Comparison of measurements of anti-PLA2R antibodies in Japanese patients with membranous nephropathy using in-house and commercial ELISA.

BACKGROUND: The prevalence of antibodies against M-type anti-phospholipase A2 receptor (PLA2R) was reported to be ~ 70-80% in early studies on idiopathic membranous nephropathy (iMN) cohorts from Western countries, China, and Korea, and ~ 50% in recent studies on two Japanese iMN cohorts. METHODS: We developed an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of anti-PLA2R antibodies, and examined sera from 217 patients with iMN, 22 patients with secondary MN (sMN), and 50 healthy individuals. All patients and healthy individuals were Japanese. The relationships between levels of anti-PLA2R antibodies and clinical parameters were analyzed. Serum samples were also tested using a standardized commercial ELISA (Euroimmun, Germany). RESULTS: In our ELISA, OD values greater than the mean + 3 standard deviation of healthy subjects were considered to be positive for anti-PLA2R antibodies. Of the patients with iMN, 33.6% (73/217) were positive, but all sMN patients were negative. Our ELISA and the Euroimmun ELISA had a high concordance (93.5%). The proportion of patients with nephrotic syndrome was significantly higher in anti-PLA2R antibody-positive patients than in antibody-negative patients (65.8 vs. 37.5%, P < 0.001). Levels of anti-PLA2R antibodies were significantly correlated with levels of urinary protein and serum albumin (P = 0.004 and P < 0.001, respectively). CONCLUSIONS: The prevalence of anti-PLA2R antibodies in our Japanese iMN cohort was lower than that in the previous studies from other countries and other Japanese institutes. The low prevalence of antibodies may be related with the characteristics of enrolled patients with mild proteinuria and undetectable antibody levels.

Macrophage C-type lectin on bone marrow-derived immature dendritic cells is involved in the internalization of glycosylated antigens.

Bone marrow-derived dendritic cells (DCs) were examined for the expression of the murine macrophage C-type lectin specific for galactose and N-acetylgalactosamine (mMGL). Flow cytometric analysis after double staining for MHC class II and mMGL with specific monoclonal antibodies indicated that mMGL was expressed on immature DCs with low to moderate levels of MHC class II and down-regulated during maturation. Immature DCs bound and internalized alpha-N-acetylgalactosaminides conjugated to soluble polyacrylamide (alpha-GalNAc polymers), whereas mature DCs and bone marrow cells did not. The two-color flow cytometric profiles indicated that the degree of alpha-GalNAc polymer bindings exactly coincided with the intensity of the binding of a mMGL-specific monoclonal antibody LOM-14. The internalized alpha-GalNAc polymers seemed to be transported to MHC class II compartments. Thus, mMGL is transiently expressed on bone marrow-derived DCs during their development and maturation and suggested to be involved in the uptake of glycosylated antigens for presentation.

Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases.

C57BL/6 mice were immunized intradermally with various doses of purified pCEP4 plasmid DNA containing full-length MUC1 cDNA (22 tandem repeats). Mice immunized with MUC1 DNA three times at weekly intervals had serum antibodies to a synthetic peptide corresponding to the tandem repeats of MUC1. The antibody titer correlated with the plasmid DNA dose. After the third immunization mice were injected intravenously with 5 x 10(5) 16-F10 melanoma cells that had been stably transfected with MUC1 cDNA (F10-MUC1-C8 clone cells). The number of lung metastatic nodules three weeks after inoculation of F10-MUC1-C8 cells was significantly lower in mice immunized with MUC1 plasmid DNA than in mice immunized with the vector DNA alone. Thus, the suppression of lung metastasis was antigen-specific. In vivo depletion of lymphocyte subpopulations by specific antibodies revealed that natural killer cells are the major effector cells responsible for the suppression of lung metastasis. CD4+ cells and CD8+ cells apparently played some roles too.