Epitopic targets for autoantibodies in systemic lupus erythematosus and Sjögren's syndrome.
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Autoantibodies↗
Biomedical subjects
E K Chan
Publications and source records attributed to E K Chan.
The anatomic relationship of the insertion of the superior lateral pterygoid muscle to the articular disc in the temporomandibular joint of human cadavers.
Confusion has existed as to whether the major insertion of the superior lateral pterygoid muscle is into the articular disc or the condyle. Five human cadaver joints were studied under the dissecting microscope. This allowed superior lateral pterygoid fibres to be selectively placed under tension to determine their point of insertion and to examine the integrity of the anterior joint capsule. All superior lateral pterygoid fibres gained either direct or indirect insertion to the condyle. A classification for the variation in insertion is suggested. The fibres of the anterior joint capsule extended from the anterior rim of the condyle to the roof of the infratemporal fossa but under the foot of the disc they blended with and became indistinguishable from disc fibres.
Cadaver↗
Macrophage-mediated islet cell cytotoxicity in BB rats.
Islet cell killing mediated by natural killer cells and T-lymphocytes in diabetes-prone (DP) and diabetic BB rats has been described, but other killing mechanisms may also be involved. Histopathologic studies suggest that macrophages are the first immune cells to infiltrate islets. To determine if macrophages are the first cells mediating islet damage, macrophage-mediated cytotoxicity was evaluated in BB rats of different ages. Splenic macrophages isolated from DP rats at 33, 100, 120, and 140 days of age showed no enhanced islet killing compared with diabetes-resistant rats. Killing at diabetes onset (121 +/- 14 days) was markedly increased (43 +/- 9.3%) compared with age-matched diabetes-resistant controls (19 +/- 8.3%, P less than .001). Islet inflammation was monitored at all time points. At 120 and 140 days of age, 9 of 11 (82%) DP rats had insulitis, and cytotoxicity was increased in 6 of 11 (55%) rats, which is similar to the number of DP rats that progress to diabetes. At 100 days, 3 of 6 (50%) DP rats again showed diabetic levels of killing, even in the absence of insulitis. These data indicate that 1) islet inflammation is dissociated from clinical diabetes onset, 2) splenic macrophages may have islet-killing potential before islet inflammation, 3) macrophage-mediated islet killing is elevated in all animals immediately after diabetes onset, and 4) macrophages, in addition to natural killer cells and T-lymphocytes, are responsible for cell-mediated islet destruction and thus are candidates for the first cellular effector to result in islet killing.
Aging↗
A 52-kD protein is a novel component of the SS-A/Ro antigenic particle.
Anti-SS-A/Ro autoantibodies are found in the sera of patients with Sjogren's syndrome (SS) and SLE. In the course of analyzing 61 SS patients for their autoantibody profiles, we found that 42 were positive for anti-SS-A by double diffusion in agarose and demonstrated precipitin lines identical to that produced by a prototype anti-SS-A serum. Further analysis of these SS-A antibody-positive sera by Western blotting of cell extracts revealed that 21 sera reacted with two proteins of 60 and 52 kD, 13 sera reacted with 52-kD protein, two detected only 60 kD, while six were nonreactive. Affinity-purified anti-60-kD and anti-52-kD antibodies reacted exclusively with their corresponding antigens. Partial proteolysis of these proteins did not reveal common degradation fragments. Thus the 52- and 60-kD proteins were found to be antigenically and apparently structurally distinct from each other. They were also distinct from 48-kD SS-B/La protein. In immunoprecipitation using labeled cell extracts, affinity-purified anti-52-kD antibodies brought down the 52-kD protein as well as the 60-kD band. In [32P]orthophosphate-labeled HeLa cell extract both antibodies precipitated the same spectrum of small RNAs (hYl-5). In indirect immunofluorescence, anti-52-kD and anti-60-kD antibodies immunolocalized in similar subcellular structures and showed similar punctate nuclear staining patterns. Western blot analysis revealed that both proteins were present in lymphocytic as well as epithelial human cell lines tested. The data above define a new antigen of 52 kD which is another component of the SS-A particle and is associated in complex formation with the previously reported 60-kD protein.
