Immunoglobulin G3 subclass production by rheumatoid synovial tissue cultures.

The cellular infiltrate in the deeper layers of the rheumatoid synovium produces a substantial amount of immunoglobulin (Ig)G. Culture supernatants of synovial tissues from 31 patients with rheumatoid arthritis (RA) undergoing joint replacement or synovectomy have been analyzed for the subclass of IgG present. IgG3 was measured by separation with Staphylococcal Protein A chromatography, precipitation with specific anti-IgG3 antibody, and differential separation of IgG3 heavy chains using polyacrylamide gel electrophoresis. IgG from RA synovial cultures contained an average of 41% IgG3 (range, 8-97%) compared with 12% IgG3 (range, 6-17%) in the serum IgG of the same patients. A group of non-RA control lymphoid tissues (four lymph nodes and five tonsils) produced 23% of total IgG as the IgG3 subclass (range, 16-35%). An average of only 9% of the synovial IgG showed aggregation compatible with IgG-rheumatoid factor (IgG-RF). Purified IgG from some of the RA synovial culture supernatants also showed significant restriction when separated by isoelectric focusing. This restriction and the enrichment for the IgG3 subclass in the IgG from RA synovial cultures suggest that either an antigen in the inflamed joint is selectively stimulating an antibody in this subclass, or that significantly differences in the catabolic rate of this subclass are found in cultures of synovial tissue when compared with that occurring in intact patients.

Analysis by two-dimensional polyacrylamide gel electrophoresis of liver ribosomal subunnit proteins obtained from free and membrane-bound polysomes of unfasted animals.

Ribosomal proteins were analyzed by means of two-dimensional gel electrophoresis. To insure that the analysis included only that fraction of the ribosome actively participating in protein synthesis, only polysomal-bound ribosomes were used. This differs from previously reported analyses of liver ribosomal proteins. The ribosomal proteins were prepared from ribosomes of polysomal origin from membrane-bound and free polysomes. Membrane-bound and free liver polysomes were isolated from unfasted mice. The polysomes were purified on hydroxyapatite under conditions known to result in polysomes and ribosomes that are active in both endogenous and synthetic mRNA translation. Moreover, this is the first time that liver ribosomal protein was obtained and analyzed from animals that have not been starved prior to sacrifice. The puromycin-released ribosomes were dissociated into subunits and ribosomal proteins were analyzed by means of two-dimensional polyacrylamide gel electrophoresis. When 100-200 mug samples of the ribosomal subunit proteins were analyzed by two-dimensional electrophoresis, approximately 32 major proteins were detected for the 60 S ribosomal subunit and 25 major proteins for the 40 S ribosomal subunit. A total of 13 "fractional" ribosomal proteins was also detected in the ribosomal subunit profiles. No differences in number or mobility of the ribosomal proteins were found between the membrane-bound and free ribosome populations. We describe a system in which all ribosomal proteins are completely solubilized and quantitatively move from the first to the second dimension gel. Thus the total sample is separated and fractionated. This procedure elimates artifacts due to incomplete solubilization of ribosomal proteins, which is common for the transfer from the first- to second-dimension gel. Therefore, a more detailed and accurate analysis is achieved.

Computed tomographic study of children with classic autism.

Computed tomographic (CT) scans were obtained for nine autistic boys aged between 9 and 16 years. All were considered to have classic childhood autism of unequivocal diagnosis, with symptoms present from infancy, and were functioning in the borderline or normal level of intelligence. They had performed poorly on tests purported to measure left hemispheric functions. There was no sign of abnormality of any kind on the CT scans or any asymmetry that might be related to lateralized cognitive functions. It is suggested that earlier reported abnormalities are a function of the inclusion of patients with a heterogeneous collection of disorders in the tested sample.

Tween 20 removes antibodies and other proteins from nitrocellulose.

