Recognition factors of Ricinus communis agglutinin 1 (RCA(1))

Ricinus communis agglutinin (RCA1) is one of the most important applied lectins that has been widely used as a tool to study cell surfaces and to purify glycans. Although the carbohydrate specificity of RCA1 has been described, the information obtained was mainly focused on inhibition of simple Galbeta1-related oligosaccharides and simple clusters. Here, all possible recognition factors of RCA1 of glycan binding were examined by enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using known mammalian Gal/GalNAc carbohydrate structural units and natural polyvalent glycans. Among the glycoproteins (gps) tested and expressed as 50% nanogram inhibition, the high-density polyvalent Galbeta1-4GlcNAc (II) glycotopes occurring in natural gps, such as Pneumococcus type 14 capsular polysaccharide which is composed of repeating poly II residues, resulted in 9.0 x 10(4), 1.5 x 10(5), 2.3 x 10(4) and 2.1 x 10(4)-fold higher affinities to RCA1 than the monomeric Gal, linear I/II and Tri-antennary-II (Tri-II). Of the ligands tested and expressed as nanomoles of 50% inhibition, Tri-II was the best, being about 2, 4, 25.6 and 33.3 times better inhibitor than Di-II, II, I (Galbeta1-3GlcNAc) and Gal, respectively. From the results of this study, it is concluded that: (a) Galbeta1-4GlcNAc and other Galbeta1-related oligosaccharides are essential for lectin binding and their polyvalent form in macromolecules should be the most important recognition factor for RCA1; (b) the combining site of RCA1 may be a groove type, recognizing Galbeta1-4GlcNAc (II) as the major binding site; (c) its combining size may be large enough to accommodate a tetrasaccharide of beta-anomeric Gal at the non-reducing end and most complementary to human blood group I Ma active trisaccharide (Galbeta1-4GlcNAcbeta1-6Gal) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc); (d) RCA1 has a preference for the beta-anomer of Gal oligosaccharides with a Galbeta1-4 linkage > Galbeta1-6 > or = Galbeta1-3; (e) configuration of carbon-2, -3 -4 and -6 in Gal are essential for binding; (f) hydrophobic interaction in the vicinity of the binding site useful for sugar accommodation increases affinity. These results should be helpful for understanding the functional role of RCA1 and for characterizing glycotopes of mammalian complex carbohydrates.     

Immunological studies of human placentae. Lectin binding to villous stroma and to trophoblastic basement membrane.

It has been demonstrated immunohistologically using cryostat sections that the lectins PHA and Con A bind to human placentae. Both lectins stain trophoblastic basement membrane (TBM), perivascular tissue and stroma. No staining of trophoblasts was observed. It has been shown by blocking experiments that the lectin binding sites on placental TBM are glycoproteins, whereas experiments involving pretreatment of placental sections with anti-collagen antisera or highly purified bacterial collagenase have indicated that lectin binding to stromal and perivascular structures is collagen-associated. The possible relation of TBM lectin-binding sites to immune response gene products is briefly discussed.     

Antigen specificity of anti-nuclear antibodies complexed to nucleosomes determines glomerular basement membrane binding in vivo

Monoclonal anti-nuclear antibodies which are complexed to nucleosomes are able to bind to the glomerular basement membrane (GBM) in vivo, whereas purified antibodies do not bind. The positively charged histone moieties in the nucleosome are responsible for the binding to anionic determinants in the GBM. We tested the hypothesis that the specificity of the autoantibodies complexed to the nucleosome influences the glomerular binding of the antibody-nucleosome complex. We induced the formation of these immune complexes in vivo, by intraperitoneal inoculation of hybridomas producing monoclonal anti-nuclear antibodies (four anti-histone, three anti-double stranded (ds)DNA and three anti-nucleosome antibodies) into nude BALB/c mice. In ascites and plasma from the mice inoculated with these hybridomas, nucleosome/autoantibody complexes were detected in comparable amounts. Immunofluorescence of kidney sections revealed that about 60% of the mice inoculated with anti-nucleosome or anti-dsDNA hybridomas had immunoglobulin deposits in the GBM, whereas only 15% of the mice with anti-histone hybridomas showed these deposits (p ≦ 0.04). In the Matrigel^®-ELISA (used as a GBM surrogate) ascites from anti-nucleosome or anti-DNA hybridomas displayed significantly higher titers (p ≦ 0.002) than ascites from anti-histone hybridomas. In conclusion, nucleosome/immunoglobulin complexes comprising anti-nucleosome or anti-dsDNA auto-antibodies do bind more frequently to the GBM in vivo than nucleosome/immunoglobulin complexes containing anti-histone antibodies. It therefore appears that the specificity of the antibody bound to the nucleosome is a critical determinant for the nephritogenic potential of the nucleosome-autoantibody complex.     