Autoantibodies↗
Antinuclear antibodies (ANAs): diagnostically specific immune markers and clues toward the understanding of systemic autoimmunity.
The convergence of studies in the clinical and basic sciences has resulted in the definitive identification of many intracellular antigens which are the targets of autoantibodies in patients with systemic lupus erythematosus, scleroderma, dermatomyositis/polymyositis, Sjogren's syndrome, mixed connective tissue disease, and drug-induced autoimmunity. Some of this new knowledge includes the identification of the Sm and RNP antigens as ribonucleoprotein particles involved in splicing of precursor messenger RNA, Scl-70 as DNA topoisomerase I, proliferating cell nuclear antigen as auxiliary protein of DNA polymerase delta, and certain antigens in myositis as aminoacyl transfer RNA synthetases. This information confirms, at a molecular level, the presence of specific profiles of autoimmune responses so that autoantibodies can be used in clinical medicine as diagnostically useful immune markers. In addition the data give compelling reasons to consider that certain autoimmune diseases are antigen-driven. Many auto-antibodies have the interesting feature of recognizing epitopes on the antigens which are active or functional sites of the molecule. It is suggested that the data provide clues to the nature of the intracellular particle initiating the immune response and may help to elucidate some of the early mechanisms of the autoimmune process.
Antibodies, Antinuclear↗
Human autoantibody-reactive epitopes of SS-B/La are highly conserved in comparison with epitopes recognized by murine monoclonal antibodies.
SS-B/La, an ubiquitous nuclear protein of 46-48 kD, is a target antigen of autoantibodies in SLE and Sjogren's syndrome and is involved in the maturation of RNA polymerase III transcripts such as 5S RNA and tRNAs. We have previously shown (14, 15) that SS-B consists of two protease-resistant domains of 23 and 28 kD, with the latter containing the RNA binding site. The epitopes of SS-B/La reactive with human autoantibodies are conserved among several mammalian species examined. BALB/c mice immunized with affinity-purified calf thymus SS-B produce IgG anti-SS-B/La antibodies, which reacted with bovine, human, and rabbit SS-B but not with mouse SS-B/La. The spleen of a mouse with the highest antibody titer was selected for fusion with P3 myeloma. Five IgG1k mAbs (A1-5) were selected by ELISA and immunoblotting. All except A3 reacted with the 28-kD domain. A1, A2, and A3 were capable of immuno-precipitating the 48-kD SS-B protein and its associated RNAs. A1, A2, and A3 also gave fine nuclear speckled staining on human, monkey, bovine, and rabbit cells that was similar in appearance to that with human autoantibodies, but in contrast to staining with human autoantibodies, they did not stain cells from rat, mouse, or rat kangaroo. It appears that human autoantibodies target highly conserved epitopes that can be distinguished from epitopes recognized by immunization-induced murine mAbs. Taken together with other data, it appears that human autoantibodies may be recognizing epitopes that are active or catalytic sites of molecules subserving important cellular functions.
Antibodies, Monoclonal↗
An immunoassay differentiating sera with antibodies to Sm alone, antibodies to Sm/RNP complex, and antibodies to RNP alone.
Combined DEAE-Sephacel and hydroxyapatite chromatography resulted in a separation of free Sm antigen from the Sm/RNP complex in rabbit thymus extracts. In Western blots, the free Sm preparation contained an immunoreactive 14-kd (D) protein, whereas the Sm/RNP complex contained, in addition to the 14-kd protein, a 68-kd-reactive protein and its putative degradation fragments. Ethidium bromide staining of these preparations separated by agarose gel electrophoresis showed that the Sm/RNP preparation contained RNA, but the free Sm preparation did not. These preparations were used as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera characterized as anti-Sm only, anti-Sm/RNP, and anti-RNP only were assayed. A quotient (Q) was derived from the ELISA optical density obtained with Sm/RNP as antigen divided by the optical density obtained with free Sm as antigen. Q values less than 4.0 characterized sera with anti-Sm only, values of 4.0-12.0 were observed in sera with anti-Sm plus anti-RNP, and values greater than 12.0 in sera with anti-RNP only. Sera with antibodies to other nuclear antigens were not reactive in this system. It has traditionally been difficult to identify sera with anti-RNP when this is present simultaneously with, and in lower concentration than, anti-Sm. With this method, such sera can be identified by their Q value, which falls in an intermediate region between a lower Q for anti-Sm and a higher Q for anti-RNP.