It has generally been assumed that the binding of most proteins to nitrocellulose is stable in the non-ionic detergent Tween 20. However, the following paper demonstrates that in immunoassays where antibodies or other proteins are bound directly to the nitrocellulose, 0.05% Tween 20 may dissociate these proteins from the membrane. The degree of dissociation appears to be dependent on the individual protein studied. Some antibodies and other proteins bind tightly to nitrocellulose and dissociation of these proteins by Tween 20 is barely detectable. In contrast, other proteins are nearly completely stripped from the nitrocellulose by the same detergent. Therefore, unless it is known from control experiments what proteins will or will not be dissociated from nitrocellulose by Tween 20, the routine use of Tween 20 in the development of Western blots, native blots and dot-blots should be discontinued.

Rapid blotting of IgG to nitrocellulose with minimal IgM contamination.

A rapid method is described for the non-electrophoretic transfer of IgG from 0.5 mm thick agarose gels to nitrocellulose. Since the agarose gels are attached to a solid support, the blot is unidirectional. However, 90% of the IgG is transferred in 10 min with no visible loss of resolution. In this procedure, less than 2% of the IgM antibodies are transferred from the gel to the nitrocellulose membrane. Therefore, this technique can be used in IgG spectrotype analysis or antigen-specific assays without the prior removal of IgM antibodies. Approximately 5-6 h are needed to run the gels, blot, and develop the protein pattern with double-antibody immunoperoxidase staining.

Site-specific immobilization of antibodies by their oligosaccharide moieties to new hydrazide derivatized solid supports.

This report describes a new method for immobilization of antibodies to solid supports. Antibodies are bound to the solid supports by covalent bonds between aldehydes generated on the carbohydrate side chains of the antibody and hydrazide groups on the solid support. The hydrazone bonds that are formed are stable at least from pH 2-10, permitting the acid elution of antigens from the affinity column. Over 25 mg of affinity-purified rabbit IgG binds per ml of solid support, with most of the bound antibodies retaining biological activity. Advantages of this new affinity support over existing technology are discussed along with procedures for the preparation and use of affinity columns containing monoclonal or polyclonal antibodies.

Characterization of immune complexes by isoelectric focusing in agarose gels.

A method is described for the characterization of immune complexes on thin-layer agarose isoelectric focusing (IEF) gels. This method involves dissociating immune complexes and then maintaining this dissociation during IEF in agarose gels containing 9 M urea. After IEF, the immune complex components can be quantitatively transferred to nitrocellulose in less than 15 min, and a variety of immunostaining procedures can be used to probe these blotted components. No loss of biological activity was detected in any of the blotted components.

Chicken anti-protein A prevents Staphylococcus aureus protein A from binding to human and rabbit IgG in immunoassays and eliminates most false positive results.

This report demonstrates that chicken anti-protein A can prevent both soluble and surface-bound Staphylococcal protein A from binding to either human or rabbit IgG. In an ELISA assay, chicken anti-protein A prevented > 98% of the soluble protein A from binding to the human IgG-Fc coat. In a blotting assay, chicken anti-protein A prevented the membrane-bound protein A from interacting with the human IgG probe. When intact S. aureus (Cowan I strain) was bound to the surface of a microassay plate, chicken anti-protein A blocked > 98% of the cell wall protein A and permitted the probing of the surface components with human IgG. In another immunoassay, rabbit anti-enterotoxin A IgG was used to measure enterotoxin A concentrations in S. aureus culture medium supernatants after soluble protein A was blocked by chicken anti-protein A. Thus, the binding of chicken anti-protein A to protein A almost completely eliminates false positive results and permits the measurement of specific antibodies or antigens in a variety immunoassays where protein A is present.

Photodynamic therapy of actinic keratosis with topical 5-aminolevulinic acid. A pilot dose-ranging study.

