Haruyoshi Yoshida

Distinct clonotypes of anti-DNA antibodies in mice with lupus nephritis.

Clonotypes of IgG anti-DNA antibodies were studied by isoelectric focusing in various autoimmune mice with or without lethal lupus nephritis. MRL/MpJ-lpr/lpr mice exhibited the most heterogeneous spectrotypes of anti-DNA antibodies in the pH range from 6.5 to 8.5, with marked variation in individual mice. Female (NZB X NZW)F1 mice expressed rather uniform DNA-binding bands composed of at least five to six distinct subgroups, having isoelectric points from 6.5 to 8.0. Male BXSB mice showed major characteristic bands confined to alkaline pH range from 7.8 to 8.5, similar to C57BL/6J-lpr/lpr mice, which showed markedly restricted bands in this region. Both AKR/J-lpr/lpr and C3H/HeJ-lpr/lpr mice expressed DNA-binding bands mostly focused between pH 6.5 and 8.2. The aging study indicated that three autoimmune mice (MRL/MpJ-lpr/lpr, [NZB X NZW]F1, and male BXSB) that developed fatal glomerulonephritis showed clonal expansion of anti-DNA antibodies throughout their life. In contrast, such age-dependent expansion of anti-DNA clonotypes was not evident in three lpr cogenic mice (C57BL/6J-lpr/lpr, AKR/J-lpr/lpr, and C3H/HeJ-lpr/lpr) that developed only mild glomerulonephritis; rather, their expression of anti-DNA spectrotypes diminished as they aged. Anti-DNA activities in renal eluates from nephritic autoimmune mice were mostly distributed in the pH range from 6.5 to 8.0, without significant concentrations in the high alkaline range of more than pH 8.0. These results suggest that there exist distinct anti-DNA clonotypes in each mouse strain and that the development of lupus nephritis does not appear to be associated with particular spectrotypes of anti-DNA antibodies. Rather, the age-dependent expansion of anti-DNA clonotypes may be a feature more characteristic of mice developing lethal lupus nephritis.

Genetic regulation of the class conversion of dsDNA-specific antibodies in (NZB×NZW) F1 hybrid

To investigate the possible effects of NZW genes on the class conversion of dsDNA-specific antibodies in NZB × NZW (B/W) F1 hybrids, we measured IgM, IgGl, and IgG2 dsDNA-specific antibodies, using the Crithidia luciliae kinetoplast immunofluorescence test, in NZB, NZW, B/W F1 hybrid, B/W F1 × NZB backcross, and B/W F1 × NZW backcross mice at 4, 7, and 10 months of age. The highest serum levels of IgM dsDNA-specific antibodies were observed in NZB mice at the ages tested; however, the amounts of IgG1 and IgG2 antibodies were scanty. In contrast, a large amount of both IgG1 and IgG2 dsDNA-specific antibodies was produced in B/W F1 hybrids, in which the serum IgM antibodies were lower than those observed in NZB mice. NZW mice were virtually negative for these antibodies. Progeny testing suggested that a combined effect of two unlinked dominant genes of the NZB strain determines the production of dsDNA-specific antibodies and that these genes only act to produce IgM antibodies. These traits are to a great degree modified by the NZW loci in B/W F1 hybrids, and a combined effect of two unlinked dominant genes leads to conversion of the class of the antibodies from IgM to IgG, which, in turn, increases the serum levels of dsDNA-specific antibodies. The F1 hybrid of C57BL/6 and NZW strains produced no dsDNA-specific antibodies, indicating that the relevant NZB predisposing genes are required for the NZW gene action. Linkage studies showed that one of such NZW genes is to some extent linked to the H-2 complex on chromosome 17, but not to Mup-1 (chromosome 4) or a coat color locus (chromosome 2). The appearance of IgG dsDNA-specific antibodies correlated well with the incidence of renal disease in B/W F1 × NZB backcross mice.

Activation of Ca-permeable cation channels by myocarditis-associated antibody in guinea pig ventricular myocytes.