Polycation binding to glomerular basement membrane. Effect of biochemical modification

The polycation hexadimethrine (HDM) binds to anionic sites in the glomerular basement membrane (GBM) and causes heavy proteinuria when infused in vivo. An in vitro assay of 3H-HDM binding to isolated dog GBM was developed, to permit further analysis of the GBM components binding HDM. 3H-HDM binding to isolated GBM was saturable, reversible in dose-dependent fashion by competing polycations, and inhibited by increasing salt concentration and low pH. The pH dependence of binding suggested that most of the HDM binds to carboxyl groups rather than to the sulfate groups of proteoglycans. Removal of heparan sulfate by heparinase or purified heparatinase had no detectable effect on HDM binding. Treatment of GBM with neuraminidase, hyaluronidase, or chondroitinase reduced binding of HDM by a maximum of 20 to 38%. However, substitution of carboxyl anions with nonionizable glycine methyl ester residues resulted in complete elimination of HDM binding. Parallel results were obtained in studies of glomerular localization of cationized ferritin (CatF), pI 8.5. After carboxyl substitution, GBM did not bind CatF; heparinase-treated GBM bound CatF in a distribution not demonstrably different from normal. Cellulose acetate electrophoresis of glycosaminoglycan fractions prepared from treated GBM confirmed that carboxyl modification did not alter the content or charge of the heparan sulfate of GBM, but heparinase treatment removed at least 90% of heparan sulfate. The results indicate that carboxyl groups are quantitatively more important than heparan sulfate for binding of HDM in vitro. Since HDM causes proteinuria in vivo, carboxyl groups may be important for maintenance of normal permselectivity.     

Interaction of Lens culinaris lectin, concanavalin A, Ricinus communis agglutinin and wheat germ agglutinin with the cell surface of normal and transformed rat liver cells.

The observation of BOREK et al. (1973) on nonagglutinability of transformed rat liver cells by Lens culinaris lectin and our ultrastructural findings of a greater mobility of the Lens culinaris lectin receptors on transformed rat liver cells as compared to normal rat liver cells (ROTH 1975) initiated the present agglutination experiments on liver cells with lectins. For agglutination assay the microhemadsorption technique after FURMANSKI et al. (1973) was used with exception of several tests on EDTA-detached cells. The transformed rat liver cells exhibited, in contrast to the findings of BOREK et al. (1973), a positive microhemadsorption with Lens culinaris lectin as well as with Concanavalin A, Ricinus communis lectin and wheat germ agglutinin whereas the normal rat liver cells became positive only after a brief trypsin treatment. The significance of the difference in agglutinability of rat liver cells with Lens culinaris lectin and the other lectins used is discussed with regard to the cell-cell interaction mediated by lectins.     

Expression of novel basement membrane components in the developing human kidney and eye

The distribution of two novel human, basement-membrane (BM) collagens has been characterized by immunohistochemical analysis of developing and mature tissue using monoclonal antibodies specific for the non-collagenous (NC1) domain of each molecule. A distribution more restricted than that of type IV collagen was observed. In the kidney, the 28K parent molecules appear relatively late, at the early capillary-loop stage of glomerular development, whereas type IV collagen is present in all BM, including those of the ureteric bud, S-form, primitive glomerulus, and vessels. Antibody to the Alport familial nephritis antigen (a 26K peptide), which is missing from epidermal BM and glomerular BM in Alport syndrome, reacted with the ureteral bud BM and all stages of glomerular BM development from the early capillary-loop stage onward, but not with BM of more primitive glomeruli (vesicles and S forms). In the human fetal eye, the collagen molecules from which the 28K NC1 peptides are derived appear later in development than type IV collagen. They are present in trace amounts in Bruch's membrane but are not detected until after birth in the retinal internal limiting membrane and cuticular and non-pigmented epithelial BM of the ciliary process. In contrast, the BM of the lens capsule and Descemet's membrane were reactive with anti-28K antibodies early in development. In all instances, the 28K peptides are detected in BM that also contain the Alport antigen, although the latter is present in some BM not containing the 28K peptides. The distribution of Alport antigen and type IV collagen in developing eye is similar to that observed in the mature eye. The 28K parent molecules appear to be expressed in concert with the maturation of the BM, coincident with fusion of glomerular endothelial and epithelial BM, whereas the lens capsule BM and Descemet's membrane contain these restricted components much earlier in gestation.     