OBJECTIVE: To examine the safety and efficacy of photodynamic therapy using topical 5-aminolevulinic acid (ALA) and red light to treat actinic keratoses (AKs). DESIGN: Actinic keratoses were treated with topical ALA (concentrations of 0%, 10%, 20%, or 30%) under occlusion for 3 hours. Before photodynamic therapy, sites were examined for fluorescence. Sites were irradiated with an argon pumped dye laser (630 nm) at fluences of 10 to 150 J/cm2. SETTING: Academic medical center. PATIENTS: Forty patients with 6 clinically typical, previously untreated AKs per patient. MAIN OUTCOME MEASURE: Complete resolution and decrease in lesion area of the AK relative to baseline evaluated at weeks 1, 4, 8, and 16. RESULTS: Three hours after ALA administration, lesions showed moderate red fluorescence. Cutaneous phototoxic effects, localized erythema and edema, peaked at 72 hours. Patients experienced mild burning and stinging during light exposure. Eight weeks after a single treatment using 30% ALA, there was total clearing of 91% of lesions on the face and scalp and 45% of lesions, on the trunk and extremities. No significant differences were observed in clinical responses with treatment using 10%, 20%, or 30% ALA. All concentrations of ALA were more effective than treating AKs with vehicle and light. CONCLUSIONS: Topical photodynamic therapy with ALA is an effective treatment of typical AKs. Complete clearing of nonhypertrophic AKs can be achieved with 10%, 20%, or 30% ALA that is easily tolerated by the patient. Lesions on the face and scalp are more effectively treated than lesions on the trunk and extremities. Hypertrophic AKs did not respond effectively.

The role of infections in the rheumatic diseases: molecular mimicry between bacterial and human stress proteins?

Infections can cause or exacerbate the rheumatic diseases in several ways, including immune cross-reactivity between bacterial heat shock proteins and similar proteins in normal human tissues. This may lead to autoimmunity in rheumatoid arthritis and systemic lupus. In addition, increased activation of the gene regulating the synthesis of a heat shock protein has been found in scleroderma fibroblasts. As an infection-induced model for other rheumatic diseases, rheumatic fever (RF), with its well-established link to prior group A streptococcal infection, will be revisited. The lessons learned from RF and other rheumatic diseases directly linked to infection will be applied to ankylosing spondylitis, rheumatoid arthritis, Sjogren's syndrome and polymyositis, for which a mounting body of circumstantial evidence suggests a probable infectious cause. The interplay of genetic susceptibility and infection with particular organisms and the implications of this new information for present and future therapy of the rheumatic diseases will also be presented.

Ribosome-membrane interactions: characterization of ribosomal proteins from loose and tight bound ribosomes.

Membrane-bound ribosomes were isolated from a post-mitochondrial supernatant fraction of mouse liver homogenate by sedimentation in a sucrose density gradient, Loose ribosomes were released from the membrane fragments with 0.5 M KCl, while tight bound ribosomes were not released. After purification of the loose and tight ribosomes subclasses, ribosomal subunit proteins were isolated and compared by two-dimensional polyacrylamide gel electrophoresis. No differences in the ribosomal protein composition was detected.

Staphylococcus aureus nasal carriage in rheumatoid arthritis: antibody response to toxic shock syndrome toxin-1.

OBJECTIVE: To determine the prevalence of Staphylococcus aureus nasal carriage and to compare antibody responses to two superantigens, staphylococcal toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA), in rheumatoid arthritis patients and normal subjects. METHODS: 88 rheumatoid arthritis patients and 110 control subjects were cultured for nasal carriage of S aureus; 62 isolates were bacteriophage typed. Twenty five patients and 11 spouses were tested for antibodies to TSST-1, SEA, and sonicate extracts of Bacteroides fragilis and Escherichia coli; 19 patients were HLA-DR typed. RESULTS: 50% of patients and 33% of normal subjects were S aureus carriers. Bacteriophage typing of isolates suggested significant differences between strains isolated from the two groups. Patients showed higher IgG (P = 0.0025) and IgA (P = 0.0372) antibody levels to TSST-1 than normal spouses and these responses were not related to rheumatoid factor titres or HLA-DR type. CONCLUSION: When compared to normals, rheumatoid arthritis patients more often carry S aureus in their nasal vestibule, carry a distinct subpopulation of S aureus strains, and have higher average antibody levels to TSST-1.