The pathogenesis of myocarditis and dilated cardiomyopathy is though to involve autoimmunological processes and myocardial calcium overload. Serum containing antiheart antibodies associated with a murine model of myocarditis increased [Ca2+]i in guinea pig ventricular myocytes only in the presence of extracellular Ca2+. The antiheart antibody-positive serum activated Ca(2+)-permeable cation channels that were insensitive to dihydropyridines and membrane stretch. The permeability sequence was Ba2+ > Ca2+ > Na+ approximately K+, and the single-channel conductance to Ba2+ was 12 pS. The channel was activated by extracellular application of the serum during on-cell recording, which suggests that a soluble intracellular messenger may be involved. The antibody-positive serum did not alter voltage-gated Ca2+ currents. We propose that excess Ca entry in myocarditis and dilated cardiomyopathy results from activation of a Ca(2+)-permeable cationic channel by the autoantibodies.

Immunopathological correlation between mesangial C3d-deposition and C3d-fixing circulating immune complexes in lupus nephritis

By a direct immunofluorescent technique, glomerular C3d deposition was examined in a total of 50 renal biopsy specimens from patients with lupus nephritis. C3d deposition was then compared with disease activity, glomerular IgG and C3c deposition, and the levels of circulating immune complexes (CIC) measured by a solid-phase anti-C3d assay. There was a good correlation between disease activity and the positivity of glomerular C3d deposits (P less than 0.001), as well as C3c deposits (P less than 0.001). Even in clinically inactive patients, a relatively high percentage (59%) of C3d deposits were positive compared with C3c deposits (17%). Mesangial C3d deposition correlated with clinical disease activity more significantly (P less than 0.005) than capillary wall C3d deposition (P less than 0.025). C3d deposits were detected in all of the 30 cases with positive C3c deposits, and moreover, in 15 of the 20 (75%) cases with negative C3c deposits. Glomerular IgG deposits were almost always associated with C3d deposits, both in mesangial areas and along capillary walls, with statistical significance (P less than 0.005, P less than 0.001, respectively). The serum levels of C3d-fixing immune complexes (IC) were significantly correlated with the positivity and intensity of mesangial C3d deposits. This study demonstrates glomerular deposition of C3d in patients with lupus nephritis and reveals a significant correlation between mesangial C3d deposition and disease activity.

Clonotypic comparison of IgG anti-DNA antibodies of healthy subjects and systemic lupus erythematosus patients: studies on heterogeneity and avidity

Qualitative characteristics of IgG anti-ssDNA antibodies were studied and compared by isoelectric focusing (IEF) and enzyme-linked immunosorbent assay (ELISA) between normal human sera (NHS) and systemic lupus erythematosus (SLE) sera. In NHS, IgG anti-ssDNA spectrotypes were observed in a high alkaline pH range (7.5 to 8.5) at physiological NaCl concentrations (0.15 M). In SLE sera the spectrotypes were found to a more intensified extent in the alkaline pH range as compared to those in NHS. With regard to avidity, analyzed by salt-dependent changes of anti-ssDNA activities, NHS showed strong ssDNA-binding bands in a wide range of pH 7.0-8.5 comparable to those in SLE sera. However, these bands became extremely weak and/or faint in pH 7.5-8.5 as the NaCl concentration was raised to 0.15 M and 0.20 M. On the other hand, SLE sera still exhibited thick binding bands at higher NaCl concentrations. This salt-dependency of these antibodies was-also demonstrated by ELISA of serum samples adjusted to contain comparable antibody levels. These findings suggest that clonotypes of IgG anti-ssDNA antibodies both in NHS and in SLE sera are essentially oligoclonal and highly cationic, and that the distinctive characteristics of these antibodies in NHS may be of low avidity, in contrast to SLE sera which exhibit high avidity.

