Anti-Ro (SS-A) antibodies from Ro (SS-A)-immunized mice.

Immunization of BALB/c mice with immunoaffinity-purified bovine or human Ro (SS-A) induces the production of antibodies reactive with Ro (SS-A). Fusion of spleen cells from the hyperimmunized mice to SP2/0 cells resulted in hybridoma cell lines that produced anti-Ro (SS-A) antibodies. Anti-Ro (SS-A) binding was established by solid-phase immunosorbent assay, immunoblotting, or RNA immunoprecipitation. Most of the anti-Ro (SS-A) antibodies bound to both human and bovine Ro (SS-A) in the solid phase, but only one of the monoclonal antibodies selectively bound to human Ro (SS-A); this suggests that there are species differences between the bovine and human Ro (SS-A) antigens. Indirect immunofluorescence studies demonstrated that most anti-Ro (SS-A) antibodies bound to cytoplasmic or nuclear HEp-2 cellular antigens, whereas others did not bind to fixed HEp-2 tissue culture cells. Nuclear staining of mouse substrates by one of the sera containing anti-Ro (SS-A) demonstrated that autoantibodies were induced by immunization with human Ro (SS-A).

Implications of anti-Ro/Sjögren's syndrome A antigen autoantibody in normal sera for autoimmunity.

We have applied a sensitive assay to analyze lupus and Sjögren's syndrome autoantibodies in 40 normal sera. Seven of these bound Ro/Sjögren's syndrome A antigen (SSA). Although this binding was 1,000-fold lower than the highest anti-Ro/SSA level measured from patients, it was inhibited by human Ro/SSA. Positive normal serum-bound Ro/SSA in Western immunoblots and binding activity was demonstrated in the F(ab')2 fragment of IgG. Affinity purification of normal anti-Ro/SSA IgG increased the specific anti-Ro/SSA binding by greater than 17-fold. This purified antibody formed a Ro/SSA precipitin and had a relative affinity for Ro/SSA identical to that of Ro/SSA precipitin-positive patients. These data demonstrate that the anti-Ro/SSA present in healthy normal donors is true autoantibody. Anti-La/Sjögren's syndrome B antigen (SSB) autoantibodies were found in 3 of the 40 normal sera, while none bound nuclear ribonucleoprotein (Sm). Finding low levels of anti-Ro/SSA and anti-La/SSB among normals may indicate that anti-Ro/SSA and anti-La/SSB occur in disease by enhancement of a preexisting immune response.

The Ro/SSA autoantigen as an immunogen. Some anti-Ro/SSA antibody binds IgG.

The rheumatic disease autoantigen, Ro/SSA, was immunogenic to a rabbit host. The heteroimmune rabbit serum bound the Ro/SSA particle in immunoblots and in an ELISA. Both the rabbit anti-Ro/SSA and a human prototype anti-Ro/SSA serum also bound IgG; and moreover, IgG inhibited both rabbit and human anti-Ro/SSA activity. Anti-IgG activity of the rabbit and human anti-Ro/SSA sera bound Ro/SSA by Western blot and solid-phase assays. In addition, purified Ro/SSA inhibited the anti-IgG activity of the anti-Ro/SSA sera from rabbit and man. Affinity purification of the IgG- and Ro/SSA-binding fractions of the rabbit anti-Ro/SSA demonstrated that both the anti-Ro/SSA and anti-IgG activities were concentrated in these fractions. These data show that Ro/SSA and IgG share epitopes that are bound by anti-Ro/SSA antibody. Inhibition experiments suggest that this antibody is found in most human anti-Ro/SSA autoimmune sera and that the epitope(s) are found in the F(ab')2 fragment of IgG.

Anti-Ro (SS-A) and anti-La (SS-B) in patients with Sjögren's syndrome.

Clinical, serologic, and genetic findings in Sjögren's syndrome patients were correlated with quantitative determinations for antibody against Ro (SS-A), La (SS-B), and nRNP (Sm) using newly developed, sensitive solid-phase assays. In 86 Sjögren's syndrome patient sera, more than 96% had anti-Ro (SS-A), and 87% had anti-La (SS-B), spanning a 4.8 log10 range of autoantibody concentration, whereas only 95% of the patients had anti-nRNP (Sm). Low levels of anti-Ro (SS-A) and anti-La (SS-B) were found in 10% and 12.5%, respectively, of the 40 normal control sera. In Sjögren's syndrome patients, the level of anti-Ro (SS-A) correlated strongly with that of anti-La (SS-B) (r = 0.80; P less than 0.0001) but not with the level of anti-nRNP (Sm). We found much higher levels of anti-Ro (SS-A) and anti-La (SS-B) in patients with purpura, leukopenia, lymphopenia, and increased polyclonal gamma globulins than in those without these conditions (between 4.3-fold and 17-fold higher; P less than 0.001 to P less than 0.05). Anti-Ro (SS-A) and anti-La (SS-B) levels correlated with the rheumatoid factor titer and with the concentrations of total globulin, IgG, and IgA, but not with the IgM concentration. The association of rheumatoid factor titer with levels of anti-Ro (SS-A) and anti-La (SS-B) occurred only in patients with primary Sjögren's syndrome. Antinuclear antibody titers correlated with levels of anti-Ro (SS-A) and anti-nRNP (Sm). HLA-DR3-positive patients had higher levels of anti-Ro (SS-A) and anti-La (SS-B).

