|Budget Amount *help
¥3,700,000 (Direct Cost : ¥3,700,000)
Fiscal Year 2004 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 2003 : ¥2,800,000 (Direct Cost : ¥2,800,000)
The Wiskott-Aldrich syndrome protein (WASP), which is defect in Wiskott-Aldrich syndrome (WAS) patients, is intracellular protein expressed in non-erythroid hematopoietic cells. Recent studies revealed that WASP interacts with numbers of intracellular molecules and plays key roles in the signal transduction and the regulation of actin polymerization, although varied clinical symptoms observed in WAS patients has not been fully elucidated from a viewpoint of WASP-deficiency.
We have established the methods to detect intracellular WASP by flow cytometry (FCM-WASP) and revealed that WAS patients showed null/very low level of intracellular WASP of lymphocytes/monocytes by FCM-WASP studies, while significant amount of WASP was detected in normal individuals. We applied the methods for the screening for WAS patient and WAS carrier, and the evaluation of the mixed chimera status in WAS patients who underwent hematopoietic stem cell transplantation. In addition, during the course of a WAS scree
ning using FCM-WASP, we happened to find a patient with WAS who possessed a small population of lymphocytes in which a spontaneous reversion of the inherited WASP gene mutation had taken place.
During these FCM-WASP studies, we have noticed that lymphocytes of control individuals showed double or a broad positive peak while those monocytes invariably showed a sharp positive peak. To study the basis for the double positive peaks (WASP^<high-bright> and WASP^<low-bright>) of control lymphocytes detected by FCM-WASP, we tried to characterize the two populations. By double/triple staining FCM-WASP methods, it was revealed following results. T cells, both CD4+ and CD8+ cells are composed of both WASP^<high-bright> and WASP^<low-bright> cells. Most of B cells (CD20+) are WASP^<low-bright> cells, most of NK cells (CD56+) are WASP^<high-bright> cells. Further characterization revealed that CD45RA+ of either CD4+ or CD8+ cells belong to WASP^<low-bright> and CD45RO+ cells of either CD4+ or CD8+ cells belong to WASP^<high-bright> cells. These results were obtained by using a-WASP antibody 3F3A5 (provided by Dr.Nelson DL : NIH/USA), whose epitopes are middle part of WASP. In case of using another a-WASP antibody (Southern Biotech. Inc), whose epitopes are N-terminal of WASP, no double positive peaks were never detected. To study any difference in quantity of WASP message or protein level, we purely purified CD3+/CD45RA+cells and CD3+/CD45RO+ cells, and then RT-PCR and Western blotting analysis were performed. The results revealed any difference in WASP message or protein level between these subpopulation cells.
Recently, the intermolecular conformational change of WASP ; active and inactive form was reported. We presumed that double positive peak of WASP observed in normal individual lymphocytes by FCM-WASP could be explained by this scenario. To prove the hypothesis, following experiments are in progress. We constructed retrovirus vector construct with very unique WASP mutatnt of L279P, which was reported as constitutively active form of WASP. We are planning transduction experiment of vector containing the mutant WASP or wild WASP to analyze using FCM-WASP. It was also reported that activated WASP is recruited to lipid raft, reorganized cell membrane functional unit. Using con-focal microscopic analysis, we are planning to analyze location of WASP in both cells belong to WASP^<high-bright> and WASP^<low-bright>. Less