The deleterious role of fibrin deposition in arthritic joints prompted us to explore the effect of the thrombin inhibition on the course of collagen-induced arthritis (CIA) in the mouse. CIA was induced in male DBA/1J mice using native chicken type II collagen. The thrombin inhibitor polyethyleneglycol-hirudin (PEG-hirudin) was given for 16 days, starting 20 days after the first immunization (preventive treatment) or at the onset of clinical signs of arthritis (curative treatment). All the mice treated with PEG-hirudin had a significantly prolonged clotting time compared with control mice. PEG-hirudin, administered in a preventive way, led to significantly reduced incidence and severity of CIA during most of the treatment period, as assessed by clinical scoring. Accordingly, histological features showed a significant diminution of synovial hyperplasia in PEG-hirudin-treated mice compared with untreated mice. There was also a significant downmodulation of the synovial proinflammatory IL-1β and IL-12p35 cytokine mRNAs in treated mice. Intra-articular fibrin, evaluated by immunohistochemistry, was significantly reduced in treated mice compared with control mice and correlated with both clinical and histological scorings. Most importantly, once arthritis was established, PEG-hirudin also showed a curative effect. In conclusion, PEG-hirudin can both prevent the onset of CIA in a dose-dependent manner and ameliorate established arthritis, suggesting that thrombin inhibition may offer a new therapeutic approach in arthritis.

Background and objective. The role of gold nanoparticles (AuNPs) in the treatment of autoimmune diseases remains vague. Therefore, the aim of this study was to determine the effect of AuNPs in the treatment of rats with established collagen-induced arthritis (CIA). Material and Methods. A total of 24 Wistar male rats with established CIA were used. AuNPs measuring 13-nm and 50-nm were prepared according to standard procedures, and their size was determined using transmission electron microscopy. These gold particles were injected intra-articularly 5 times a week, 12 injections in total. Body and organ weight, arthritic profiles based on paw swelling, histological changes in the joints and internal organs, blood indices, and serum oxidative products were investigated. Results. An examination of the course of the experimental disease and a subsequent histological analysis as well as hematological studies revealed a nontoxic effect of AuNPs on the vital organs. The treatment of the rats with established CIA by 13-nm and 50-nm gold nanoparticles decreased joint swelling by 49.7% (P<0.002) and 45.03% (P<0.01), respectively. That corresponded to the decrease in statistically significant histological changes in articular tissues. AuNPs showed their antioxidant effect by increasing the level of antioxidant enzyme catalase. Conclusions. The continuous intra-articular administration of AuNPs not only reduced the inflammation, joint swelling, and development of polyarthritis, but also reduced histological changes in articular tissues without toxic effects on the internal organs. The results obtained disclose the role of AuNPs as antioxidant agents.

Objective. To determine the conditions for synovial accumulation of protoporphyrin IX (PpIX) and photodynamic therapy (PDT)-induced synovial cytotoxicity in vitro and in vivo.Methods. Synovial tissues were obtained from mice with antigen-induced arthritis (AIA) and incubated with different concentrations of 5-aminolevulinic acid hexyl ester (h-ALA), a PpIX precursor. Following photoexcitation, cell death in synovial tissues was evaluated by Sytox green fluorescence. PDT was performed after intraarticular injection of h-ALA into the knee joints of mice with AIA, and its effect on joint inflammation was assessed by technetium scintigraphy and histology. Synovial biopsy samples were obtained from patients with osteoarthritis (OA; n ‫؍‬ 9) and rheumatoid arthritis (RA; n ‫؍‬ 7) and studied for PDT-induced cytotoxicity in vitro.Results. Conversion of h-ALA to PpIX was observed in inflamed synovium in mice and humans. Cytotoxicity was confirmed by Sytox green staining in samples subjected to PDT. In the AIA model, injection of affected knees with h-ALA prior to PDT led to a statistically significant reduction of joint damage in the irradiated joints. The preferential transformation of h-ALA to PpIX in inflammatory tissues was confirmed in human synovial biopsy tissues, where RA samples showed higher tissue concentrations of PpIX following incubation with h-ALA than did OA samples. Fluorescence microscopy showed that PpIX was localized to the synovial lining layer, endothelial cells, and macrophages and induced cell death after PDT.Conclusion. Our findings suggest that PDT based on the accumulation of PpIX in the synovial membrane may be a rational basis for photodynamic synovectomy in arthritic diseases.

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The inflamed synovium of rheumatoid arthritis exhibits many features typical for neoplastic tissue implying that the photodynamic therapy might be an efficient modality for chronic poliarthritis. The accumulation of endogenously produced porphyrins after administration of exogenous 5-aminolevulinic acid (ALA) in a rabbit model of rheumatoid arthritis was evaluated by fluorescence spectroscopy. Independent of the way, intravenously or intra-articularly, in which ALA was administered to the experimental animals, the highest fluorescence intensity of endogenously produced porphyrins was detected in the tissues of the inflamed joints. Besides, the application of ALA had a systemic sensitising effect on the whole organism of rabbits. The highest amount of endogenously produced porphyrins in the inflamed joints measured from the surface of the skin above the synovium tissues was detected 1-3 h after the administration of ALA. Fluorescence measurements performed on the tissue specimens ex vivo showed the predominant accumulation of porphyrins in the synovium of the inflamed joints. The fluorescence of porphyrins was also observed in the cartilage tissues taken from knee joints. However, the fluorescence spectra features indicated that the composition of porphyrins detected in the cartilage tissues was different than that in the synovial tissues. The selective accumulation of porphyrins in the inflamed synovial tissues stands up for the application of photodynamic therapy in the treatment of rheumatoid arthritis and implies the possibility to use optical non-invasive methods based on fluorescence detection of endogenously produced porphyrins for diagnostics of inflamed tissues.

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the factors possibly infl uencing the outcomes of this disease.

Physiological and endocrine maintenance of a normal human growth hormone (hGH) concentration is crucial for growth, development, and a number of essential biological processes. In this study, we describe the preparation and characterization of magnetic nanoparticles coated with a gold shell (MNPs-Au). The optimal surface concentration of monoclonal anti-hGH antibodies (m-anti-hGH) on magnetic nanoparticles, as well as conditions that decrease non-specific interactions during the magneto-immunoassay, were elaborated. After the selective recognition, separation, and pre-concentration of hGH by MNPs-Au/m-anti-hGH and the hGH interaction with specific polyclonal biotin-labeled antibodies (p-anti-hHG-B) and streptavidin modified horseradish peroxidase (S-HRP), the MNPs-Au/m-anti-hGH/hGH/p-anti-hGH-B/S-HRP immunoconjugate was formed. The concentration of hGH was determined after the addition of 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide substrate solution for HRP; the absorbance at 450 nm was registered after the addition of STOP solution. The developed sandwich-type colorimetric magneto-immunoassay is characterized by a clinically relevant linear range (from 0.1 to 5.0 nmol L−1, R2 0.9831), low limit of detection (0.082 nmol L−1), and negligible non-specific binding of other antibodies or S-HRP. The obtained results demonstrate the applicability of the developed magneto-immunoassay for the concentration and determination of hGH in the serum. Additionally, important technical solutions for the development of the sandwich-type colorimetric magneto-immunoassay are discussed.