Moritaka Gotoh

Distinguishing β2‐glycoprotein I dependent (systemic lupus erythematosus type) and independent (syphilis type) anticardiolipin antibody with Tween 20

Summary We investigated whether or not the use of Tween 20 could help to distinguish β2‐glycoprotein I (GPI) independent anticardiolipin antibody (aCL) (syphilis‐type aCL) from GPI‐dependent aCL (SLE‐type aCL) in a GPI‐dependent/ independent aCL ELISA. aCL was positive in all 16 SLB patients arid all 15 syphilis patients, who were positive for aCL in the standard ELISA. in the GPI‐independent ELISA with Tween 20. GPI‐dependent aCL was detected in 12/16 SLE patients by the GPI‐dependent ELISA with Tween 20. aCL was not detected in any of the syphilis patients by GPI‐dependent ELISAs. On the basis of these results, we recommend that Tween 20 should be used in ELISAs to distinguish GPI‐dependent aCL from GPI‐independent aCL.

Resistance to activated protein C activity of an anti‐/2 ‐glycoprotein I antibody in the presence of β‐glycoprotein I

Some researchers claim that lupus anticoagulant‐positive plasma may cause a false‐positive reaction in the test for activated protein C (APC) resistance, a hereditary thrombophilic state characterized by abnormal factor V, which frequently causes venous thrombosis, We investigated whether anti‐/32 ‐glycoprotein I antibody (aGPI), which has recently come to be regarded as an anti‐cardiolipin antibody (aCL) itself, might have an effect on the APC resistance test.

Anti-endothelial cell antibodies to the endothelial hybridoma cell line (EAhy926) in systemic lupus erythematosus patients with antiphospholipid antibodies

The endothelial hybridoma (EAhy926) cell line was employed to clarify whether antiphospholipid antibodies (aPA) [lupus anticoagulant (LA), antiprothrombin antibody (aPT) and/or anticardiolipin antibody (aCL)] and anti‐endothelial cell antibodies (AECA) are identical, and establish whether β2‐glycoprotein I (β2‐GPI) is needed for reactivity of aPA to endothelial cells. Ig‐G AECA was positive in 9/30 SLE patients with aPA (30.0%) and 10/22 SLE patients negative for aPA (45.5%). Ig‐M AECA was positive in one SLE patient with aPA and one SLE patient without aPA. AECA‐positivity was not significantly different among unfixed, TNF‐stimulated and fixed EAhy926. The influence of β2‐GPI on the reactivity of serum to EAhy926 was minimal, and absorption experiments of serum with cardiolipin‐liposome/β2‐GPI or phosphatidylserine‐liposome/prothrombin gave little evidence of cross‐reactivity of aPA and AECA. The results of our study suggest that aPA and AECA may have partially cross‐reacted, but were different antibodies. However, further study is needed to clarify the clinico‐pathological significance of AECA.

Molecular analysis of chronic eosinophilic leukemia with t(4;10) showing good response to imatinib mesylate

A 38-year-old Japanese man was referred to our hospital in June 2003 for treatment of acute respiratory failure with severe eosinophilia. Idiopathic hypereosinophilic syndrome had been diagnosed in 1994. However, karyotypic examination of bone marrow cells revealed that chromosomal translocation with t(4;10)(q12;p11) had occurred in 2000, and chronic eosinophilic leukemia was diagnosed. At admission, the patient’s respiratory condition was extremely serious, and mechanical support was necessary. Despite treatment with steroid pulse therapy and cytarabine, the blood eosinophil count did not decrease, and the patient’s respiratory condition worsened. After obtaining informed consent, we administered imatinib mesylate at a dose of 200 mg/day for 2 days and 100 mg/day for 3 days. The blood eosinophil count decreased dramatically over 5 days, and the patient’s condition rapidly improved, such that the patient could be discharged. In this case, we performed molecular analysis using peripheral blood. The FIP1-like 1 (FIP1L1)-platelet-derived growth factor receptor α (PDGFRα) fusion transcript was found with the reverse transcriptase polymerase chain reaction analysis. In this case, eosinophilia was possibly caused by constitutive activation of tyrosine kinase produced by the FIP1L1-PDGFRa fusion transcript.

