% specificity and 65% observed (D:4/l0; E: 5/10), may be due to a/the higher dispersion detected positive predictive value of lupus anticoagulant. CONCLUSION: in P than in P:N. In attempt to the present results, it would be useful to add Assessment of anticardiolipin and anti-beta2-glycoprotein I antibodies are the P:N neutralization to the series of tests to evaluate the presence of LA in valuable diagnostic tools for the work-up of patients with suspected OA, improving the sensibility and specificity for the dRVVT. antiphospholipid syndrome, particularly when determination of lupus anticoagulant is not available. However these ELISA methods do not substitute the functional clotting assay oflupus anticoagulant Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 EtIhlandCrd Ossmcn ipd es Potm psat"om PREVALENCE OF IGG ANTI-PHOSPHATIDYLETHANOLAMINE STUDY OF ASSOCIATION BETWEEN CLINICAL 627 (Ape) ANTIBODIES IN SLE MANIFESTATIONS AND ANTIPHOSPHIOLIPID ANTIBODIES IN A COHORT OF PATIENTS WITH THE APS J. Kucal, L. Olsen', K. Dierl, C. Fink', D. Wagenknecht2, J. McIntyre2, L. Lopez' M.A. Aguirre, R. Roldan, A. Escudero, M. Romero, P. Font, E. Collantes 'Corgenix, Inc., Westminster, CO, 2Saint Francis Hospital and Health Hospital Reina Sofia, Reumatologia Cordoba, Spain Centers, Beech Grove, IN, USA Objective: To analyse the association between lupus anticoagulant (LA) and High levels of antibodies to phosphatidylethanolamine (aPE) have been anticardiolipin antibody (aCL) and the clinical features, in a cohort of reported in antiphospholipid syndrome (APS) patients, somc with negativc patients with antiphospholipid syndrome (APS). Patients and Methods: aCL. We studied the prevalence ofIgG aPE antibodies in SLE patients. Seventy-three patients (57 females, 16 males, median age: 38.5(16.1 years), ELISA microwell plates were coated with purified egg PE and blocked with fulfilling the classification criteria for APS (Sapporo criteria, 1999) were porcine gelatin. Samples were diluted in PBS containing adult bovine included. Twenty-five patients (34.2%) had primary APS, 39 (53.4%) plasma (cofactor source). Cut-off was established at the mean OD + 2SD of secondary APS and 9 (12.3%) APS secondary to lupus-like syndrome. IgG 200 blood donors. SLE patients were classified into 3 groups: 1.- 36 SLE and IgM aCL isotipes IgG were measured by standard ELISA and LA by unselected (consecutive patients) showed a mean OD of 0.995 with 38.8% DRVVT and confinnatory tests. Qualitative differences were calculated by positives;I.- 50 SLE (APL-) selected for the absence of serologic/clinical chi-squared test or Fisher's exact test when necessary. Results: Thirty-nine manifestations of APS and showed a mean of OD of 0.950 with 22% patients (53.4%) presented arterial thrombosis, 24 patients (32.9%) venous positives; III.- 40 SLE (APS+) classified as secondary APS with a mean OD thrombosis and 28 patients (38.4%) fetal loss. Other clinic features were of 1.189 and 42.5% positives. The mean OD ofthe 200 healthy controls was thombocytopenia in 22% of patients, haemolytic anaemia in 2.7%, epilepsy 0.595 with only 3% positives. IgG aPE levels and prevalence within the 3 in 5.5% and ulcers in 11% of patients. The following table shows clinical association in these patients: Thrombosis aCL groups of SLE patients were statistically different (p<0.001) than the and serological Arterial aCL LA-50% Venous Thrombosis aCL controls. The highest mean level was observed with secondary APS patients IgG=79.5% IgM-42.9% aCL with 42.5% presenting IgG aPE antibodies. Only 3 patients in group 1, 2 in IgG=60.9% aCL IgM=34.8% LA=71.4% Fetal loss aCL lgG-64.3% IgM-57.1% LA=53.8% Arterial thrombosis was significantly associated group II and I in group III were aPE positive and aCL negative. In contrast, aCL isotype (p<0.05) and venous thrombosis with LA positive the majority of aPE positive (82%0% and 65% in groups I, II, III with IgG respectively) were anti-B2GPI negative. These results confirm that IgG aPE (p<0.05). No other association was noticed. Conclusion: The IgG aCL was the most prevalent antibody in our cohort of APS patients. A antibodies are prevalent in SLE and secondary APS. IgG aPE detection isotype should be considered in SLE patients with suspected APS without anti- significant association between arterial thrombosis and aCL IgG and B2GPI antibodies. between venous thrombosis and LA was found. COMPARISON OF THREE ELISA METHODS FOR THE IONIC STRENGTH EFFECTS ON TESTS FOR LUPUS DETECTION OF ANTI-B2GPI ANTIBODIES ANTICOAGULANT K. Dier, T. Buckncr, L. Olsen, C. Fink, L. Lopez T. Exner, J. Low, J. Joseph Corgenix, Inc., Westminster, CO, USA Haematology Department, St. Vincents Hospital, Sydney, Australia Antibodies to B2GPI have been reported to be more specific for thrombosis It has been shown that some, if not most, lupus inhibitors are sensitive to than classic anticardiolipin antibodies and very useful in the diagnosis of ionic strength and lose activity when increasing levels of salts, eg sodium antiphospholipid syndrome (APS). We evaluated the performance of three chloride, are added to a clotting test system such as a kaolin clotting time commercial IgG anti-B2GPI ELISA methods. A group of 22 selected APS test. However anticardiolipin antibodies bind beta 2GP I associated with serum samples were tested by each method. All assays performed within phospholipid in ELISAs independently of high ionic strength. If the latter their expected quality control specifications. Method 1 classified 16 as antibodies function as coagulation inhibitors then they should also be positive (73%, mean value = 87.9 units), Methods 2 classified 10 (45%, unaffected by sodium chloride levels. Thus our hypothesis was that lupus mean = 43.5 units) and Method 3 classified 12 (54%, mean = 65.0 units). inhibitors which lose anticoagulant activity, relative to others, on the When the individual values from Method I were compared with Methods 2 addition of sodium chloride may be less dependent on beta 2GPI than on lhe and, coefficients of correlation (r) of 0.432 and 0.588 were obtained. other known LA 'cofactor, prothrombin. This might provide an additional Methods 2 and 3 showed a correlation of 0.765, however, these two methods method for subtyping prothrombin-dependent lupus inhibitors which some detected 6 and 4 less positives than Method 1. Four samples that classified researchers claim may be clinically less important as a thrombotic risk factor as positive with the three methods differed significantly in values and than those dependent on beta 2GPI. We tested 48 specimens positive for interpretation (i.e. weak versus strong positive). Since the majority of APS lupus inhibitors according to ISTH criteria. Kaolin clotting time tests were patients with history of thrombotic cpisodes should present with these carried out using kaolin made up either in low ionic strength buffer or in antibodies, Method 1 had the best perfonnance by identifying more patients buffered 0.1 2M sodium chloride solution. As previously noted, most lupus (73%) as positive. In addition, Method I was tested using a proposed inhibitors were significantly weaker in the presence of sodium chloride, chimeric IgG monoclonal antibody standard (HCAL), which resulted as though there was considerable variability. Specimens which were least