Protein synthesis in rheumatoid synovial tissue.

1. Function of synoviocytes and other cells in the synovium A. Histologic Considerations 1. Electron microscopic studies 2. In vivo and in vitro phagocytosis studies 3. Fluorescent antibody staining B. Culture techniques 1. Problems posed by study of isolated cells 2. Long-term explant cultures 3. Advantages of short-term incubations of synovial fragments 4. Isolation of immunoglobulins C. Non-Immunoglobulin Products of the Synovium 1. Products of normal synovium 2. Alterations induced by rheumatoid arthritis II. The Local Immune response in Rheumatoid Synovitis A. Evidence for Active Immune Stimulation 1. Meditators of cellular immunity in synovial fluid 2. Effect of synovectomy 3. Type and amount of immunoglobulin produced B. Local Commitment of Antibody Response 1. Effect of exogenous immunization 2. Rheumatoid factors. 3. Pepsin agglutinators C. 1. Relative enrichment for IgG-3 subclass 2. Increase in lambda-light chain composition III. Pathogenetic Considerations in Rheumatoid Arthritis A. Comparison of Rheumatoid versus Experimental Immune Synovitis 1. Chronic synovitis as a local immune response. 2. Role of cartilage complexes in substaining chronic synovitis B. Significance of the Restriction in the Immunoglobulin Response in Rheumatoid Arthritis 1. Analogy with other disease states in man 2. Common antigen in RA?

Tissue-specific ribosomal protein composition.

Membrane-bound and free polysomes from murine liver and kidney were isolated under identical conditions and their ribosomal proteins were compared by two-dimensional gel electrophoresis. The results demonstrate that these ribosome subpopulations are quantitatively and qualitatively similar except for the presence of one additional protein in the kidney-bound polysomal fraction.

Analysis by two-dimensional polyacrylamide gel electrophoresis of the in vivo phosphorylation of ribosomal proteins derived from free and membrane-bound polysomes.

Analysis of in vivo phosphorylation of mouse liver ribosomal proteins was performed by two-dimensional polyacrylamide gel electrophoresis following 32P-injection. Our method is special and differs from other eukaryotic systems reported in that all proteins separated on the first dimension gel are completely solubilized, moving quantitatively to the second dimension gel. Only ribosomes from polysomes were used, ensuring analysis of ribosomes actively engaged in protein synthesis. We resolved sixty-five distinct proteins from ribosomes from membrane bound or free polysomes. In both cases radioautography revealed similar labeled patterns with one highly phosphorylated ribosomal protein and five marginally labeled spots.

Inhibition of the streptavidin-biotin interaction by milk.

Nonfat dried milk is routinely used as a blocking agent and diluent in immunoassays. However, the data presented in this paper demonstrate that milk contains an inhibitor of the biotin-streptavidin interaction. It is recommended that milk be dialyzed or used at lower concentrations when it is employed as a diluent of streptavidin.

Site-directed immobilization of glycoproteins on hydrazide-containing solid supports.

Methods are described for the preparation and use of solid supports containing hydrazide functions for the immobilization of glycoproteins specifically through the oligosaccharide moieties. The solid supports are prepared from commercial "active ester" agarose by reaction with hydrazine hydrate. Glycoproteins are oxidized with sodium periodate, resulting in the production of aldehydes on the oligosaccharide moieties. Oxidized glycoprotein is then reacted with the hydrazide-derivatized solid support to produce stable hydrazone linkages. Data are presented for the optimization of binding of oxidized glycoprotein to hydrazide-derivatized agarose. Agarose hydrazide/glycoprotein gels were shown to be stable from pH 3 to 10 and activity studies using immobilized avidin show that this method of immobilization results in an increased "specific activity" of bound protein when compared with standard methods of immobilization.