Resistance to activated protein C in systemic lupus erythematosus patients with antiphospholipid antibodies

To the Editor: Dahlback et al. (1) have recently reported three families who are thrombophilic due to an as yet uniuentified “cofactor” abnormality to activated protein C (APC), namely resistance to APC. Some researchers (2) confirmed that its prevalence rate, especially among thrombophilic patients, was higher than any other known hereditary causative abnormalities for venous thrombosis and suggested that the poor response to APC might be the most important hereditary cause of venous thrombosis. Antiphospholipid antibodies (aPL) (3), sometimes detected in patients with systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome, are also known as causative autoantibodies to arterio-venous thrombosis; however, its true mechanism of action is still remained to be clarified. It seems highly possible that a risk of thrombotic event may be further increased in patients positive for both aPL and resistance to APC. However, to our knowledge, there is no report concerning this point. This fact prompted us to investigate a prevalence rate of resistance to APC in SLE patients with aPL. The studied subjects were 50 SLE patients who fulfilled the ARA diagnostic criteria for SLE. They have been maintained on < 10 mg/day of prednisolone and were not taking any drugs to influence this study for at least 7 days. Fifteen of them had previous history of arterio-venous thrombosis, habitual abortion/fetal loss, thrombocytopenia and/or biological false positive reaction to syphilis (BFP). aPL (anticardiolipin-, antiphosphatidyl serine-, antiphosphatidyl inositol-, antiphosphatidic acid-antibody) was measured previously reported ELISA method (4). Resistance to APC in plasma was measured using test kit (Coatest APC Resistance, Chromogenix AB, Sweden). Resistance to APC was judged positive when the ratio of clotting time of the sample/ control was < 2 according to the instruction of the manufacturer. Twenty-five of 50 SLE patients ( 5 0 2 ) ) were positive for at least one of aPL. Twelve of 15 SLE patients with previous history of aPL related complications were positive for aPL. Resistance to APC was initially positive in 3 of 50 SLE patients. Two patients (patient A, female, 42 years old, patient B, female, 49 years old) were repeatedly positive for resistance to APC at different test occasions, and thus 2 patients were finally diagnosed as definitely positive cases for resistance to APC. They were also positive for aPL (negative for lupus anticoagulant), but neither of them had a previous history of thrombosis other than habitual abortion (patient A) and thrombocytopenia (patient B). Thus we have confirmed the existence of resistance to APC in 2 SLE patients with aPL. But since they had no previous history of thrombosis, we could not confirm our hypothesis that the co-existence of APC-resistance in patients with aPL was an additional risk factor for thrombotic event. Svensson et al. (5) reported that the probability of a person positive for resistance to APC in families with thrombophilia being free of thrombosis at the age of 45 was approximately 59%. Provided this holds true, our 2 SLE patients could be said to be still at risk for thrombosis; hence, we need further follow-up studies to record whether they develop venous thrombosis in the near future. Since not all patients with aPL necessarily develop thrombosis, additional facto(s), such as resistance to APC may play an important role along with aPL for developing thrombosis in this clinical setting.

Circulating intercellular adhesion molecule‐1 and soluble interleukin 2‐receptor in patients with systemic lupus erythematosus

To the Editor: It is well known that clinical complications, such as thrombosis and fetal loss, are sometimes seen in patients with systemic lupus erythematosus (SLE), especially in those found to be positive for antiphospholipid antibody (aPL) (1). Intercellular adhesion molecule1 (ICAM1) serves in intercellular adhesion by binding to its ligand, leukocyte integrins lymphocyte functionassociated antigen 1 (LFA-1) and complement receptor type 3, which allows participation in many immunological functions. ICAM1 is expressed primarily in endothelial cells, and its surface expression may be augmented in association with cell activation and/or the production of various cytokines. Thus, it seems possible that ICAM-I is a marker reflecting endothelial activation and/or injury (2). We measured serum ICAM-1 level in patients with SLE to investigate a possible correlation with aPL positivity. Furthermore, we measured serum soluble interleukin-2 receptor (sIL2-R), known as lymphocyte activation marker (3), to clarify whether or not there was any correlation between the levels of ICAM-1 and sIL2-R in SLE patients. The subjects were 48 patients with SLE who ful-

