A distinct pathway of cell-mediated apoptosis initiated by granulysin.

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

Detection of anti-phosphatidylethanolamine antibodies using flow cytometry.

BACKGROUND: The finding that lupus anticoagulant (LA) is significantly associated with anti-phosphatidylethanolamine (PE) activity has led to great interest in its relation to the clinical features of the antiphospholipid syndrome (APS). Considerable variability has, however, been reported in the prevalence of anti-PE antibodies in APS patients using enzyme-linked immunosorbent assay (ELISA) methodology. The lack of standardization and differences in technique may in part explain these discrepancies. PE binds variably to different types of microtiter wells, reflected in the consequent detection, or lack of detection, of anti-PE antibodies. This study describes the use of flow cytometry as an alternative method for the detection of anti-PE antibodies. METHODS: Six LA-positive plasma samples were used in this original study. Polystyrene beads were coated with PE overnight. These were subsequently incubated with patient plasma. Both IgG and IgM binding were detected by flow cytometry using a cocktail of fluorescently labelled anti-human Ig isotypes. RESULTS: When these results were compared with those from ELISA, flow cytometric analysis provided an apparent enhanced detection of anti-PE antibodies. It was found that 6/6 were IgM anti-PE positive by flow cytometry, whereas 5/6 were IgM by ELISA; 2/6 negative for anti-cardiolipin antibodies by ELISA were positive by flow cytometry; and 2/6 positive for antiphosphatidylcholine antibodies in cytometry were negative by ELISA. CONCLUSIONS: With appropriate quantification, this method may be more sensitive than ELISA in detecting anti-PE antibodies in plasma samples of patients with APS.

Microtitre plate and assay buffer alter detection of antiphosphatidylethanolamine antibodies in lupus anticoagulant positive plasma.

The ELISA for detection of anti-phosphatidylethanolamine antibodies in patients with lupus anticoagulant was studied. The influence of microtitre plate types, antigen concentration, antibody dilution, and buffer system was evaluated. Human albumin in the blocking buffer gave greater numbers of positive plasma samples than bovine serum. Comparison of simultaneous plasma and serum samples showed only minor variation in binding characteristics.

Phospholipid specificity of autoimmune and drug induced lupus anticoagulants; association of phosphatidylethanolamine reactivity with thrombosis in autoimmune disease.

OBJECTIVE: To assess the phospholipid specificity of lupus anticoagulants (LAC) in autoimmune and drug induced LAC positive patients, and to determine their relevance with the clinical feature thrombosis. METHODS: Thirty-five plasma samples with LAC were tested. Of these, 22 samples were from patients with autoimmune disease: 12 had systemic lupus erythematosus (SLE) and 10 primary antiphospholipid syndrome (APS). These were compared with 13 patients with drug induced LAC. Antiphospholipid (aPL) activity was tested by inhibition of LAC activity in a modified coagulation assay using the phospholipids: egg phosphatidylethanolamine (PE), 20% phosphatidylserine/80% phosphatidylcholine (PS/PC), and 15% PS/65%PC/20%PE mixture. Anticardiolipin antibodies (aCL) were measured in all samples by ELISA. RESULTS: In the autoimmune group, 95% were positive for reactivity to PE, 68% for PS/PC, 68% for PS/PC/PE, and 77% aCL. Of the drug induced group, 69% were positive for reactivity to PE, but only 23% for PS/PC, 46% PS/PC/PE, and 23% aCL positive. In the autoimmune patient group, 64% had thrombotic episodes, all of which had anti-PE activity, and 64% had associated anti-PS activity. No drug induced patient had episodes of thrombosis. CONCLUSION: Autoimmune induced LAC may contain a broader range of phospholipid specificities than those from drug induced patients. These data thus identify another potential reason why patients with drug induced LAC appear to be at less risk of developing APS than those with autoimmune LAC.