strong positive (124 units). Although all of these assays use purified human affected by ionic strength were not substantially different in their reactivity B2GPI, similar testing procedures, cut-offs and possibly similar reporting with cardiolipin or prothrosnbin in conventional ELISAs. Thus the ionic units, the standardization and clinical performance seems to be different. strength effect appears to be a general phenomenon among LA and cannot These results demonstrate inter-method variability for detecting aB2GPI be used to subclassify these antibodies. antibodies and the need for better standardization. LLPS Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lGbihiki OMnanAtionppidArtibocles Poit*sGts 628 A NOVEL FLOWCYTOMETIC METHOD TO DETECT CONCENTRATION OF PHOSPHOLIPID IN DRVVT REAGENTS AUTO-ANTIBODIES RELATED TO PREGNANCY LOST MAY AFFECT THE DIAGNOSIS OF LUPUS ANTICOAGIULANT (LA). S. Shail, G. Filuxian', R. Shabo', Y. Shoenfeld2 1. Jennings', S. Kitchen', T.A.L. Woods', I. Cartwright2, F.E. Preston' 'BioShafLtd, Tel-Hannan, Nesher, 2Chaim Sheba Medical Center, Tel-Hashomer, Israel 'UK NEQAS (Blood Coagulation), 2University ofSheffield, UK Objective: Evaluate the application of a flowcytometric method to detect The DRVVT is the most widely used screening test for LA in the UK, and auto-antibodies related to pregnancy lost as compared to routine ELISA. kits obtained from 10 different commercial sources are now used. Materials and Methods: Flowcytometric analysis was performed using a Differences in the composition of DRVVT reagents have previously been Flowcytometer supplied with Argon lazer, measuring emission of 515, 540 describcd. Wc have mcasured phospholipid conccntrations of both 'test' (low and 600nm, analyzing particles size and a new developed auto-antibodies concentration) and 'confirm' (high concentration) reagents for 5 different kits (BioShaf- Israel) for Ig level of anti: Thyroglobulin, Thyroid DRVVT kits (American Diagnostics, Gradipore, Instrumentation peroxidase, Cardiolipin, Phosphatadilserin, Prothrombine, beta-2- Laboratory, Thrombosis Reference Centre and Unichrom) together with an Glycoproteinl and ANA (dsDNA, Jo-1, Sm, Sm/RNP, SSA, SSB, and Sol- in-house method, using Bell & Alton reagent and washed platelet was 70). Routine ELISA analysis of sera were performed using commercial kits. preparations. The relative concentration of phospholipids similar for The precision of the methods was analyzed by perforning sensitivity, each reagent, with an average 48% phosphatidylcholine, 30% specificity, linearity and recovery studies. Eighty serum samples were phosphatidylserine and 22% phosphatidylethanolamine. However, the final the test analyzed in parallel, by both methods and the statistical McNemar's test was concentration of total phospholipid in system varied markedly, with applied to determine whether there was a significant difference between the test reagent concentration of two kits more than twice that in the confirm Flowcytometer based kit analysis and two separately routine analysis and reagent of another kit (range 4.5ug/ml to 26.lug/ml for test reagents, between the two routine analysis themselves. Results: The flowcytometric 11.8ug/ml to 327.8ug/ml for confirm reagents). Perfonnance of each kit was method to screen auto-antibodies using the BioShaf set of kits is both more compared in a multicentre study comprising samples spiked with precise and more accurate than the routine ELISA analysis. The monloclonal antibodies and from a LA positive patient. Significant flowcytometer based analysis agrees with a given ELISA routine analysis to correlation (r>0.85, P<0.03) was seen between normalized test/confinn ratios and the phospholipid concentration in confirm reagents for 2 out of 3 the sane degree that two separate ELISA analyses agree with each other. Conclusions: Auto-antibodies screening based on Flowcytometric method spiked sanples and the LA positive sample, with higher ratios obtained using the BioShaf kits can replace the routine analysis based on ELISA using reagents with higher phospholipid concentrations. Five out of eight screening and leads to a better evaluation of auto-antibodies level. centres using a kit with a low phospholipid concentration in the confinn reagent obtained a false negative result with a plasma sample containing monoclonal antibodies against prothombin. We conclude that differences in phospholipid concentration may affect sensitivity and specificity of DRVVT methods. OMEGA-CARBOXYL VARIANTS OF 7-KETOCHOLESTERYL METHODOLOGICAL DIFFERENCE BETWEEN DIFFERENT ESTERS ARE LIGANDS FOR BETA2-GLYCOPROTEIN I ANTICARDIOLIPIN ELISAS: THE USE OF PURIFIED BOVINE B2GPI K. Kobayashi', Q. Liu', T. Yasudal, D.R. Voelker2, T. Koike3, E. Matsuura' C. Biasini Rebaioli, M. Frassi, M. Spunghi, B. Abissoni, E. Ervas, F. A. Tincani, G. Balestsieri 'Okayama University, Graduate School ofMedicine and Dentistry, Allegri, Okayama, Japan, National Jewish Medical & Research Center, Brescia Hospital, Italy Denver, CO, USA, 3Hokkaido University, Graduate School ofMedicine, Background: anticardiolipin (aCL) ELISA is known to be a complex test. Sapporo, Japan Part of the problems may be related to the different amount of beta 2 Beta2-glycoprotein I (beta2-GPI) is a major antigen for anticardiolipin glycoprotein I (b2GPI) or to its specific features. In fact, fetal calf serum, antibodies (aCL, Abs) present in patients with antiphospholipid syndrome that is the main source of the b2GPI present in the microtitre plates, may (APS). We recently reported that beta2-GPI specifically binds to oxidized differ in different preparations. Objective: to evaluate the performance of a LDL (oxLDL) and that the beta2-GPI's major ligand, oxLig-1 is 7- new aCL Varelisa assay (Pharmacia) containing purified bovine b2GPI ketocholesteryl-9-carboxynonanoate (J. Lipid Res. 42: 697, 2001). In the instead of fetal calf scrum. Matcrial and Methods: 73 serum samples present study, we demonstrated that omega-carboxylated oxysterol esters are randomly chosen among those referred to our Institution because of critical for beta2-GPI binding. A novel ligand, namely, oxLig-2, was most autoimmune diseases were tested by Varelisa Assay by our in-house aCL likely to be 7-ketocholesteryl-12-carboxy (keto) dodecanoate. Liposome ELISA performed routinely in the last 18 years. 