Detection of Beta-2-Glycoprotein-l-Dependent Antiphospholipid Antibodies and Anti-Beta-2-Glyco-protein-l Antibody in Patients with Systemic Lupus Erythematosus and in Patients with Syphilis

We investigated whether or not antiphospholipid antibodies (aPLs; antiphosphadidylserine antibody, aPS; antiphosphatididylinositol antibody, aPI; antiphosphatidic acid antibody, aPA, and antiphosphadidylethanolamine antibody, aPE) were beta 2-glycoprotein-I (GPI)-dependent antibodies like anticardiolipin antibody (aCL) in patients with systemic lupus erythematosus (SLE). None of the patients with syphilis or healthy controls was positive for any GPI-dependent aPL. By contrast, GPI-dependent aCL (40%), aPS (20%), aPI (18%), aPA (12%) and aPE (8%) were detected in patients with SLE. Among these, 4 patients were negative for aCL, but positive for aPS. Those who were positive for more than 2 types of aPL, along with lupus anticoagulant, had a high incidence of arteriovenous thrombosis, fetal loss, thrombocytopenia and biological false-positive reaction to syphilis. From these findings we conclude that GPI-dependent aPLs, other than aCL, are present in patients with SLE, and we should examine more than 2 types of aPL, such as a combination of aCL and aPS, to avoid overlooking aPL. Furthermore, we confirmed that GPI-independent aPL was not rare in SLE patients, but the clinical significance of this type of aPL in this clinical setting is unclear.

Plasma concentrations of total/free and functional protein S are not decreased in systemic lupus erythematosus patients with lupus anticoagulant and/or antiphospholipid antibodies

SummaryWe conducted an investigation to clarify whether or not the levels of total, free, and functional protein S and C4-binding protein (C4bp) in plasma are decreased in systemic lupus erythematosus (SLE) patients, especially those with antiphospholipid antibody (aPL), which is known to be a causative factor of such complications as habitual abortion and arteriovenous thrombosis. Fifty patients with SLE were recruited as subjects of the study. Serum aPL (anticardiolipin, antiphosphatidyl serine, antiphosphatidyl inositol, and antiphosphatidic acid antibodies) were measured by ELISA. Lupus anticoagulant was determined by aPTT, KCT, and diluted RVVT. Furthermore, plasma concentrations of total, free, and functional protein S and C4bp were measured. There were no significant differences in the mean levels of total, free, or functional protein S and C4bp between aPL-positive, aPL-negative SLE patients, and the healthy controls. From these results, we concluded that the protein S level is not the sole factor causing complications, and that other factor(s) may be involved in the induction of such complications in this clinical setting.

Negligible synergistic effect of β2‐glycoprotein I on the reactivity of antioxidized low‐density lipoprotein antibody to oxidized low‐density lipoprotein

We conducted this study to investigate whether antioxidized low‐density lipoprotein (a‐oxLDL) is an antibody to cryptic and/or neo‐antigen on β2‐glycoprotein I (GPI), which is introduced by binding to anionic phospholipid, similar to that of GPI‐dependent anticardiolipin antibody (aCL) employing a‐oxLDL ELISA. We found that no significant optical density differences existed among systemic lupus erythematosus patients, including cases with aCL and/or lupus anticoagulant positivity, before and after the addition of GPI. Our results suggest that a‐oxLDL is not an antibody to denatured GPI, but rather to oxLDL.

INFLUENCE OF TWEEN 20 ON THE DETECTION OF β2-GLYCOPROTEIN I-DEPENDENT ANTICARDIOLIPIN ANTIBODIES

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