K. Thoss

Attenuation of murine antigen-induced arthritis by treatment with a decoy oligodeoxynucleotide inhibiting signal transducer and activator of transcription-1 (STAT-1)

The transcription factor STAT-1 (signal transducer and activator of transcription-1) plays a pivotal role in the expression of inflammatory gene products involved in the pathogenesis of arthritis such as various cytokines and the CD40/CD40 ligand (CD40/CD40L) receptor-ligand dyad. The therapeutic efficacy of a synthetic decoy oligodeoxynucleotide (ODN) binding and neutralizing STAT-1 was tested in murine antigen-induced arthritis (AIA) as a model for human rheumatoid arthritis (RA). The STAT-1 decoy ODN was injected intra-articularly in methylated bovine serum albumin (mBSA)-immunized mice 4 h before arthritis induction. Arthritis was evaluated by joint swelling measurement and histological evaluation and compared to treatment with mutant control ODN. Serum levels of pro-inflammatory cytokines, mBSA-specific antibodies and auto-antibodies against matrix constituents were assessed by enzyme-linked immunosorbent assay (ELISA). The transcription factor neutralizing efficacy of the STAT-1 decoy ODN was verified in vitro in cultured synoviocytes and macrophages. Single administration of STAT-1 decoy ODN dose-dependently suppressed joint swelling and histological signs of acute and chronic arthritis. Delayed-type hypersensitivity (DTH) reaction, serum levels of interleukin-6 (IL-6) and anti-proteoglycan IgG titres were significantly reduced in STAT-1 decoy ODN-treated mice, whereas mBSA, collagen type I and type II specific immunoglobulins were not significantly affected. Intra-articular administration of an anti-CD40L (anti-CD154) antibody was similarly effective. Electrophoretic mobility shift analysis (EMSA) of nuclear extracts from synoviocytes incubated with the STAT-1 decoy ODN in vitro revealed an inhibitory effect on STAT-1. Furthermore, the STAT-1 decoy ODN inhibited the expression of CD40 mRNA in stimulated macrophages. The beneficial effects of the STAT-1 decoy ODN in experimental arthritis presumably mediated in part by affecting CD40 signalling in macrophages may provide the basis for a novel treatment of human RA.

Influence of cyclosporin A on cytokine levels in synovial fluid and serum of rats with antigen-induced arthritis

The effects of the T-cell-directed immunosuppressant cyclosporin A (CsA) on the developmnnt of antigen-induced arthritis (AIA) in rats as well as on cytokine levels in synovial fluid and serum were determined. The treatment with CsA effectively inhibited the chronic phase of arthritis as demonstrated by decreased joint swelling and reduced histological arthritic score. In animals with AIA the level of IL-6 in the synovial fluid and serum is increased, showing good correlation with the severity of the disease. The CsA treatment reduced IL-6 levels to normal. IL-1, IL-2 and TNF-α do not seem to be directly involved in the pathogenic mechanisms of chronic arthritis.

Immunomodulation of rat antigen-induced arthritis by leflunomide alone and in combination with cyclosporin A

The effects of the new immunomodulating isoxazol derivative leflunomide, in comparison with cyclosporin A, on established antigen-induced arthritis in rats as well as serum antibody levels were determined. When treatment with leflunomide, at concentrations from 2.5 to 10 mg/kg/d, was started on day 3 of arthritis, the acute and chronic phases of arthritis were effectively inhibited. This was demonstrated by decreased joint swelling and reduced histopathological arthritis score at the end of experiment (day 26). Furthermore, the treatment resulted in a significantly reduced level of serum antibodies to the matrix components collagen type I, type II and proteoglycans. Neither leflunomide nor cyclosporin A, at doses of 1 mg/kg/d, had an effect on the severity of arthritis and antibody levels. However, when both drugs were used together, at these non-effective doses, the histopathological score of chronic arthritis was significantly reduced. The results of our experiments demonstrate that leflunomide has a strong suppressive effect on both acute and chronic phases of antigen-induced arthritis and formation of autoantibodies in rats. Furthermore, orally administered doses of leflunomide were as effective as doses of cyclosporin A given intraperitoneally. The combination of sub-effective doses of leflunomide and cyclosporin A resulted in significant inhibition of chronic arthritis.