Ro (SS-A) antibody and antigen in a patient with congenital complete heart block.

Congenital complete heart block is closely associated with the presence of anti-Ro (SS-A) autoantibodies. Quantitative solid-phase assays for Ro (SS-A) autoantigen and autoantibody have established the presence of Ro (SS-A) in cardiac tissues and have been used to evaluate an informative pedigree. The propositus we describe here had complete congenital heart block and showed anti-Ro (SS-A) binding of 13-fold less than his normal HLA-identical twin sister. Both had identical titers of antinuclear antibody. These data support the hypothesis that anti-Ro (SS-A) may be directly involved in the pathogenesis of congenital complete heart block.

Immunologic and structural studies of the lupus/Sjögren's syndrome autoantigen, La/SSB, with a monoclonal antibody.

La/SSB is a small nuclear RNA protein against which precipitating autoantibodies are made in many patients with systemic lupus erythematosus or Sjögren's syndrome. The recent purification of La/SSB has made structural and immunologic studies possible. Consequently, a mouse hybridoma antibody (La1) was raised, after immunization and fusion, that reacted with bovine La/SSB. Results of inhibition tests with tissue extracts and fluorescent antinuclear antibody tests demonstrated that La1 reacted with bovine extracts and cells, but not with those from human, mouse, or rabbit sources. La1 reacted in Western blot and in an adapted anti-La/SSB enzyme-linked immunosorbent assay with only the 41-kD bovine La/SSB peptide and not with the smaller 29-kD bovine La/SSB peptide. RNA gels showed that La1 bound the La/SSB particle that contained the predominant La/SSB RNA species near 90 nucleotides as well as the minor RNA species, both of which were bound by the human autoimmune anti-La/SSB serum. A solid-phase assay for human autoimmune anti-La/SSB antibody using La1 was more sensitive for the detection of human anti-La/SSB than was a comparable assay using purified La/SSB, and showed that anti-La/SSB is present in nearly all Ro/SSA precipitin-positive sera. Thus, this study demonstrates that monoclonal antibody can be raised against La/SSB; that the protein moiety of bovine La/SSB differs from human, mouse, and rabbit at an epitope on the 41-kD La/SSB peptide; that the RNA bound to the La1-reactive particle was as heterogeneous as that binding the anti-La/SSB autoimmune serum; and that anti-Ro/SSA and anti-La/SSB are closely associated.

Changes in the relative distribution and properties of smooth endoplasmic reticulum subfractions isolated from the livers of rats fed 2-acetylaminofluorene.

A marked increase in the amount and relative proportion of fraction II of the smooth endoplasmic reticulum (SER II) and a concomitant decrease in the SER I fraction were observed in the liver of male Sprague-Dawley rats fed diets containing 0.05% (w/w) 2-acetylaminofluorene (AAF) for various lengths of time (3-18 weeks). The amount of rough endoplasmic reticulum (RER), which remained at control levels after 3 weeks of AAF feeding, i.e. before the appearance of hyperplastic nodules (HPN), was clearly decreased after 18 weeks of continuous AAF-feeding (HPN present). When the rats were subjected to 4 or 5 cycles of interrupted AAF feeding, similar increases in SER II were also observed both in the homogenates of HPN-containing livers, as well as in the homogenates of HPN dissected out from the surrounding liver tissue. SER II, the predominant microsomal membrane fraction in the livers of rats fed AAF for 3 weeks, showed the highest level of induction of epoxide hydrolase. A marked elevation of the manganese-dependent enhancement of polysome binding in vitro was also observed in SER II from the livers of AAF-fed rats.

Sialic acid metabolism in rats undergoing chemically induced hepatocarcinogenesis in specific dietary states.