The use of the radioimmunoassay in the characterization of antibodies to basement membrane collagen.

This study describes the use of the radioimmunoassay for the characterization of antibodies to basement membrane (type IV) collagen from bovine anterior lens capsule. The immunogen was extracted from calf anterior lens capsules by limited pepsin digestion and injected into rabbits. The antisera were characterized using gel diffusion, haemagglutination and the radioimmunoassay in which 125I-labelled types I, II, III, and IV bovine collagen were employed. In the direct radioimmunoassay there was no reaction with either native or denatured types I, II or III bovine collagen, whereas there were high titres towards both native and denatured type IV bovine collagen. Radioimmune inhibition studies using unlabelled types I, II, III and IV bovine collagen, collagenase digested and repepsinized type IV collagen showed that there was marked inhibition by either native, denatured or repepsinized type IV collagen, and slight inhibition by native type I collagen; native type II and type III, denatured types I, II and III, and collagenase digested type IV collagen had no inhibitory effects.     

Enzyme-linked immunosorbent assay for circulating anti-glomerular basement membrane antibodies.

An enzyme-linked immunosorbent assay for the quantitative determination of anti-glomerular basement membrane antibodies in human sera, which is both sensitive and reproducible, is described. The test detected circulating antibodies in each of seven patients with active anti-glomerular basement membrane disease, whilst sera from 42 patients, with a variety of other glomerulonephropathies, were negative by the test. It has also been possible to demonstrate a good correlation between the levels of circulating anti-glomerular basement membrane antibodies and the clinical course of disease in one patient with Goodpasture's syndrome.     

Antigens of the human glomerular basement membrane

Conclusion The intent of the previous discussion has been to acquaint the reader with the recent expanding knowledge in matrix biochemistry and immunopathology as it pertains to the human GBM. However, it must be emphasized that the GBM antigens, which have yet to be characterized, may outnumber those that are currently defined. SDS-PAGE analysis of GBM isolated by detergent solubilization or sonication reveals at least fifteen different polypeptide components [39]. Of a panel of nine monoclonal antibodies developed following immunization of mice with human GBM, eight stain mature and/or fetal GBM by immunofluorescence microscopy, but only two react by ELISA with currently defined components of basement membrane [98]. Furthermore, current understanding of the mechanisms by which matrix macromolecules participate in such human pathological states as diabetic nephropathy, nephrotic syndrome, sclerosis, and immune complex glomerulonephritis is still quite limited. It is obvious that much is yet to be learned and considerably more is to be gained from continued research in this area.     

Antiglomerular basement membrane antibodies in human sera: detection by a modified micro-ELISA

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to human glomerular basement membrane has been developed. Special emphasis has been put on the choice of microtiter plates which were coated with a collagenase digest of human glomerular basement membrane. Results differed markedly between the different microtiter plates. Best results were obtained with a flexible polyvinylchloride microtiter plate with flat wells (Dynatec). This plate exhibited the highest positive/negative ratio and the lowest intraassay standard deviation. Optimal conditions for each step in the ELISA have been determined. The assay proved to be specific, sensitive, and reproducible. Circulating antibodies in each of 11 patients with active antiglomerular basement membrane disease were detected by the ELISA, while sera from patients with various renal and nonrenal diseases were negative by the test.     

Immunological characterization of human glomerular basement membrane antigens.

Normal human glomerular basement membrane (H-GBM) was solubilized by collagenase and subjected to crossed immunoelectrophoresis with rabbit antibodies against H-GBM. Seven precipitates appeared with the mobility of alpha, beta, and gamma globulins. Only two of these precipitates might be specific for GBM, since the other precipitates disappeared after absorption of the antiserum with liver and placenta. In normal human urine one precipitate, cross-reacting with one of the H-GBM precipitates, was found; this precipitate could also be demonstrated in human placenta and liver.     