30 normal sera from healthy binding to macrophages was significantly increased depending on the blood donors were tested as controls. Diagnosis and clinical features of the ligand's concentration, in the presence of beta2-GPI and an anti-beta2-GPI patients were derived from the clinical notes taken by an independent Ab. A synthesizcd variant, 7-ketocholesteryl-13-carboxytridecanoate observer. Results: aCL Varelisa results were related to those of our in-house (13-COOH-7KC), also showed a significant interaction with beta2-GPI and aCL ELISA (IgG r = 0.7983, p<0.0001; IgM r =0.6245; p<0.0001). a similar binding profile with macrophages. Methylation of the carboxyl Specificity and sensitivity for thrombosis episode of IgG aCL Varelisa was function diminished all of the ligand interactions specific for beta2-GPI and 66% and 61% respectively, for pregnancy loss was 76% and 63%. IgG aCL anti-beta2-GPI Abs, in TLC-ligand blot analysis, ELISA, and in vitro uptake detected by our in-house ELISA showed 77% and 61% by macrophages. Thus, omega-carboxyl variants of 7-ketocholesteryl esters specificity/sensitivity for trombosis episodes atnd for pregnancy loss 76% can mediate anti-beta2-GPI Ab-dependent uptake of oxLDL by and 59%. Comment: according to our limited experience, the new Varelisa macrophages, and autoimmune atherogenesis linked to beta2-GPI assay perfonnance is comparable to that of our classical assay. interaction with oxLDL. LLPS Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 1ahk1mnairgi Owe= olnqilcEpdAtibocies osl_ AstaicE IMMUNOLOGICAL EVALUATION IN WOMEN WITH DRUG ABUSE INITIATES SLE IN A PATIENT WITH PRIMARY 629 SPONTANEOUS ABORTIONS APS G. Kortebani, L. Rodriguez Rilo, A.B. Mazzolli G. Pohla-Gubo, M. Wieser CISER, Buenos Aires, Argentina Department ofDernatology, General Hospital Salzburg, Austria Object of study: To evaluate obstetric background, autoimmune diagnosis, Heroin abuse is known to cause several immunological abnormalities associations and treatment of women consulting for spontaneous abortions including autoantibody production (1). We report about a patient with between 1996 and 2000 Subjects and Methods: 501 women with previous primary APS and consecutive fatal SLE after starting heroin abuse. A 27 yo history of spontaneous abortion were included. Clinical histories and the man was first seen in our hospital with cellulitis in January 2000. 2 months main outcome measures were serum levels of antiphospholipid antibodies later he was seen with fever of unknown origin and severe pain on his soles. (APA) to cardiolipn (CL), lupic anticoagulant (LAC), phosphatidylserine An autoantibody-screening revealed positive ANA, ENA, dsDNA as well as (PS), phosphatidic acid (PA), phosphatidylinositol (PI) antithyroid positive ACA (IgM/IgG). Furthermore the LA was also positive and he was antibodies (ATA) and complement fractions (C3, C4). In cases of primary diagnosed to have a primary APS with pseudovasculitis. No renal, spontaneous abortions, human leukocyte antigens were characterized in each hematologic, cardiac or neurologic involvement was found. Oral couple. Results: Mean age: 33.16 +/- 4.4 years (range 21-44). Mean anticoagulation caused some relief but he was not compliant in taking his previous abortions: 2.42 +/-1.1l(range 1-8), 70.5% were considered primary medication and missed regular controls. One year later (July 2001) he spontaneous abortions and 29.5% secondary. Abortions: 80% occurred returned with severe pain on the left leg, an ulcer on the tip of the toe and during first trimester. Clinical findings: Raynaud syndrome 80%, arthralgias in the wrists. ANA, ENA and dsDNA-autoantibodies were again haematoma 51%, headache 53% and hypocomplementemia 46%. positive and for the first time the complement level for C4 was decreased. Autoantibodies: CL 49%, PS24%, PA12%, P115%, LAC32%, ATA21%. IgM ACA were borderline and IgG-ACA were weakly positive. As he did Diagnostics: antiphospholipid syndrome APS 72.65% infection 8.78%, not fulfill the ACR criteria for systemic involvement he again was informed thyroiditis 7.58%. Among 364 APS:18.4% were associated with infection, to take the anticoagulants regularly but did not receive immunosuppressive 13.73% with autoimmune thyroiditis and 11.5% with hypothyroidism. HLA drugs. In September 2001 he was seen again because of ureterolithiasis, a was investigated in 47% couples. 23% matched more than three antigens. 59 scrotal abscess and necrotizing vasculitis on the lower limbs. At that time women were sensitized with paternal leukocytes. Over 501 patients, 53% the abuse of different drugs including i.v. heroin became evident. ANA, got pregnant: 86% delivered newly boms and 14% relapsed in abortions. ENA and dsDNA-autoantibodies were markedly elevated and the IgM- and Normal newly borns: mean gestational age: 37 +/-3 weeks, mean weight: IgG-ACA raised. 3 months later he was again admitted to our hospital with 2938+/-678 g. Combined treatment with ASA, corticosteroids, LMWH or severe clinical signs of SLE including glomerulonephritis with renal failure, IVIG, were prescribed. Conclusions: The autoimmune factor (APS + pericarditis, heart failure and hemolytic anemia. All treatments failed and he thyroiditis) reached the higher diagnostic percentage: 80.23% died within 4 weeks (January 2002). Nikolova et al. (1) studied 10 heroin addicts and found that 4 were ANA positive. 3 patients were IgM-ACA positive and I of them had acute renal failure in the course of heroin coma and APS (deep vein thrombosis). This patient was also positive for Sm and dsDNA. APS and SLE are known to be triggered by different causes and the evolution of APS into SLE is observed more frequently. Our patient developed a fulminant SLE on the basis of a primary APS. We suggest that the incompliance in taking the anticoagulants as well as the heroin abuse were the main causes for this outcome. (1) Nikdlova et al.: The prevalence of some autoantibodies in heroin abusers (in press). USE OF MONOCLONAL ANTIBODIES AS STANDARD MATERIAL FOR ANTI-BETA 2GPl, ANTI-PROTHROMBIN AND ANTI-PHOSPHOLIPIDS IMMIJNOASSAYS ANTIPHOSPHOLIPID, ANTIPROTEIN ANTIBODIES AND K Rodolfo, A. Guilloteau, S. Le Floch, V. Quignon, M. Grimaux, HOMOCYSTEINE AS RISK FACTORS FOR A FIRST B.J. Woodhams PREGNANCY LOSS BETWEEN 8 AND 10 WEEKS OF PREGNANCY: A MATCHED CASE-CONTROL STUDY Serbio SA (Diagnostica Stago), Gennevilliers, France J.-C. Gris"23, E. Mercier', I. Quere3, H. Dechaud3, M. Hoffet4, Persistently elevated levels of anti-Phospholipids (APA), anti-B2GPI and S. Rippart-Neveu4, P. Mares4, T.V. Pemeger5 anti-Prothrombin antibodies are associated with an increased risk of thrombosis and foetal loss. Standardisation of these assays is based on the 'Laboratoire d'Hematologie, University Hospital, Nimes, 2Laboratoire use of pooled positive patient plasma. The quantity of these is limited and d'Hematologie, Faculte de Pharmacie, Montpellier, 'EA 2992, Montpellier, the specificity and affinity of the standard changes when the pool changes. 'Departement de Gynecologie-Obstetrique, University Hospital, .MJimes, This has complicated standardisation between assay types and laboratories. France, 5Quality of Care Unit, Geneva University Hospitals, Geneva, For Lupus anticoagulanit tests, iiionoclonial antibodies against B2GP1 and Switzerland have been used as reference material to improve intra and inter Prothrombin, We performed a 1:1 case-control study in 743 normal patients with a first (1,2). We propose to use a similar monoclonal laboratory precision pregnancy loss between 10 and 12 weeks of pregnancy, which corresponds antibodies as standard in APA, anti-B2GPI and anti-Prothrombin system to the momment where the placeita replaces the yolk sac as the source of 1, 27G7 and 28F4 respectively). Using immunoassays (using clones 22A1 blood supply to the embryo. Controls were normal women with a provoked we batches of calibrators these antibodies are able to produce lyophilised abortion between 10 and 12 weeks. Cases and controls were matched for same (for either anti APA, or anti- with the activity. Each batch anti-B2GPI age, total number of pregnancies and time elapsed since abortion. We tested can 100 arbitrary units’ activity Prothrombin) be adjusted to have the presence of lupus anticaogulant (LA) and anti-cardiolipin (aCL), anti- with the same affinity and specificity. As demonstrated with Lupus phosphatidylethanolamne(aPE), anti-beta2-glycoprotein, anti-annexin V Anticoagulant tests, monoclonal antibodies are also a valuable tool for the (aAnV), anti-tissue-type plasminogen activator(atPA), anti-thrombo- standardisation of immtnological assays. Their universal acceptance will modulin(aTM), anti-Tissue Factor IgG and IgM antibodies, and result in reduced interlaboratory variation. (l) Amout, J. et al 'lhromb homocysteinaemia (HCY). Using multivariate adjusted conditionnal Haemost 1998; 79 955-958. (2) LeQuerrec, A. et al Thromb Haemost regression analysis, LA, aCL-IgM, aPE-IgM, aAnV-IgG, atPA-IgG, and 2001 ; 86: 584-589. HCY are independent risk factors for a first fetal loss in a concentration- dependant way. There is an apparent justification to consider homocysteinaemia and some antiphospholipid or antiprotein antibodies in nonnal women with a first pregnancy loss between 8 and 10 weeks of gestation. Wm Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 llhktenric QWesnalfiouiospidAdribocles PosetPiais 630 RELEVANCE OF ANTI PROTHROMBIN ANTIBODIES IN OPTIMIZATION OF PHOSPHATIDYLSERINE DEPENDENT PATEINTS WITH VENOUS AND/OR ARTERIAL THROMBOSIS ANTI-PROTHROMBIN ELISA B. Montarulil, A. Vaccarino2, C. Foli2, S. Platcrotil, M. Saittal, A. Ambrozic, T. Kveecr, P. Zigon, B. Bozic L. Cavagnal, M. Bazzan2 University Medical Centre, Ljubljana, Slovenia 'Laboratorio Analisi, Ospedale Evangelico Valdese, 2Dipartimento di Aim: To optimize the standard anti-phosphatidylserine/prothrombin aPS/PT Ematologia e Malattie Trombotiche, Ospedale Evangelico Valdese, Torino, ELISA Procedure: 1. coating phosphatidylserine on medium binding plates Italy overnight, 2. blocking, 3. washing and application of prothrombin, Arterial and venous thrombosis and recurrent miscarriages are the most 4. washing and appLication of sera, 5. washing and addition of conjugate, frequent and clinically relevant complication of patients with 6. washing and addition of substrate. Our modification was in the antiphospholipid antibodies (aPL). The role of anti prothrombin antibodies simultaneous application of PT and serum samples (joining steps 3 and 4). (aPT) as risk factors of thrombosis has not yet been clearly established. In The methods were quantitatively compared using decreased concentrations the present study we investigated the association of aPT with venous and of PT. The sensitivity was estimated by the ratio of PT concentrations arterial thrombosis. We determined the prevalence of aPT in 105 patients yielding the same optical density at 405 nm (OD405). A qualitative with history of venous thrombosis (66 cases), arterial thrombosis (35 cases), comparison was performed to ascertain if aPS/PT of the same specificity both arterial and venous thrombosis (4 cases) and in 105 sex and age were detected by both methods. Results: The analytical sensitivity of the matched controls. aPT were assayed by a commercial ELISA method. modified ELISA was about four times higher than of the so called standard Prevelence of IgG and IgM aPT was higher in patients with arterial method. Plots ofOD405 values for each sample ran parallel in the range of thrombosis (aPT IgG: 34% versus 6%, p < 0,0002; aPT IgM: 28% versus PT concentrations above 2.5 mg/L. All sera positive by the standard method 4%, p< 0,0004) and venous thrombosis (aPT IgG: 21% versus 6%, p <0,02; showed significant bindings also by the modified method, and additionally, aPT IgM: 22% versus 4%, p< 0,0001) than in controls. In the group of OD values measured by the modified method were 2 to 8 times higher than patients negative for Lupus Anticoagulant, anti cardiolpin and anti beta2 by the standard method. Conclusions: The analytical sensitivity of an Glycoprotein I the prevalence of aPT were significantly higher in patients aPS/PT ELISA can be increased by simultaneous addition of PT and sera to with arterial and or venous thrombosis than in control subjects (aPT IgG: wells. The most likely explanation for the lower sensitivity of the standard 16% versus 3%, p <0,005; aPT Ig;: 11% versus 1%, p <0,008). Univariate method is the loss of PT during washing in phase 4. Similarly, a higher PT analysis revealed that IgG and IgM aPT were significant risk factors for concentration in the modified method enables a more efficient competitive arterial thrombosis (odds ratio 8.6 and 4.8 respectively), and venous inhibition of antibodies binding directly to PS, thus lowering the non- thrombosis (odds ratio 9.9 and 6.7, respectively). In conclusion these results specific binding. showed that aPT IgG and IgM are correlated with history of venous and arterial thrombosis. MONOCLONAL ANTIBODIES AGAINST THE COMPLEX OF CORRELATION BETWEEN LEVEL OF ESTROGEN AND PHOSPHATIDYLSERINE AND PROTHROMBIN FROM PATIENTS ANTIPHOSPOLIPID ANTIBODY SUBCLASSES IN SLE WITH THE APS D. Martinovic, M. Radic, L. Giunio, D. Fabijanic K Ichikawa, T. Honta, T. Atsumi, S. Jodo, Y. Amasaki, T. Koike University Hospital Split, Department ofInternal Medicine, Split, Croatia Hokkaido University, Graduate School ofMedicine, Sapporo, Japan Introduction: Correlation between estradiol and antiphospholipid antibodies Purpose: Anti-prothrombin (PT) antibodies have been detected in patients (aPL) has already been established, as well as correlation between aPL with the antiphospholipid syndrome (APS). Anti-PT antibodies may be subclasses and specific clinical manifestations of antiphospholipid syndrome classified into 2 categories. One is antibodies against the complex of PT and (APS): IgM and hemolytic anemia, IgG and thrombosis, thrombocytopenia, phosphatidylsenine (aPS/PT). The other is antibodies against PT bound to fetal loss, IgA and thrombosis. Estradiol stimulates immunoglobulin oxygenated polystyrene (aPT). Recently we reported that aPS/PT were synthesis and enhances production of antibodies and autoantibodies. correlated with the manifestations of APS and the presence of lupus Relation between estradiol and specific aPL subclasses has not been anticoagulants (LA). To investigate the specificity and biological activity of investigated. Patients and methods: levels of estradiol and IgA, IgG, IgM aPS/PT, we aimed to establish monoclonal aPS/PT from APS patients. subclasses of aPL have been correlated in 33 female patients with systemic Mcthods: Monoclonal aPS/PT were established from peripheral blood B lupus erythematosus (SLE) and positive aPL (>16 GPL). Results: Positive cells from APS patients by combined methods of EBV transformation and correlation was found between elevated serum titer of estradiol and elevated somatic cell hybridization. The specificity of aPS/PT was studied by solid titers of IgA and IgM subclasses of aPL (p<0.005). Correlation was phase enzyme immunoassays. To clarify LA like activity of aPS/PT, the confirmed by multiple regression analysis (p 0.023). Conclusion: Results effects of established monoclonal aPS/PT on thrombin generation were point to significant association between estradiol and IgA and IgM studied. Results: Four IgM monoclonal aPS/PT (HG-4, KE-6, KF-5, and subclasses of aPL, which could shed more light on the mechanism of onset KF-6) were established. Three of them (HG-4, KF-5, and KF-6) did not bind of aPL and their relation with clinical expression of SLE. either to PS or to PT on the oxygenated plates. KE-6 bound to PT on the oxygenated plates. None of them bound to PT on non-oxygenated plates or to fragment 1 of PT. HG-4, KE-6, and KF-5 inhibited the thrombin generation. Conclusions: Our results suggested that aPS/PT is different from aPT and that autoinmmune aPS/PT may recognize the cryptic epitopes or neoepitopes that are exposed when PT binds to PS. aPS/PT had LA like activity. These monoclonal antibodies can be used as a useful tool to clarify the pathophysiology ofAPS. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lahIraiaci sstmanfioqhipdAiboodes ostwPrisAUM APS IN MALE LUPUS CYTOKINES AND THE APS 631 N.G. Kljukvinal, S.V. Shekshina3, A.A. Baranov2, E.L. Nassonov3 M. Ehrenfeld, H. Amital, Y. Shoenfeld 'Medical Academy, Moscow, 2Medical Academy, Yaroslavl, 31nslitute of Chaim Sheba Medical Center and Tel-Aviv University, Tel-Hashomer, Israel Rheumatology RAM4S, Moscow, Russia Since our description of the experimental model of APS, we have The aim of this study was to examine the prevalence and clinical features of investigated the role of IL-3 in APS. Interleukin-3 and GM-CSF, were antiphospholipid syndrome (APS) in male lupus. Materials and methods: shown to promote placental development and fetal growth,findings which We have studied 75 male patients (pts) (mean age 28,9 yrs, mean disease led us to assume that cytokine abnonnalities would be involved in the duration,9 yrs) fulfilled 1982 ARA criteria for systemic lupuserythematosus pathogenesis of APS. Mice injected with mrIL-3, showcd a dramatic (SLE). IgG and IgM anticardiolipin antibodies (aCL were measured by reduction in the rate of fetal resorption, and a correction of the ELISA according to international standardized method. Also the serum thrombocytopenia. Thus, IL-3 was found to be effective in the prevention of levels of C-reactive protein (CRP) in 44 pts with SLE and 40 healthy donors recurrent fetal loss of APS. Leukotrienes and later low-dose aspirin were by ELISA were studied. Results: 53% of pts had an elevated level of aCL. shown to stimulate IL-3 production, both in vivo and in vitro. This The characteristic features of APS in male lupus were venous thrombosis mechanism explains the beneficial effect of low-dose aspirin in preventing (16 pts), thrombocytopenia (II pts) and coronary artery occlusions with the cliniical iiianifestations of APS, in animal and human studies. myocardial infarction (5 pts).). 29 pts (39%) fulfilled criteria for definite (17 Ciprofloxacin, was found to enhance the production of various cytokines, pts) or probable (12 pts) APS (D. Alargon-Segovia et al., 1992). 10 from 29 including IL-], IL-2, Interferon-ganma, GM-CSF and IL-3. Ciprofloxacin pts (34%) with SLE and secondary APS had aCL-associated manifestations was able to decrease pregnancy loss, and lead to an improvement in the (such as recurrent venous thrombosis, epilepsy, chorea) at onset disease. clinical manifestations of APS. Though the exact mechanism of action of These symptoms preceded clinical features of SLE during few years (range Ilr3 in APS is not completely understood, our studies provided a deeper 1-10 years). 13 (29,5%) of 44 pts had raised levels of CRP (above 15 mg/l). insight into the inter-relationships, between the two, as well as on the IL-3 6 pts had concomitant infection (4 from 6 with high CRP levels). modulating agents such as low-dose aspirin, which now comprise part of the Thrombotic events, especially arterial thrombosis and elevated IgG and IgM conventional therapeutic measures used in APS. aCL levels were significantly more frequent in CRP-positive than negative pts. Concelusiotn: Our results demonstrate a high prevalence of APS in male lupus. Raised level of CRP may be a useful matker of thrombosis in lupus patients without infection. CYTOKINE GENE POLYMORPHISM IN APS DOES THE MATERNAL DOSE RESPONSE TO LOW MOLECULAR WEIGHT HEPARIN VARY DURING PREGNANCY? C.G. Echenique', M.B. Rodriguez Cardozo', J.U. Scher', G.R. Bierfass', A LONGITUDINAL PROSPECTIVE STUDY D.S. Femandez Romero', J.O. Latino2, M. Pesaresi2, A.M. Di Lonardol V.C. Sephton', R Farquharson', J. Toppin ', C. Cowan', S. Quenby', 'Immunology Unit, 2Tocogynecology Division, Hospital Dr. C. G. Durand, D. Back2, C. Toh Buenos Aires, Argentina 'Obstetrics and GynaecologyDepartment, Liverpool Women's Hospital, The cytokine networks exert effects on the regulation of immune responses. 'Pharmacology Department, 'Haematology Department, Royal Liverpool Pro-inflammatory and anti-inflammatory cytokines are believed to influence University Hospital, UK the pathogenesis of autoimmune diseases but the reports presently available To if low molecular weight heparin on this subject are not conclusive. Polymorphisms in the promoter region Objectives: determine (LMVH) as defined by are known to influence the amount ofproduced cytokine. Aim: Contributing provides an appropriate level of thromboprophylaxis, gestation. to this discussion we examine weather cytokine gene polymorphisms in pharmacokinetic differences in anti-Xa activity with advancing activity following a bolus sc dose of patients carrying antiphospholipid syndrome had any genotypic differences Aims: To assess serial serum anti-Xa gestation points followed by a with control individuals and any predictive value in assessing the immmne Dalteparin 5000 inits at three standardised non- pregnant measurement in a longitudinal manner. Methods: A group of status of patients. Patients and Methods: We analyzed the gene women with inherited or acquired thrombophilia associated polymorphism of TNF-alpha, TGF-betal, IL-10, IL-6 e IFN-ganma twenty-nine 0 promoter regions in a group of twelve APS patients with history of abortions pregnancy loss were recruited. Anti-Xa serum activity was assessed at using QIAGEN extracted DNA by PCR-SSP (CYTGEN ̵ 1;0ne hours and then, 12 hours, post bolus dose of Dalteparin 5000iu sc, at four (12, 24,36 weeks gestation and 6 weeks Lambda,Inc.). Afterwards, we compared the same polymorphisms with a standard time points after and control group of ninety healthy individuals. Results: Frequency of IL-10 (- postpartum). A dose response curve for anti-Xa activity was generated clearance was calculated from the area under the curve (AUC). Using 1082) G (-819)C / (-819)C (-592)C, high producer genotype was present in for the normnal group (13 percent) and not in the APS group. The same is seen analysis of variance, the group AUC means were then compared with the frequency of IL-10 (-1082)A (-819)C / (-819)C (-592)C, low gestation and time post injection. Results: The peak anti-Xa level occurred producer genotype. When comparing TGF-betal codon 1OC/25G, we found at 4 hrs in pregnancy compared to 2 hrs in the non-pregnant state. At 2 hrs the mean anti-Xa level for 12 and 36 weeks gestation were it in thirteen percent of normal group, while none of the APS group post injection displayed it. TNF-alfa and IFN-gamma did not show differences between significantly less than in the non pregnant state. (P= 0.03). At 36 weeks gestation, the mean anti-Xa levels, at and 12 hours, fell outside the both groups. Conclusion: Preliminary results of our ongoing study shows non-pregnant, prophylactic range. Compared to the non- that the presence of IL-10 (-1082) G (-819)C / (-819)C (-592)C, high recommended, producer genotype and the absence of TGF-betal 1OC/25G codon pregnant state, the AUC decreases with advancing gestation. Conclusions: (intennediate producer) could be protective against the apparition of Differences in the pharmacokinetics of low molecular weight heparin during antiphospholipid syndrome. pregnancy were observed, with an overall reduction in anti-Xa activity. On the basis of this study, the implications for pregnancy thromboprophylaxis, using extrapolation ofnon-pregnant data becomes questionable. Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 lOitmarW inArtssan riqvdAribocies PftPseftcn 632 PROSPECTIVE OBSERVATIONAL STUDY OF BONE MINERAL UNFRACTIONATED VERSUS LOW-MOLECULAR-WEIGHT DENSITY DURING PREGNANCY: LOW MOLECULAR WEIGHT HEPARIN IN TREATMENT OF APS DURING PREGNANCY HEPARIN VS CONTROL A. Ruffatti', M. Ermani2, M. Favarol, A. Calligarol, C. Radul, M. Tonellol, A.J. Carlin, R.G. Farquharson, S.M. Quenby, J. Topping, W. Fraser P. Grypiotis', T. Del Ross', S. Todesco' Miscarriage Clinic, Liverpool Women's Hospital, UK 'Division ofRheumatology, 2Division ofNeurology, University ofPadova, Italy, Aim: To assess the effect of long-term low molecular weight heparin on bone mineral density during pregnancy. Methods: 55 women with recurrent The rationale for using heparin to prevent antiphospholipid (aPL)-related miscarriage and Antiphospholipid Syndrome were followed through obstetric complications is based on its prophylactic activity of matcenal pregnancy in an ethically approved prospective observational study. All thrombosis and on the observation that aPL alter the hemostasis of placental women had dual energy x-ray absorptiometry (DEXA) scans at the lumbar bed, producing thrombosis and infarction which may cause fetal wastage. spine (L1-L4) performed within 6 months prior to conception and in the We compared the efficacy and safety of unfractionated heparin (calcium immediate postnatal period, within 6 weeks of delivery. Dalteparin was heparin) with those of a low-molecular-weight heparin (nadroparin) by commenced after a positive urine pregnancy test and continued throughout prospectively analysing 30 and 17 pregnancies of patients affected by anti- pregnancy and after delivery until 6 weeks postpartum. A group of 20 phospholipid syndrome (APS) according to recently defined criteria (1999). volunteers with recurrent miscarriage, not requiring any treatment Calcium heparin and nadroparin-treated patients had pregnancy morbidity intervention, acted as controls and were monitored in an identical fashion. respectively in 25 (83.3%) and 9 (52.9%) cases, vascular thrombosis alone Results: Patient characteristics of age, parity, number of live births, or associated with pregnancy morbidity in 5 (16.6%) and 8 (47%) cases, breastfeeding, total dose of LWMH and mean gestation at delivery were not anticardiolipin G antibodies in 27 (90%) and 15 (88.2%) cases, significantly different between cases and controls. Both groups showed a anticardiolipin M antibodies in 2 (6.6%) and 5 (29.4%) cases and lupus similar loss in lumbar spine (LI -LA) bone mineral density by the end of the anticoagulant in 3 (10%) and 4 (23.5%) cases. Both treatments were started pregnancy (LMWH 4.17% or -0.049 g/cm3 CI -0.036 to -0.062 vs Control when a sonogram demonstrated a live embryo, continued until delivery and 3.56 % Or -0.043 g/cm3 CI -0.027 to -0.059). However the difference in stopped at the end of puerperium. The daily dosage of calcium heparin bone loss between the groups was not statistically significant ( 0.00185 varied between 15,000 and 37,500 U subdivided in three doses, while that of g/cm3 p=0.88 Cl 0.0124 to 0.00865 ). No patient suffered vertebral fracture. nadroparin varied between 75 and 150 anti-Xa U/kg subdivided in two Conclusion: Bone loss associated with long-term low molecular weight doses. The following parameters were compared: mean age, matemal and heparin use is not significantly different to that which occurs physiologically fetal complications, favourable outcome, mean gestational age at delivery, during pregnancy. cesarean delivery rate, mean birth weight, and neonatal intensive care unit admission rate. The statistical comparison performed by Fishers exact test and Mann-Whitney U test showed no significant difference between calcium heparin and nadroparin-treatod patients. Both treatments were not complicated by heparin major side effects. We conclude that nadroparin shows the same efficacy and safety of calcium heparin in preventing aPL- related obstetric complications of patients with clinical and serological features of APS. EVOLUTION AND RESPONSE TO TREATMENT IN PATIENTS WITH POSITIVE ANTICARDIOLIPIN ANTIBODIES LOCALIZATION OFT CELL EPITOPES ON PROTHROMBIN IN JJ. Alegre-Sancho, C. Fernindez-Carballido, J.A. Romin-Ivorra, PATIENTS POSITIVE FOR ANTIPROTHROMBIN ANTIBODIES 0. Viana, P. Molina, M.J. Ripoll, 1. Martinez, S. Fernandez T. Sumida, K. Yoshida, Y. Ohnishi, A. Tsuitsumi Hospital Universitario Dr. Peset, Yalencia, Spain Department ofInternal Medicine, University of Tsukuba, Japan To describe clinical with Objective: features and evolution in patients Objective: Prothrombin (PT) is one of the major autoantigen in patients with with anticardiolipin antibodies (ACA) Patients and Methods: 61 patients APS. Prothrombin is a 74 kDa molecule, consisting of Fragment-I (Fl) ACA were referred to our to 2001 to rule out the positive Unit from 1995 domain and the prethrombin-l (pre-1) domain, and the presence of presence of Antiphospholipid syndrome (APS). The following data were antiprothrombin antibodies is closely related to history of thrombosis. Since collected at onset: age, sex, type and titers of ACA, thrombocytopenia, the production of anti-protein antibodies is driven by antigen specific T clinical features (livedo reticularis, pregnancy losses, thrombotic events, cells, the analysis of T cell epitopes may lead to autoantigen specific neurologic abnormalities, skin lesions, Raynaud's phenomenon and others) immunosuppressive therapies. We wished to determine the localization of and treatment prescribed. Patients were screened for the presence of APS or common T cell epitopes of PT. Methods: All patients included in this study other connective tissue diseases (CTD) criteria. Clinical and analytical were positive for antiprothrombin antibody, as measured by enzyme at were also recorded. Results: were findings follow-up Most of the patients immunoassay. PT was digested with thrombin, and Fl and pre-1 domains women mean years Mean time of follow-up (80,3%); age: 40,2 (s.d.: 15 y). were purified. Peripheral blood mononuclear cells (PBMC) from 15 patients was 22 months. ACA isotypes: IgG 74,5%, IgM 69,1%. Clinical features: with APS or SLE were tested for T cell responses to full length PT, Fl or Asymptomatic 18%9% had thrombocytopenia, 31,1% previous pregancy pre-1. Cell proliferation was measured by BrdU uptake method (Cell losses (1st trimester: 26,2%, 50% of them >3 abortions; 2nd trimester:3,3%; proliferation ELISA). Results: 1) Positive T cell reponses were observed in 3rd trimester, 6%), 24,6% thrombotic events (arterial,8%, venous,8% and 6 patients. Among these patients, PBMC from 2 patients responded to Fl, 3 both 5%); 11,5% livedo reticularis;,8% cutaneous ischemic lesions; 11,5% to pre-1, and PBMC from I patient responded to both Fl and pre-1. 