Katsuhiko Sudo

Quantitation of C3 nephritic factor of alternative complement pathway by an enzyme-linked immunosorbent assay.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C3 nephritic factor of the alternative pathway of complement (NeFA). Incubation of the NeFA-positive serum (patient KS serum) with normal human serum (NHS) in Mg-EGTA resulted in the formation of C3-B-IgG complex. No complex was formed in EDTA. At first this was detected as three types of complexes: C3-IgG, B-IgG and B-C3, by the combination of antibodies. The reaction mixture in Mg-EGTA was filtered through an ACA 22 column, from which the complexes were eluted in the same part as the first protein peak. When IgG purified from KS serum was incubated with NHS in Mg-EGTA, B-C3 complex increased in proportion to the dose of IgG. These results indicated that only one kind of complex consisting of IgG, C3 and B (IgG-C3-B) was generated by the addition of NeFA to NHS. Serum NeFA could be quantified as the titer of B-C3 complex formed after its incubation with NHS in Mg-EGTA. Using the ELISA method, NeFA was positive in five out of six patients with membranoproliferative glomerulonephritis (MPGN) type II and in only one of 17 with MPGN type I. Titers obtained by the new method were in good accordance with those by C3 conversion and C3bBb stabilization assays for NeFA, and the new method was more exact and simple than the conventional methods.

Membranous Nephropathy Associated with Nodular Sclerosing Hodgkin’s Disease

A 61-year-old man developed steroid-resistant nephrotic syndrome in which renal biopsy showed membranous nephropathy. Six months after the initial presentation, nodular sclerosing Hodgkins disease was diagnosed. Cyclophosphamide, vincristine, prednisolone and procariazine chemotherapy was administered, after which proteinuria gradually decreased resulting in complete remission in the course of 7 months. A second renal biopsy, which was undertaken 4 months after complete remission, revealed significant histological improvement with disappearance of immune deposit.

Limitation of kidney biopsy in detecting crescentic lesions in IgA nephropathy.

Osamu Hotta, MD, Department of Nephrology, Sendai Shakaihoken Hospital, 3-16-1 Tsutsumimachi, Sendai 981 (Japan) Dear Sir, Whether cellular crescentic lesions are present or not is one of the important factors in predicting the prognosis of patients with IgA nephropathy (IgAN) [1], and consequently affects the choice of therapeutic interventions. However, cellular crescents are usually distributed in a focal manner, especially in the milder or early phase of IgAN. Therefore, the accuracy in detecting crescentic lesions may depend to a large extent on the size of the biopsy specimen [2]. In an attempt to clarify the limitation of kidney biopsy in detecting cellular crescentic lesions in IgAN, we investigated the relationship between the size of biopsy specimens and the frequency of the presence of cellular crescentic lesions in 206 subjects with IgAN. Renal biopsy specimens were obtained by surgical (184 cases) or percutaneous (22 cases) needle biopsy. Two specimens were obtained from each patient. About two-thirds of each specimen from the same patient was processed for light microscope (LM) study. The remaining one-third of each specimen was processed for immunofluorescence and electron microscope study. The number of glom-eruli and the presence or absence of cellular crescentic lesions were examined in two sections of each specimen for LM study from the same patient. In addition to the relationship between the incidence of the presence of cellular crescentic lesions and the total number of glomeruli in the biopsy specimen, the relationship between the incidence of cellular crescentic lesions in one of two specimens (the smaller one and the larger one) and the number of glomeruli in the smaller specimen was examined. The incidence of cellular crescentic lesions in one of the two specimens was determined by the following formula: the number of subjects in whom cellular crescentic lesions were present in one specimen and absent in the other/the total number of subjects in whom cellular crescentic lesions were present, including those with lesions present in both specimens and those with lesions present in only one of the two specimens. The relationship between the total number of observed glomeruli in two specimens and the incidence of the presence of cellular crescentic lesions is shown in figure 1. The incidence of the presence of cellular crescentic lesions increased with the total number of glomeruli. When the number of glomeruli is > 30, the incidence of the presence of cellular crescentic lesions reached approximately 60%. The relationship between the number of glomeruli in the smaller specimens and the incidence of cellular crescentic lesions in one of the two specimens is shown in figure 2. In total, of 206 subjects, 105 contained cellular crescentic lesions in both or either specimen. The presence of

Quantitation of C4 nephritic factor by an enzyme-linked immunosorbent assay

We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C4 nephritic factor (C4NeF). Incubation of the C4NeF-positive serum from patient M.I. with normal human serum (NHS) in the presence of human aggregated IgG (AHG) resulted in the formation of stable C4-C2 complex. No complex was formed in EDTA or under the condition free of AHG. The reaction mixture was filtered through an ACA 22 column, from which the C4-C2 complex was eluted at the first protein peak. When IgG purified from M.I. serum was incubated with NHS and AHG, C4-C2 complex also increased in proportion to dose of the purified M.I. IgG. These results show that C4NeF in M.I. serum stabilizes C4b2a convertase of the classical complement pathway, and is quantified by the ELISA. C4NeF activity was measured, using the ELISA method, in patients with various glomerular diseases, and found elevated in three of 24 patients with membranoproliferative glomerulonephritis (MPGN) type I and slightly but distinctly positive in seven of 24. No C4NeF was detected in two C3 nephritic factor-positive patients with MPGN type II and six with active systemic lupus erythematosus. The new method was more simple and quantitative than C4b2a stabilization assay for C4NeF.

Soluble ELAM-1 Is Elevated with the Progression of IgA Nephropathy but Not with That of Polycystic Kidney Disease

Osamu Hotta, MD, Department of Nephrology, Sendai Shakaihoken Hospital, 3-16-1 Tsutsumimachi, Aoba-ku, Sendai, Miyagi, 981 (Japan) Dear Sir, The endothelial leukocyte adhesion molecule (ELAM)-1 ‚ a member of the selectin family with a lectin-like N-terminal domain [1], binds granulocytes, monocytes and a subset of memory T cells [2-4]. ELAM-1 is expressed only on vascular endothelium, predominantly on postcapillary venules [5-7]. Expression of ELAM-1 on vascular endothelium can be observed in the proximity of cells producing inflammatory cytokines such as interleukin-1 and the tumor necrosis factor, stimuli which, in vitro, are known to induce an upregulation of adhesion [5]. Recently, a quantitative sandwich ELISA for ELAM-1 in the fluid phase (soluble ELAM-1, sELAM-1) has been developed [6]. The expression of ELAM-1 has been described only on activated endothelial cells, not on other cell types; therefore, levels of ELAM-1 in serum may provide a basis for the assessment of endothelial damage or activation [6, 7]. We examined the level of s-ELAM-1 in patients with IgA nephropathy (IgAN), and those with polycystic kidney disease (PCK), paying special attention to its correlation with the decline of renal function. A total of 38 patients were enrolled in the present study: 24 with IgAN, and 14 with PCK with varying degrees of renal function. Twenty-seven healthy adult volunteers served as normal controls for s-ELAM-1 assay. They were determined healthy by biochemical tests and urinalysis. All blood samples were obtained in the morning (07.00-09.00) at Sendai Shakaihoken Hospital. In 5 patients with IgAN, blood samples were obtained twice: before steroid therapy and 1