IgE receptors on human lymphocytes. I. Identification of the molecules binding to monoclonal anti-Fc epsilon receptor antibodies

The present study indicates that the surface molecules from Fc epsilon receptor-bearing human lymphoblastoid cells binding to monoclonal antibodies to Fc epsilon receptor (mAbER) are identical to those binding to IgE. mAbER identify three components in the Nonidet-P40 cell lysate of surface-iodinated RPMI 8866 cells with approximately 65-95 kDa, 45 kDa and 37 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and autoradiography. The same pattern is observed under nonreducing conditions, except for the low mol. wt component which displays an apparent molecular mass 31 kDa. When the labeled and solubilized membranes are adsorbed on IgE-Sepharose the 45-kDa component is always found in the eluate, whereas the 65-95-kDa component is occasionally detected and the low mol. wt component is rarely found. The binding of these molecules to IgE-Sepharose is inhibited by soluble IgE, mAbER but not by an unrelated mAb; reciprocally, the binding to mAbER-Sepharose is prevented by mAbER and to some extent by IgE. To improve the stability of IgE-Fc epsilon R complexes, biotinylated IgE was cross-linked on surface iodinated intact cells, which were then solubilized and adsorbed on avidin-Sepharose. Under these conditions, it was clearly shown that IgE binds to the same three surface molecules as mAbER. The isoelectric point of the high mol. wt component ranged from 4.2 to 4.4 as compared to 5.1-5.2 for the 45-kDa and the 37-kDa components. The observation in the Western blot assay that both the 65-95-kDa and the 45-kDa molecules react with 9 different mAbER indicates that these molecules have a polypeptide sequence in common.     

Transplacental transmission of antibodies to tubular basement membrane in guinea-pigs with autoimmune tubulointerstitial nephritis.

The offspring of female guinea-pigs with tubulo-interstitial nephritis were studied for possible passive transfer of disease. Whereas no immune deposits were seen on or before day 30 of gestation, IgG was detected in the tubular basement membrane (TBM) of fetuses at and after day 44. Serum of offspring contained antibodies to TBM, albeit in much lower titres than found in circulation of the mother guinea-pigs. No histopathological changes were seen in fetal kidneys. Thus, autoantibodies induced by heteroimmunization of pregnant guinea-pigs may be transmitted to offspring in the last third of the gestation period and can bind to fetal TBM. However, this transfer of antibodies does not cause disease.     

Mercuric chloride-induced anti-glomerular basement membrane antibodies in the rat: genetic control.

Mercuric chloride induces anti-glomerular basement membrane antibodies in the Brown-Norway rat. Various other inbred rat strains (Lewis, Wistar AG, August, PVG/c) were not found to be able to produce such antibodies under the same experimental conditions. Hybrids (F1, F2 and F1 x LEW) were bred from Brown-Norway and Lewis rats and injected with mercuric chloride. It has been demonstrated that the induction of anti-glomerular basement membrane antibodies by mercuric chloride in these crosses is under genetic control. The response was found to depend on two or three genes one of which was H-1-linked. The negative results obtained with L.BN congenic rats were in complete agreement with this conclusion.     

Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase

The aim of this study was to evaluate the response of osteoblastic cells to the composite of Ricinus communis polyurethane (RCP) and alkaline phosphatase (ALP) incubated in synthetic body fluid (SBF). RCP pure (RCPp) and RCP blended with ALP 6 mg/mL polymer (RCP+ALP) were incubated in SBF for 17 days. Four groups of RCP were tested: RCPp, RCP+ALP, and RCPp and RCP+ALP incubated in SBF (RCPp/SBF and RCP+ALP/SBF). Stem cells from rat bone marrow were cultured in conditions that allowed osteoblastic differentiation on RCP discs and were evaluated: cell adhesion, culture growth, cell viability, total protein content, ALP activity, and bone-like nodule formation. Data were compared by ANOVA or Kruskal-Wallis test. The group RCP+ALP was highly cytotoxic and, therefore, was not considered here. Cell adhesion (p = 0.14), culture growth (p = 0.39), viability (p = 0.46) and total protein content (p = 0.12) were not affected by either RCP composition or incubation in SBF. ALP activity was affected (p = 0.0001) as follows: RCPp < RCPp/SBF < RCP+ALP/SBF. Bone-like nodule formation was not observed on all evaluated groups. The composite RCP+ALP prior to SBF incubation is cytotoxic and must not be considered as biomaterial, but the incorporation of ALP to the RCP followed by SBF incubation could be a useful alternative to improve the biological properties of the RCP.     