2) T cell transient ischemic attacks; 24,6% migraine (MRI performed in 67% of them epitopes and B cell epitopes seemed to be located on the same domain in 4 with one pathological findings consistent with ischemic infarctions in patients, but not in the other 2 patients. 3) No significant association was was thrid);,8% Raynaud's phenomenon. APS diagnosed in 25 patients observed between localization of T cell epitopes and HLA-DRB I alleles or (41%); 3 patients fulfilled criteria for another CTDs (I SLE, I Sj6gren and history of thrombosis. Conclusion: T cell epitopes of PT in patients positive 1 spondyloarthropathy). IgM ACA isotype was more frequently found in for antiprothrombin antibodies differ among individuals. patients with APS and pregnancy losses. Treatment: Antiplatelet therapy was used in 65,6% of patients, acenocoumarin in, 9% and both treatmcnts in 13,1%; 9 patients no treatment. Evolution: 62,3% showed a good clinical response to treatment (clinical improvement/no recurrences). ACA tumed negative in 36,1%; but didn't decrease or oscilate in the majority. Conclusions: Almost half of the patients submitted to our Unit because positive ACA had APS; most of them being women. IGM ACA was most frequent in our APS patients (with or without other ACA isotypes). Pregnancy losses, thrombotic events and migraine were the predominant clinical findings. The majority of patients responded to antiplatelet/anticoagulant therapies. UPS Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 IDJ r 1tG ss hipdan odassm _mt Ps~~~MMO-o.tdo 633 FUNCTIONAL CHARACTERIZATION OF CD8+ T SUPPRESSOR FREQUENCY OF MALIGNANT TUMOURS IN A SERIES OF 238 CELLS PATIENTS WITH SLE AND APS G. Filaci, M. Fravega, S. Negrissi, D. Fenoglio, M. Basso, F. Indiveri M. Mora', J.I. Bemardino', L. Diez', A. Robles', P. Lavillal, N. Sastrel, A. Gil Aguado2 Department ofInternal Medicine, University ofGenoa, Italy 1La Paz University Hospital. Madrid. Spain, 20n Behalf of The APS Study This study concemns a subset of CD8- Ts cells that is generated in vitro by Cooperative Group incubation of purified CD8+ cells with IL2, GM-CSF and monocytes. To To the frequency and epidemiological characteristics of verify whether their generation and suppressor activity depend on antigen Aim: evaluate and Methods: A presentation, purified CD8+ cells were incubated in the presence of tumours in patients with SLE and APS. Patients to a prospective follow allogeneic monocytes. In this condition, both generation and expression of retrospective analysis of charts from 1975 2000 and SLE or APS. Results: Twenty three suppressor activity occurred, demonstrating that these functions do not up till March 2002 of 238 patients with require antigen presentation. When CD8+CD28+ and CD8+CD28- subsets malignant tumnours in 20 patients were diagnosed (8,4% of the series). The tumours described were CIN (6), VIN, thyroid (2), prostate (2), were separately purified generation of suppressor cells was possible only tongue, acute leukaemia (2), Hodgkin disease, from the latter subpopulation. To better define the cytokines involved in endometrium, breast (3), were no differences in CD8+ Ts cells generation, we tried to generate them in the presence of melanoma, bladder, colon and lip epidermoid. There cancer were Most of patients blocking anti-ILIO, anti-IL4 or anti-TGFbeta niAb. We found that the anti- sex distribution, Patients with older (p<,0001). ILl0 mAb abolished their generation. TCR Vbeta repertoire of circulating in the series were PAPS (26%). In the malignancy group the diagnose more frequent was SLE + APS (40%). Three patients from the cancer group had CD8+ cells and CD8+ suppressor cells from the same subject were for the syndrome. The 90% of patients with cancer and comparatively analysed to understand whether CD8+ Ts cells represent an also criteria Sjogren 71% of patients without cancer were aAP positive. Mortality was higher in oligoclonal or a polyclonal population. No cxpansion of cloncs bearing group. patients with cancer died and only 3% died in the specific TCR Vbeta chains was observed in CD8+ Ts cells. In conclusion, the tumour 40% of group The 80% of patients with cancer and the 61,4% we demonstrate here that CD8+ Ts cells originate from a circulating general (p<,0001). without cancer had ever been treated anytime with steroids. No differences CD8+CD28- cell pool, that the differentiation of these cells in suppressors with other immunosuppressive treatments were found. Conclusions: occurs exclusively by cytokine conditioning without need for antigen Neoplasms seem to be more frequent in patients with aPL. The use of recognition, that a complex cytokine network (including ILIO) is required steroids may be related with the occurrence of tumours in these patients. for their generation, and that likely every CD8+CD28- clone can be a source of CD8+ Ts cells since no particular restriction to specific TCR Vbeta chains could be demonstrable in this cell subset. RESTRICTED T CELL RECEPTOR BETA-CHAIN USAGE BY T LONGITUDINAL ANALYSIS OF MARKERS OF IMPAIRED CELLS AUTOREACTIVE TO BETA2-GLYCOPROTEIN I IN TROPHOBLAST INVASION AND MATERNAL ENDOTHELIAL PATIENTS WITH APS DYSFUNCTION IN APS PREGNANCIES M. Kuwanal, K. Yoshida', T. Araii, J. Kaburaki2, Y. Kawakami', S. Stone"2, B.J. Hunt?-, P.T. Seed', K. Parmas9, K. Langford4, Y. Ikeda' M.A. Khamashtal, L. Poston 'Keio University, 2TEPCO Hospital, Tokyo, Japan 'Maternal and Fetal Research Unit, 2Lupus Pregnancy Clinic, 3Department ofHaematology, 'Department ofObstetrics, We recently identified T cells that are autoreactive to beta2-glycoprotein I Guy's and St Thomas'Hospitals, London, UK (beta2GPI) and promote antiphospholipid antibody production in patients with antiphospholipid synidrome (APS). In this study, T-cell receptor (TCR) Objective: Impaired placentation may complicate antiphospholipid beta-chains of beta2GPI-reactive T cells were examined in 8 beta2GPI- syndrome (APS) pregnancies. Insulin-like growth factor binding protein-I responders, including S APS patients and 3 healthy individuals, using PCR (IGFBPl) at the decidua-trophoblast interface acts as a modulator of insulin- and single-strand conformation polymorphism (PCR-SSCP) combined with like growth factor-I (IGF-I). Raised maternal IGFBP1 concentrations in in vitro stimulation of peripheral blood T cells with recombinant beta2GPI. growth restricted or pre-eclamptic pregnancies may contribute to reduced The TCR V-beta segments that expanded oligoclonally after stimulation spiral artery trophoblast invasion. Antiphospholipid antibodies may also with beta2GPI varied among responders, but the Vbeta7 and V beta8 cause endothelial cell activation (ECA). We evaluated serum concentrations segments were commonly detected in 6 and 4 responders respectively. of markers of IGF-I and IGFBPI and markers of ECA in APS pregnancies. Analysis of the CDR3 sequence of beta2GPI-reactive T cells revealed Methods: Longitudinal blood samples were collected 4-weekly from 8 limited diversity, and all Vbeta7+ TCR had an amino acid motif of weeks gestation (28 treated primary APS and 19 control women). Serum TGxxN/Q or minor variations. The Vbeta8+ TCR had another motif, IGF-I and IGFBPl were measured by immunoradiometric assay. PxAxxD/E. Surprisingly, an identical