A Comparative Immunologic Study of IgA Nephropathy

A conglutinin binding assay has been used to detect circulating immune complexes (CIC) containing IgA, IgG, or IgM in sera from patients with IgA nephropathy. IgA class CIC were detected in 40.7% of patient. IgG class CIC were detected only in patients with glomercular IgG deposits. IgM class CIC were detected more often in patients with glomerular IgM deposits than in patients without glomerular IgM deposits. These results demonstrate an association between the immunoglobulin in CIC and those in glomerular deposits. CIC were not detected in sera from most patients with IgA nephropathy by a Clq binding assay, however, since this assay does not detect IgA class CIC. Immunoelectronmicroscopic studies of IgA nephropathy have shown that C3 deposits are localized to the same areas as IgA deposits. In conclusion, we suggest that mesangial IgA deposits are composed of immune complexes and may be derived from CIC.

A case of 17α-hydroxylase deficiency syndrome associated with right adrenal tumor

A 35-year-old woman, who had been hypertensive for about 17 years and had lacked menarche, showed hypokalemia, low plasma cortisol and aldosterone levels, suppressed renin activity, and marked elevation of plasma corticosterone. The patient was diagnosed as having 17 alpha-hydroxylase deficiency from functional studies. In addition, a right adrenal tumor was found by adrenal venography. Adrenal venous sampling showed that this tumor might be secreting corticosterone and possibly also deoxycorticosterone (DOC). The genotype was 46,XY, so she was diagnosed as having male pseudohermaphroditism. Right adrenalectomy and contralateral adrenal biopsy were done. The retained testicles were removed. Dexamethasone administration normalized the blood pressure and serum potassium. This is the first report of 17 alpha-hydroxylase deficiency with a right adrenal tumor.

Expression of human atrial natriuretic polypeptide gene in cos 7 cells

Cos 7 cells transfected with human atrial natriuretic polypeptide (hANP) gene with SV40 enhancer and replication origin sequences expressed hANP gene. The expressed RNA was indistinguishable from native hANP mRNA and the transcribed protein seemed to be properly processed to alpha-hANP and beta-hANP. This system provides a useful approach to investigate the processing of hANPs and the structure-function relationship of amino acid sequences of hANPs.

High Immune Complex Level in Healthy Middle Aged Females

The pathological roles of immune complexes in various diseases have been extensively documented (3, 8, 10, 11). Recently various methods have been devel opedto detect circulating immune complexes (CIC) (4) and even though normal values of CIC have been given in various reports, very little is known about the incidence and characteristics in normal subjects (1, 6). The formation of CIC itself is not a pathologic process but it serves to clear antigens, and the dynamics of CIC is assumed to be dependent on clearance by the reticuloendothelial system. Still it is not clear what type of CIC is pathogenic. Here we report a distribution study in different ages and sexes in a large sample taken from the normal population and solve basic parameters of the assay system. Clq was purified from fresh pooled sera of young males by the method of Yonemasu and Stroud (13). Purity was checked by 4.5% SDS polyacrylamide disk

Detection of IgA binding site by flow cytometry with fluorescent microspheres.

To establish a sensitive method to detect IgA Fc receptors (Fc alpha R) on human and murine lymphoid cells, fluorescent microspheres (FMS) were used in a flow cytometric assay. The following three assays with FMS were tested and compared with a conventional indirect Fc alpha R assay using FITC-labeled anti-IgA: (1) direct cell binding assay with murine myeloma IgA(MOPC315)-coated FMS(IgA-FMS assay), (2) indirect assay with TNP-BSA-coated FMS which bind to cells preincubated with MOPC315 IgA bearing anti-TNP activity (TNP-BSA-FMS assay), and (3) indirect assay with anti-IgA coated FMS after preincubation of the cells with IgA(anti-IgA-FMS assay). In these three assays for Fc alpha R using FMS, the binding of IgA to the cells was not affected by purified IgM or IgG preparations. In both indirect assays using TNP-BSA-FMS and anti-IgA-FMS, sharp and dose dependent IgA binding was obtained at lower IgA concentrations ranging from 4 to 125 micrograms/ml as compared with the conventional indirect assay. The background MFI levels in all these FMS assays remained as low as those in the conventional assay. These findings suggest that FMS coupled with TNP-BSA or anti-IgA antibodies is suitable for the detection of Fc alpha R on both murine and human T cells.