The sialic acid content and the activities of the enzymes responsible for the activation, transfer, and hydrolysis of sialic acid were determined in the livers of male Sprague-Dawley rats maintained on diets containing the hepatocarcinogen 2-acetylaminofluorene (AAF). The incidence of hyperplastic lesions was modulated by dietary fat and by the presence of a synthetic antioxidant. The purified diets utilized differed in the amount and degree of unsaturation of the lipid component; they contained either 20% corn oil, 18% coconut oil + 2% linoleic acid, or only 2% linoleic acid, and each diet was prepared either with or without 0.3% butylated hydroxytoluene (BHT). The supplementation of diets with BHT greatly retarded the development of hyperplastic nodules compared to unsupplemented diets. The AAF-treated rats and age-matched controls fed AAF-free diets were killed after a 12 to 17-week period of dietary treatment, and the livers were removed for histological and biochemical studies. Significant increases in sialic acid content, and in the activities of CMP-sialic acid synthetase and neuraminidase were observed in the livers from AAF-treated rats. The BHT supplementation of the AAF-containing diets resulted in livers with lower levels of sialic acid and CMP-sialic acid synthetase activities. The chemical and enzymatic changes observed in AAF-treated rats are consistent with an increased turnover of sialoglycoconjugates during the carcinogenic process.

Effects of dietary fat and alpha-tocopherol on gamma-glutamyltranspeptidase activity of 7, 12-dimethylbenz(alpha)anthracene-induced mammary gland adenocarcinomas.

The levels of gamma-glutamyltranspeptidase (GGTP) (EC 2.3.2.2) were measured in 7, 12-dimethylbenz(alpha)anthracene-induced mammary adenocarcinomas, in control mammary gland tissue and in sera from tumor-bearing and control rats. The carcinogenic process was modulated by diets differing in the type and amount of fat with and without alpha-tocopherol supplementation. Rat mammary adenocarcinomas showed significantly elevated (up to 25-fold) levels of GGTP when compared with adjacent histologically-uninvolved tissue or with mammary tissue of control rats. GGTP activity in sera from tumor-bearing rats was also elevated up to 4-fold than in the corresponding controls. Histochemical studies of the frozen section of mammary adenocarcinomas indicated that GGTP was localized in neoplastic ductal epithelial cells. In tumor rats on alpha-tocopherol supplemented diets, GGTP activity in the adenocarcinomas was mainly in the particulate (membrane-bound) fraction. In contrast, the tumor rats receiving alpha-tocopherol-deficient diets, the total GGTP activity was distributed in both particulate and cytosolic fractions, suggesting an altered membrane-GGTP interaction. The levels of GGTP in control mammary gland and sera of control rats from the low fat dietary groups were up to 7-fold higher than the corresponding control values in either of two high-fat groups. These high levels of GGTP in the serum and tissues of animals from the low fat dietary group are consistent with lower tumor incidence through enhanced carcinogen detoxification.

Sialic acid metabolism in rats undergoing chemically-induced mammary gland carcinogenesis in specific dietary states.

The enzymes responsible for the activation, transfer and hydrolysis of sialic acids were investigated in female rats with mammary adenocarcinomas induced by administration of a single oral dose (10 mg) of 7,12-dimethylbenz[alpha]anthracene. The carcinogenic process was modulated by the levels and degree of unsaturation of the dietary lipids. Tumor incidence was highest in rats fed a diet containing 20% corn oil, intermediate with 18% coconut oil plus 2% linoleic acid, and lowest in the group receiving a diet with 2% linoleic acid. Sialyltransferase and CMP-N-acetylneuraminic acid synthetase activities were higher in tumors than in control mammary glands. Neuraminidase activity, on the other hand, was higher in control tissue than in tumors. In addition to these tumor-related effects, comparison of the enzyme levels in mammary tissues from control animals of the 3 dietary groups revealed the presence of diet-related effects on sialic acid metabolism. In the livers of tumor-bearing rats, only minor changes of enzyme activities were detected.

Isolation and characterization of glycoproteins from canine tracheal mucus.

Three homogeneous glycoproteins were isolated from reduced and S-carboxy-methylated canine tracheal pouch mucus by gel filtration and ion-exchange chromatography. Initial fractionation was carried out on Sephadex G-200; chromatography of the excluded Sephadex G-200 fraction on Bio-Gel A-15 m yielded two high molecular weight glycoprotein fractions. Following rechromatography on the same column, the main fraction behaved as an electrophoretically homogeneous high molecular weight (581 600) glycoprotein, with a high carbohydrate content (80%) and a single amino-terminal amino acid (arginine). Ion-exchange chromatography (DEAE-cellulose) of the included Sephadex G-200 fraction yielded two electrophoretically homogeneous glycoproteins of lower molecular weight (20 800 and 24 600, respectively). A single amino-terminal amino acid, glycine and alanine, respectively, was detected for each glycoprotein. Chemical analysis of these three glycoproteins revealed the presence of fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid and sulfate monoester. The high molecular weight glycoprotein had a higher hexose, sialic acid and sulfate content, per mg of protein, than the low molecular weight glycoproteins. The results of the alkaline borohydride treatment indicated that the majority of the carbohydrate chains of these glycoproteins are linked to the protein core through O-glycosidic bonds involving N-acetylgalactosamine and serine or threonine.