Autoantibodies↗
Monoclonal autoantibodies to nuclear antigens from murine graft-versus-host disease.
Systemic lupus erythematosus-like graft-versus-host (GVH) disease was induced in 10-week-old male (C57BL/10 X DBA/2) F1 mice by the intravenous injection of spleen and thymus cells (2:1) from 10-week-old male DBA/2 mice. GVH mice were bled at regular intervals 1 month after injection. Antibody to nuclear antigens (ANA) were detected by immunofluorescence using HEp-2 cells as substrate, and antibody to histones and DNA were detected by enzyme-linked immunosorbent assay (ELISA). The titer and frequency of ANA were found to relate directly to the number of donor cells injected. In order to determine the spectrum of ANA in GVH disease, mice were reinjected with optimum cell numbers (120 X 10(6], and splenocytes from two mice with high titer ANA were fused to mouse myeloma cell line P3/X63Ag8.653. Hybridomas were analyzed for ANA by immunofluorescence and ELISA. Sixty-eight clones were found which secreted ANA. Of these, 59% produced antibody to double-stranded DNA, single-stranded DNA, and/or histones and the remainder gave a variety of nuclear immunofluorescence patterns including speckled, homogeneous, nuclear matrix, and nucleolar. This study indicates that GVH disease provides an excellent source of splenocytes for the production of ANA-producing hybridomas as well as a model for the study of autoimmunity.
Animals↗
The small nuclear ribonucleoprotein SS-B/La binds RNA with a conserved protease-resistant domain of 28 kilodaltons.
SS-B/La is a nuclear protein of 48 kilodaltons with two structural domains of Mr 28,000 and Mr 23,000 generated by proteolytic cleavage. UV irradiation was used to cross-link preexisting intracellular La-RNA complexes. Subsequent protease digestion and diagonal gel electrophoresis showed that the RNA-binding site resided in the nonphosphorylated, methionine-rich 28-kilodalton domain.
Autoantigens↗
Synergistic lipid-lowering effects of alfalfa meal as an adjuvant to the partial ileal bypass operation.
Partial ileal bypass (PIB) and ingestion of alfalfa are both known to lower plasma cholesterol (C) levels. During an 18-week period, the combined effects of both were studied in four randomized groups of rabbits receiving C-free, hypercholesterolemia-inducing, semisynthetic diets 3 weeks after sham or PIB surgery. The diets, with or without alfalfa, had similar overall compositions of fat, protein, carbohydrate, and fiber. We measured blood C, triglycerides (T), and lipoprotein fractions of both C and T at biweekly intervals. In vivo liver and small-bowel synthesis of C, fatty acids (FA), and nonsaponifiable lipids (NSL) were determined with radioactive 14C-acetate at the end of the study. The results were evaluated by means of analysis of variance using unweighted cell means. The combined PIB and alfalfa modalities significantly lower C levels in serum, plasma, low-density lipoproteins, and high-density lipoproteins by 66%, 71%, 85%, and 35%, respectively. However, due to alfalfa, a significant increase of 49% was observed in plasma T when both treatments were combined. Liver FA synthesis was significantly decreased (65%) with PIB and increased (161%) with alfalfa; when the two treatments are combined, a nonsignificant response was observed. Similarly, this inverse relationship for PIB and alfalfa was seen for C and NSL synthesis. Small-bowel FA synthesis was significantly decreased (72%) by the combination of PIB and alfalfa. We conclude that alfalfa suppresses, in part, the physiologic rebound effect of PIB surgery by increasing hepatic C and NSL synthesis; inversely, PIB surgery inhibits the additive effect in the liver synthesis of FA produced by alfalfa. Alfalfa and PIB alone, and synergistically, decrease total small-bowel lipid synthesis, specifically that of FA. Alfalfa is an effective adjuvant to PIB for reducing total and lipoprotein C fractions.
Analysis of Variance↗
IgG subclasses of autoantibodies in systemic lupus erythematosus, Sjogren's syndrome, and drug-induced autoimmunity.
