S-27 1 Cardeza Foundation for Hematologic Research, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA, USA; 2 Rheumatology Division, Cerrahpasa Medical Faculty, University of Istanbul; 3 Electro-Neurophysiology Research and Application Centre, Univer- sity of Istanbul, Istanbul; 4 Rheumatology Division, Gulhane School of Medicine, Ankara, Turkey. Ayhan Dinc, MD, Associate Professor; Toshiro Takafuta, MD, PhD, Assistant Professor; Dongsheng Jiang, MD; Melike Melikoglu, MD; Guher-Saruhan Direskeneli, MD, Associate Professor; Sandor S. Shapiro MD, Professor. Please address correspondence to: Ayhan Dinc, MD, Rheumatology Division, Gulhane School of Medicine, 06010 Etlik, Ankara, Turkey. E-mail: adinc@gata.edu.tr Received on June 26, 2002; accepted in revised form on November 25, 2002. Clin Exp Rheumatol 2003; 21 (Suppl. 30): S27-S30. © Copyright CLINICAL AND EXPERIMEN- TAL RHEUMATOLOGY 2003. Key words: Behçet’s disease, anti- endothelial antibodies, endothelium. ABSTRACT Objective. Circulating antibodies that bind to human endothelial cells cul - tured in vitro have been detected in a variety of diseases, including Behçet’s disease. In this disorder the reported prevalence of AECA has varied widely. One likely source of variability is the ELISA assay itself, in which differing conditions and reagents have been used in different reports. Methods. We have re-examined the fre - quency of AECA in 132 Turkish Behçet’s patients and 50 healthy Turk - ish controls, comparing several dif - ferent methods of preparing the target endothelial cells. Human umbilical vein endothelial cells (HUVEC) were used either: 1) fresh and non-treated, 2) fixed, or 3) TNF -stimulated. All stages of the procedures were per - formed at room temperature. Results. In Behçet’s patients, using fresh, non-treated HUVEC, 17 of 130 (13.1%) and 9 of 132 (6.8%) sera were positive for IgG- and IgM-AECA, respectively. However, among 50 nor - mal controls, 2 (4.0%) had IgG-posi - tive and 4 (8.0%) had IgM-positive ELISAs under the same conditions. The difference in the frequency of positives between patients and controls was not statistically significant. Fixed HUVEC and TNF -treated HUVEC gave simi - lar results as well. When group means were examined, only the mean for IgG- AECA determined with TNF -stimulat - ed HUVEC reached statistical signifi - cance. Conclusion. The discrepancy between our data and earlier reports in the lit - erature probably reflects the method - ological differences alluded to, and highlights the difficulties in interpret - ing ELISA assays for AECA. Introduction Behçet’s disease (BD) is a condition of unknown etiology, which is characteri- zed by recurrent orogenital ulceration, skin lesions and an inflammatory arth- ritis, as well as by vasculitic involve- ment of the major peripheral arteries and the veins and vessels of the central nervous system (1). The vascular invol- vement of BD includes thrombophle- bitis, seen in about 25% of patients,sel- dom if ever with embolization, and ar- teritis with occlusion and aneurysm formation (2). This has suggested the existence of a hypercoagulable state in BD patients, and has raised the possi- bility of a role for the endothelium in the pathogenesis of BD. Indeed, previ- ous studies in Behçet’s disease have been interpreted to show evidence of endothelial dysfunction (3-5), and sev- eral studies have found AECA in 18- 37% of BD patients (6-12). However, a number of methodological differences exist between these studies, making it difficult to compare their results. Some studies, for example, were performed at 37ºC, a temperature at which surface- bound IgG can be internalized (13), while others have not taken into ac- count the possible occurrence of hete- rophil antibodies that might have aug- mented the apparent specific binding in ELISA (14). Such variations have sug- gested a need for standardized tests for AECAs (15). In the present study, we measured AECA by ELISA in a large group of Behçet’s patients and controls, and examined the significance of sever- al variables in the methodology. Materials and methods Source of sera Sera were obtained from 132 Turkish patients with BD attending three cen- ters in Turkey (Cerrahpasa and Istanbul Medical Faculties, University of Istan- bul and Gulhane School of Medicine, Ankara) and from 50 healthy Turkish controls. All patients fulfilled the ISG criteria for diagnosis of BD (16). The clinical features of the patients are shown in Table I. The control group consisted of 22 fe- males and 28 males,with a mean age of 27.7 ± 8.4 years (range 19-49 years). Anti-endothelial cell antibodies in Behçet’s disease A. Dinc 1,4 , T. Takafuta 1 , D. Jiang 1 , M. Melikoglu 2 , G. Saruhan-Direskeneli 3 , S.S. Shapiro 1