|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1999 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1998 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Primary crescentic glomerulonephritis is rapidly progressive and poorly prognostic. The treatment depends mainly on glucocorticoid hormone and imuunosuppressants. Animal models play pivotal roles to establish new strategy for therapy of intractable of intractable diseases. In this study, I tried to raise severe combined immunodeficiency (SCID) mice carrying perinuclear anti-neutrophil cytoplasmic antibody (P-ANCA) and crescentic glomerulonephritis (GN).
1. Association between P-ANCAGN and HLA class II genotypes.
Utilizing PCR-RFLP and PCR-SSO, I determined HLA class II genotypes of 34 Japanese patients with P-ANCAGN. I found significant of P-ANCAGN with HLA-DRB1ィイD1*ィエD10803 (Odd's ratio 2.5 p<0.01) and with HLA-DQB1ィイD1*ィエD10601 (Odd's ratio 26 p<0.01), DRB1ィイD1*ィエD10803 and DQB1ィイD1*ィエD10601 are in linkage disequiribrium in Japanese population.
2. Detection of human P-ANCA in patients and mice.
Solid phase enzyme linked immunoadsorbent assay was used to detect human P-ANCA. In brief, pur
ified myeloperoxidase (MPO) was immobilized on a plastic microtiter plate, sample serum was applied, wells were washed extensively, alkaline phosphatase labeled goat anti-human IgG or IgA antibody was added, and PNPP was added as substrate. The titers determined at my lab were well correlated with those from SRL laboratory.
3. MPO specific T cell proliferation.
Peripheral lymphocytes were cocultured with purified MPO for 7 days, and the proliferation was assayed by uptake of tritiated thymidine. No T cell specific response was demonstrated.
4. SCID mouse producing P-ANCA.
Peripheral lymphocytes from P-ANCAGN were transferred to 8-week-age SCID mice. The body weight, systolic and diastolic blood pressure, urine proteinuria, human IgG and IgM, and human P-ANCA were monitored every two weeks. The mice were sacrificed at 12 weeks of follow-up. Human IgG and IgM were detected in mice with human lymphocytes, but P-ANCA only in those with patient lymphocytes. The mice died before end point, and all were mice transferred patient lymphocyte. No mice showed proteinuria or hypertension suggesting P-ANCAGN, Pathological examination is under investigation.