The IgG subclasses displayed by autoantibodies were examined in patients with systemic rheumatic diseases. Solid-phase assays performed with purified antigens were combined with a set of four mouse monoclonal antibodies specific for each human subclass to provide quantitative data for all the major autoantibody specificities. IgG1 accounted for an average of 55% of the total antibody activity to native and denatured DNA, Sm antigen, and histone and constituted significantly more anti-SS-B and anti-nRNP (84% and 92%, respectively). The remaining antibody activity consisted largely of IgG3, and this subclass was particularly prominent with anti-histone and anti-Sm in patients with systemic lupus erythematosus. In contrast, IgG2 constituted 3 to 12% of the anti-native and anti-denatured DNA and less than 5% of the anti-SS-B/La activity in only three patients with Sjogren's syndrome. IgG2 was essentially undetectable in antibodies to Sm and RNP antigens. IgG4 was also uncommon, although this isotype was significantly more prevalent in anti-histone from patients treated with procainamide showed that the isotype distribution of anti-histone and anti-denatured DNA remained remarkably constant. However, during periods of large increases in autoantibody activity, a shift from predominantly IgG3 to predominantly IgG1 occurred, consistent with the interpretation that there might be a sequential activation of heavy chain constant regions as the immune response matures. The disproportionately high levels of IgG1 and IgG3 displayed by all the autoantibody specificities examined may indicate that a common immunogenic feature of autoantigens or a common control mechanism underlies the regulation of autoantibody expression.
Antibodies, Monoclonal↗
Antibodies against small nuclear ribonucleoproteins immunoprecipitate complexes containing ts110 Moloney murine sarcoma virus genomic and messenger RNAs.
Small nuclear ribonucleoproteins (snRNPs) are believed to play a role in processing premessenger RNAs. In this study, snRNPs were immunoprecipitated from extracts of cells infected with ts110 Moloney murine sarcoma virus (ts110 MoMuSV). Both the unspliced 4.0 kb and the spliced 3.5-kb ts110 MoMuSV specific RNA species were found in the immunoprecipitates obtained with monoclonal antibody anti-Sm and polyclonal anti-Sm, anti-(U1) RNP and anti-La sera. Although only a portion of the total ts110 RNAs was present in these immunoprecipitates, immune recognition by the anti-snRNPs was specific and not due to contaminating anti-RNA (at least for the anti-Sm sera) or, to anti-viral protein activities. Genomic 8.3-kb RNA and subgenomic 3.0-kb spliced env mRNA from Moloney murine leukemia virus (MoMuLV) infected cells as well as the cellular actin mRNA were also detected in immunoprecipitates obtained with the same antisera. The fact that pre-mRNAs and mature mRNAs of different origin can be recovered from immunoprecipitates formed with anti-snRNP sera establishes their tight association and confirms the role of snRNPs in mRNA processing.
Actins↗
Epitopes, structural domains, and asymmetry of amino acid residues in SS-B/La nuclear protein.
SS-B/La is a conserved cellular phosphoprotein of 46 to 48 KD that is the target antigen of autoantibodies in sera of patients with Sjogren's syndrome and systemic lupus erythematosus. SS-B/La is also known to be associated with certain small cellular and viral RNA, including adenovirus VAI and VAII RNA. Two relatively protease-resistant domains (X and Y) were defined in SS-B from HeLa cells by using human autoantibodies as reagents. Domain X, a methionine-containing nonphosphorylated 28 KD polypeptide, was found to be resistant to partial digestion with six different proteases. Similar domains were also found in calf and rabbit SS-B. Domain Y, a 23 KD polypeptide, was detected after limited digestion with S. aureus V8 and trypsin. This domain contained little if any methionine, but all the detectable phosphorylated amino acids. Among 16 anti-SS-B sera tested by immunoblotting, 11 (69%) were reactive with both domains, three (19%) only with domain X, and two (13%) only with domain Y. These results showed that there are at least two distinct antigenic epitopes on the 46 to 48 KD SS-B/La protein, each located on a separate structural domain. The asymmetric distribution of methionine and phosphorylated amino acid residues in SS-B/La show striking similarity to the two reported domains of the adenovirus 72 KD DNA-binding protein, and raises questions concerning functional similarities that await investigation.
Antigens↗
Effect of cationic proteins on the glomerular deposition of anionic proteins and immune complexes.