Significance of cell-mediated and humoral immunity in the acute and chronic phase of antigen-induced arthritis in rabbits

In the course of antigen-induced arthritis of rabbit cell-mediated and humoral immune responses were repeatedly tested in order to prove their significance for the acute and chronic phase of inflammation. The arthritis was monitored during the progression of the inflammation by means of the joint swelling and at the end of experiments by histological evaluation of synovitis and cartilage degradation. Following the arthritis induction a strong increase of specific antibodies and of circulating immune complexes was evident. The correlations between antibody levels and joint swellings confirmed that the local formation of immune complexes is responsible for the initiation and perpetuation of arthritis. In the early phase after immunization the responsiveness of lymphocytes to antigenic and mitogenic stimulation was increased, in the late chronic phase of arthritis proliferative responses of lymphocytes to cartilage matrix components were revealed. No direct correlations could be demonstrated between any cell-mediated immune response and the severity of arthritis. The hyperreactivity of cell-mediated immunity is suggested to be responsible for the transition of the acute arthritis into the chronic stage. The deficiency of an effective suppression results in the activation of B-lymphocytes with increased production of antibodies, maintaining the inflammatory process for a long time. Under these conditions the release of cartilage matrix components during the acute joint reaction induces autoimmune responses against cartilage, which could contribute to the chronification of arthritis and to cartilage degradation.

Electron microscopic demonstration of cell surface carbohydrates by means of peroxidase and ferritin complexes of the Lens culinaris lection.

SummaryThe use of Lens culinaris lectin for electron microscopic detection of D-mannose, D-glucose and N-acetyl-D-glucosamine like sites on tumor cells, erythrocytes, erythrocyte ghosts, cultured rat liver cells and various tissues of mice is demonstrated. In addition to Lens culinaris lectin-peroxidase reaction (LcL-po reaction) the preparation of active Lens culinaris lectin-ferritin conjugate are described and the specificity of cytochemical reactions are demonstrated. Furthermore experiments by immuno freeze-etching are reported for topological analysis of the lectin receptors.

Different immunological mechanisms contribute to cartilage destruction in antigen-induced arthritis

Antigen-induced arthritis in guinea pigs was used as a model to investigate the pathogenic mechanisms responsible for cartilage destruction in chronic joint inflammation. The activation of macrophages, their effects on cartilage metabolism, and the development of autoimmunity to cartilage constituents were studied during the progression of arthritis. The results show that in arthritic animals the macrophages are systemically activated, with a peak in the early phase of inflammation. Interleukin 1, produced by the activated cells, suppresses the proteoglycan synthesis in cartilage explants and cultured chondrocytes and increases the proliferation of the cells in vitro. During the progression of arthritis humoral and cell-mediated immune responses to collagen type II and cartilage proteoglycans occur correlating with the severity of arthritis. It is concluded that different immunological mechanisms may be involved in cartilage destruction during antigen-induced arthritis. Mediator-induced metabolic reactions dominate in the early phase, whereas autoimmunity to cartilage might play an essential role in later phases of arthritis.

High-dose clodronate therapy prevents joint destruction in chronic antigen-induced arthritis of the rat but inhibits bone formation at the axial skeleton

Abstract:Objective: To investigate the effects of clodronate on clinical disease activity, inflammatory alterations and cartilage destruction, periarticular and axial bone volume and bone turnover in chronic antigen-induced arthritis (AIA; day 28).¶Methods: Rats with AIA were treated with clodronate (5 mg/kg/day continuously; 20 mg/kg/day intermittently or high-dose with 300 mg/kg 3 hours after arthritis induction +20 mg/kg/day continuously, respectively). Joint pathology was examined by histology. Bone volume and cellular turnover parameters of the right tibia head and the third lumbar vertebra were evaluated by histomorphometry. The findings were compared with those of healthy controls, sham-treated AIA and AIA treated continuously with 250 μg/kg of dexamethasone.¶Results: All three therapy regimens with clodronate resulted in a significant reduction of joint swelling, histopathological inflammatory changes and cartilage destruction in comparison with sham-treated AIA. The antiinflammatory effect of high-dose clodronate was comparable with dexamethasone. The intermittent administration of 20 mg/kg/day of clodronate completely prevented periarticular bone loss by reduction of bone resorption without affecting bone formation at the periarticular and axial bone. Both continuous treatment with 5 mg/kg/day of clodronate and high-dose clodronate therapy partially prevented periarticular bone loss and reduced parameters of bone formation at the axial bone to values below those of healthy controls.¶Conclusion: High-dose clodronate therapy exerts an excellent preventive effect on clinical disease activity and on joint destruction in AIA. However, continuous treatment with high doses of clodronate may result in a low turnover state of bone remodelling.¶

The use of fluorescein isothiocyanate labelled lectins for immuno-histological demonstration of saccharides. III. Studies by use of Ricinus communis lectin and wheat germ agglutinin.