Vbeta7+ TCR was used by beta2GPl- Plasminogen activator inhibitor type 1, tissue plasminogen activator, soluble reactive T cells in 3 independent APS patients. There was no apparent intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-I difference in the TCR beta usage betweeni APS patienits anid lealthy and soluble thrombomodulin were determined by ELISA. Results: 3 APS responders. Some of the Vbeta7+ TCR with the TGxxN/Q motif were also pregnancies miscarried, 3 were complicated by IUGR and pre-eclampsia used by beta2GPI-specific CD4+ T-cell clones responsive to an and 1 by placental abruption. Six women with APS suffered thrombotic immunodominant epitope containing the major phospholipid-binding site. events. Birthweight in the APS group was 2867+/-914g (mean+/-SD) Depletion of Vbeta7+ or Vbeta8+ T cells from the peripheral blood (control: 3492+/-527g, p=0.02) and gestational age at delivery 36.5+/-5.2 mononuclear cell cultures significantly inhibited in vitro anti-beta2GPI weeks (mean+/-SD) (control: 40.3+/-0.7 weeks, p=0.002). IGFBPI and antibody production in response to beta2GPI. Our results indicate IGF-I concentrations increased with gestation in both groups but IGFBPI preferential usage of TCR beta chains by beta2GPI-reactive T cells. These was significantly higher throughout in the APS group (v control p=0.004). TCR beta chains can be reasonable targets for TCR-based immunotherapy No significant differences in concentrations of ECA markers between APS in APS patienits. and control pregnancies were observed. Conclusion: Serum concentrations of IGFBP-1 are abnonnal in the APS group and may reflect abnornalities in trophoblast invasion. Despite poorer pregnancy outcome, there was no evidence of ECA in treated APS pregnancies. (Supported by Tommy's, the baby charity and Lupus UK). Wus Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015 Illhktianaiui nAossen oqhpdArtiboces Poste 1s 634 TESTING FOR ANTIBODIES AGAINST OTHER PHOSPHOLIPIDS VENOUS VS. ARTERIAL / ARTERIOLAR APS SUBSETS THAN CARDIOLIPIN IMPROVES THE DIAGNOSTIC OF THE ANTIPHOSPHOLIPID-ANTIBODY-SYNDROME Jean-Charles Piette Pitie-Salpetriere, Paris, France R. Woehrlel, M. Oppermann', T. Matthias2, K. Helmkei Hopitul In patients with the Antiphospholipid Syndrome (APS), recurrences of 'Municipal Hospital Munich-Bogenhausen, Rheumatology and Clinical thrombotic events, when they occur, strongly tend to affect venous sites Immunology, Muenchen, 2Aesku.Lab Diagnostika, Wendelsheim, Germany when the initial event was venous thrombosis or pulmonary embolism, and Methods: We examined 20 sera from patients with an Antiphospholipid- arterial sites when the initial event affected arteries. However, 10-15% of Antibody-Syndrome (APS, meeting the criterias of Sapporo) and 45 sera patients develop both venous and arterial events. Heart valve involvement from individuals with thrombotic events of other origin than APS (control is frequently disclosed in patients with ischemic cerebral events. This patients). The ELISA-test-system we used detects IgG- and IgM-class allowed a first categorization between venous 'vs arterial APS subsets. antibodies against beta-2-glycoprotein-I (b2-GP-I) and six phospholipids: Cardiolipin (CL), Phosphatidyl-Serine (PS), Phosphatidyl-Inositol (PI), Microvascular involvement has been recently included in the Sapporo Sphingomyelin (SM), Phosphatidyl-Ethanolamine (PE) and Phosphatidyl- criteria for definite APS. Beside Catastrophic APS, microvascular involvement usually manifests as livedo and/or renal thrombotic Choline (PC) without cofactor and in combination with b2-GP-I both in and SLE-related APS. Recent data rcspccitively. Results: Significant differenices (chi-squarc-test) between microangiopathy, Primary indicate that these features are closely associated with "classical" arterial APS-patients and the controls were found in 21 out of the 26 performed manifestations ofthe APS. assays: IgG-class-antibodies against CL with cofactor were positive in 71 % out of the APS-patients, 64 % had elevated antibodies against b2-GP-I, PS Finally, the term "arterial / arteriolar" subset is suggested to qualify and PI (all IgG-class, the latter two with cofactor). In sera from the 4 APS- patients presenting these frequently clustered clinical items: arterial stroke, patients which were negative for antibodies against CL in the used test transient ischemic attack, seizures, permanent non-infiltrated irregular fine system, TgM-class-antibodies against further phospholipids could be livedo, heart valve lesions, APS nephropathy, and systemic hypertension. detected: PS in 2, PI in 1, SM in 1, PC in I (each without cofactor) and PE On a biological ground, this "arterial / arteriolar"APS subset seems to be with and without b2-GP-l as cofactor in 1. Thus, testing for further characterized by a high prevalence of antibodies directed to beta2-GPI. phospholipids increased the sensitivity to 88%. None of the control patients Large ongoing studies will help to improve the delineation of APS subsets, had positive test results in the 26 assays performed, giving a specificity of which might be relevant for therapeutic guidelines in daily practice. 100 % for every assay. Conclusions: Individuals with typical clinical symptoms ofthe APS tested negative in the commonly performed assays for Cardiolipin-antibodies should be tested for antibodies against further phospholipids, especially Phosphatidyl-Serine, to improve the laboratory diagnostic of the APS. NEW VARELISA ANTI-CARDIOLIPIN AND ANTI-Pr GLYCOPROTEIN 1 ANTIBODY ASSAYS IN THE SCREENING OF ANTI-PHOSPHOLIPID SYNDROME B. Gilburd, M. Blank and Y. Shoenfeld Centerfor Autoimmune Diseases, Department ofMedicine "B", Sheba Medical Center, Tel Hashomer, Israel Introduction: Phospholipid-based assays, especially the traditional anti-cardiolipin assay (ani-CL), are the most widely used screening tests performed for the ahtiphospholipid antibodies determination in sera of patient with autoinurune diseases. In the past years however, growing interest has been paid to the determination of antibodies in patients with antiphospholipid syndrome (APS) directed to P2- Glycoprotein 1 (P2GPI), independently of the presence of phospholipids. Objective: To evaluate the sensitivity and specificity of the traditional anti-CL or anti-P2GPI in a cohort of patients with primary (PAPS) and secondary anti phospholipid syndrome (SAPS) and other rheumatic diseases. Methods: The IgG, IgM, IgA anti-CL and anti-p2GPI antibodies were determined in 186 sera of patients with APS and other rheumatic diseases using Varelisa diagnostic kits (Pharmacia & Upjohn Diagnostics GmbH & Co.KG, Germany). Results: The results arc shown in Table 1. Conclusions: The combination of anti-CL and anti- B2GPI assays significantly (p<0.05) increase sensitivity of anti-phospliolipid antibody determination. In addition, the anti-B2GPI assay was found to be more specific than anti- CL in anti- phospholipid antibody determination. Table I Frequcncy ofAnti-CL and anti-B2GPI antibodies in the sera ofpatients with autoimmune diseases. Patients Anti-CL Anti- Ant-CL + Anti- (GMA) B2GPI(GM,A) B2GPI (G ,A) PAPS (n=66) 71.2% 50% 81.8% SAPS (n=60) 63.3% 53.3% 70% RA (n=30) 10%/e 0% 10%/e SS (n=30) 13.3% 0% 13.3% bpM Downloaded from lup.sagepub.com at University Library Utrecht on March 17, 2015