When 3 mg of cationized human IgG (cIgG, pI greater than 9) was injected intravenously into mice and followed 15 min later by 3 mg anionized bovine serum albumin (aBSA, pI = 4), deposits were detected along the capillary loops and in the mesangium. Additional infusion of immunoaffinity-purified rabbit anti-BSA antibodies 1 h later led to their deposition at the same location. Intravenous injection of an equivalent amount of preformed BSA-anti-BSA or aBSA-anti-BSA immune complexes into mice with glomerular planted cIgG gave rise to few deposits. Similarly, the perfusion of preformed aBSA-anti-BSA complexes in isolated rat kidneys with planted cIgG led to granular deposition of both, but the intensity of staining was less than when sequential perfusions were used. Cationic macromolecules bound to the glomerulus may lead to the deposition of anionic macromolecules and anionic immune complexes.
Animals↗
Epitopes and structural domains of a RNA-binding nuclear protein, SS-B/La: similarities with adenovirus DNA-binding protein.
SS-B/La is a conserved cellular phosphoprotein of 46/48 KD molecular weight which is the target antigen of autoantibodies in sera of patients with Sjögren's syndrome. Two relatively protease-resistant domains (X and Y) were defined in the SS-B antigen from HeLa cells. Human autoantibodies to SS-B were used as reagents. Domain X is a methionine-containing, non-phosphorylated 28 KD polypeptide and is resistant to partial digestion with six different proteases. Domain Y is a 23 KD polypeptide which contains little if any methionine, but all the detectable phosphorylated amino acids. These results demonstrate that there are at least two distinct antigenic epitopes on the 46/48 KD SS-B protein, each located on a separate structural domain. The asymmetric distribution of methionine and phosphorylated amino acid residues in SS-B show striking similarity to two reported domains of the adenovirus 72 KD DNA-binding protein, which also have separate methionine and phosphorylated amino acid distributions.
Adenovirus E2 Proteins↗
Characterization and purification of lupus antigen La, and RNA-binding protein.
HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.
Antigens↗
Gastritis after gastric bypass surgery.
To determine the preferable reconstruction of gastrointestinal continuity after gastric bypass, we studied by endoscopic, chemical, and histologic analyses 28 randomly selected patients with a loop gastroenterostomy, a loop gastroenterostomy plus enteroenterostomy between the afferent and efferent loops, and a Roux-en-Y anastomosis. Total bile acid levels for the three groups were: 5092 +/- 1673 mumol/L, 1638 +/- 581 mumol/L, and 404 +/- 384 mumol/L, respectively. The incidence of gastritis by endoscopy was 71% in the standard loop bypass, 45% in the enteroenterostomy group, and 13% in the Roux-en-Y group. Histologic abnormalities were present in 86% of the patients who underwent standard loop bypass, in 91% of those with an additional enteroenterostomy, and in 63% of the Roux-en-Y group. There was poor correlation of symptoms and objective findings. In our study Roux-en-Y reconstruction after gastric bypass, in comparison with loop gastroenterostomy or loop gastroenterostomy with an additional enteroenterostomy, is less likely to result in bile in the stomach, endoscopic changes, and histologic abnormalities.
Adult↗
Suppression of weight gain by glucagon in obese Zucker rats.
Glucagon has been shown to lower blood lipids and to decrease food intake and body weight in short-term studies in man and animals. There is evidence of decreased secretion of glucagon in human obesity. The Zucker obese rat suffers from a genetic type of obesity and has an absolute reduction in circulating glucagon concentration. The effect of long-term administration of glucagon on the body weight in obese Zucker rats was studied. Glucagon caused a marked (-20%) reduction of body weight in obese Zucker rats with no change in feed intake. Urine glucose, urea nitrogen, creatinine, and ketone content, as well as serum triglyceride, cholesterol, alkaline phosphatase, creatinine, and insulin levels remained unchanged. Weights of perirenal fat, kidneys, and heart also remained unchanged. However, glucagon injection in obese Zucker rats caused significant decrease in serum glucose, and increases in SGOT, liver weight, and liver lipid and glycogen content. Further investigations are needed concerning the safety of chronic glucagon administration for weight control.
Adipose Tissue↗