Cryostat sections of heart, skeletal muscle, spleen, liver, cartilage, brain, nerve, tongue, esophagus and salivary gland from man, rabbit, rat and guinea pig were stained with fluorescein isothiocyanate labelled Ricinus communis lectin and wheat germ agglutinin. The stained sections were examined by fluorescence microscopy. The general pattern of fluorescence is very similar to that obtained with labelled Lens culinaris lectin and Concanavalin A. Particularly the lectins stain connective tissue structures of the organs. The staining intensity in these structures was weaker by wheat germ agglutinin as compared to the other lectins.

Untersuchungen zum immunhistochemischen Verhalten der senilen Plaques des menschlichen Gehirnes

Senile plaques showed an intensive fluorescence with thioflavine S caused by the deposition of amyloid. From these results fluorescence-immune histochemical studies of senile plaques with antihuman-gamma globuline serum and anti-human fibrin serum were carried out on ten brains. Forty brains were selected from autopsies of normally aged men without mental diseases. About 5–10% of senile plaques of each brain showed in the central cores positive immunohistochemical reactions whereas the fluorescence in their peripheral zones appeared less intensive or was totally absent. Furthermore, the walls of some meningeal and cerebral vessels were also positive with these methods, independent of the plaque-like degeneration of arteries and capillaries (Scholz). It is assumed that the majority of senile plaques has no relation to the blood vessels and, therefore, the plaques arise locally in brain tissue. The findings of immunhistochemical positive plaques are due to an exudation of serum. An immunological genesis of senile plaques is discussed; it is concluded that the senile plaque is a particular form of senile amyloidosis due to insufficient regulatory mechanisms of glia cells. Die intensive Thioflavin-S-Fluorescenz seniler Plaques weist auf Amyloidablagerungen hin. Davon ausgehend wurden immunhistochemische Untersuchungen an 10 Gehirnen mittels Anti-Human-Gammaglobulin- und Anti-Human-Fibrin-Serum vorgenommen. Die Fälle wurden dem Obduktionsmaterial 40 physiologisch gealterter Menschen entnommen. 5–10% der senilen Plaques des Gehirnes ergeben zentral positive immunhistochemische Reaktionen, wogegen die periphere Zone weniger intensiv fluoresciert oder reaktionslos ist. Darüber hinaus zeigen Wandungen einiger meningealer und cerebraler Gefäße unabhängig vom Vorliegen einer drusigen Entartung (Scholz) positive Befunde. Die Mehrzahl der senilen Plaques kann daher keine Beziehungen zu Blutgefäßen aufweisen und muß folglich lokal im Hirngewebe entstehen. Die positiven immunhistochemischen Reaktionen sind auf eine Serumexsudation zurückzuführen. Die immunologische Genese der Plaques wird diskutiert, und es wird die Schlußfolgerung gezogen, daß die senilen Plaques eine besondere Form der senilen Amyloidose auf dem Boden zellulärer Regulationsstörungen darstellen.

Histochemical lectin affinity technique by means of FITC-labeled serum protein fractions

SummaryA two-step affinity technique is described for light microscopic demonstration of the Concanavalin A, Agaricus bisporus lectin and Ricinus communis lectin binding sites by means of various FITC-labeled human and rabbit serum protein fractions. Experiments for the visualization of the Lens culinaris lectin and the Pisum sativum lectin binding sites gaves negative results. The technique consist of two reaction steps which involve the incubation of tissue sections in the lectins followed by the visualization of receptor-bound lectins with FITC-labeled serum protein fractions basing on their carbohydrate content. The specificity of the technique could be demonstrated by the addition of the hapten or by incubation in the FITC-labeled serum protein fractions only. In contrast to the direct or indirect staining methods only very small amounts of purified lectins are necessary.