Monoclonal antibody to human basement membrane collagen type IV

We have developed a hybridoma cell line which secretes monoclonal antibody to human basement membrane type IV collagen. The monoclonal antibody secreted by this hybridoma has been obtained in large amounts by either concentrating it from culture supernatants or from the ascites fluid of mice bearing the hybridoma tumour. This monoclonal antibody to type IV collagen does not cross-react with other types of collagens, including types I, III and V, as determined by an enzyme-linked immunosorbent assay (ELISA) and by immunohistochemical staining of corneal and lens sections. Descemet's membrane of mouse, rabbit and human corneal endothelium and lens capsule, both rich in type IV collagen, bind the antibody when stained immunohistochemically. By the indirect precipitation technique, the antibody is found to bind more than three peptides in the basement membrane collagen-rich fraction of human placenta. Based on the observations of other investigators, these peptides are probably derived by proteolysis of the larger polypeptides, since the purification steps in involve extensive pepsin digestion.     

Induction of anti-glomerular basement membrane antibodies in the Brown-Norway rat by mercuric chloride.

Anti-glomerular basement membrane (GBM) antibodies were induced in the Brown-Norway rat by mercuric chloride. The existence of anti-GBM antibodies was suspected because of the immunofluorescent linear pattern. It was proved because eluted antibodies from kidneys and circulating IgG had an anti-glomerular basement membrane activity. A glomerular basement membrane antigen modified by HgCl2 is probably responsible for the appearance of such antibodies. The previous demonstration of the occurrence of an immun complex type glomerulonephitis, in outbred Wistar rats, under the same experimental conditions, suggests the existence of a genetic control for this type of immune response.     

Assessment of invasion in breast lesions using antibodies to basement membrane components and myoepithelial cells.

This paper describes immunostaining of consecutive sections from 15 cases of fibrocystic change of the breast (including 2 examples of intraductal papilloma), 4 ductal carcinomas-in-situ and 17 invasive carcinomas (4 tubular, 1 papillary, 2 lobular and 10 infiltrating ductal, NOS) with antisera to components of the basement membrane (BM), type IV collagen and laminin, and with the muscle antibodies actin and muscle-specific actin. A simple digestion technique was developed to improve the clarity of BM staining with these antibodies. The BM stains facilitated identification of small invasive foci through breaks in the BM in 2 of the cases which had been reported as pure intraductal carcinoma. Tubular carcinomas were surrounded by abnormal, fragmented, and focally discontinuous BM, a feature which could be used to distinguish this well-differentiated breast carcinoma sub-type from sclerosing adenosis, in which individual acini were invariably surrounded by a continuous BM. BM staining emphasized the fibrovascular core of intraductal papillomas, whereas the BM layer was absent in intraductal, cytologically malignant, papillary projections. Similarly, myoepithelial cells, stained with antisera to muscle actins, were identified in a continuous layer surrounding benign epithelial proliferations. These immunohistochemical staining techniques may thus assist the diagnostic histopathologist in differentiating between benign epithelial proliferations of the breast and well-differentiated invasive breast carcinoma, and in identifying foci of microinvasive carcinoma.     

Identification of Goodpasture antigens in human alveolar basement membrane.

Goodpasture (GP) antigens, protein components reactive with human autoantibodies against glomerular basement membrane (GBM), were identified in human alveolar basement membrane (ABM) using an enzyme-linked immunoassay (ELISA), Western blotting and immunoprecipitation. All six anti-GBM antisera studied, three obtained from patients with glomerulonephritis and pulmonary haemorrhages (i.e. GP syndrome), and three from patients with glomerulonephritis alone, distinctively reacted with collagenase-digested (CD) ABM. Very cationic 22-28 kD and 40-48 kD components were detected by blot analysis combined with two-dimensional gel electrophoresis. These proteins showed some similarities to GP antigens in human GBM with respect to the monomer-dimer composition and charge distribution. Inhibition ELISA revealed that the binding of anti-GBM antisera to CDGBM decreased when they were pre-incubated with CDABM, suggesting that the anti-GBM antisera recognized the same epitope(s) on the GBM and ABM. Heterogeneity of the GP antigens in human ABM was demonstrated by blotting; monomeric antigens were absent or at low levels in the CDABM of three out of 10 normal individuals. In immunoprecipitation, anti-GBM antisera from patients with and without pulmonary haemorrhage showed different reactivities with CDABM. The former antisera precipitated both monomeric and dimeric components, but the latter did not. The observations of variation in monomer-dimer composition of ABM, and the different binding of anti-GBM antisera to it may explain why only some patients with anti-GBM nephritis have lung involvement.