Crocus sativus Extract as a Biological Agent for Disease-Modifying Therapy of Collagenase-Induced Mouse Model of Osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is an age-related joint disease that involves the degeneration of cartilage and is the most prevalent form of arthritis, affecting a large part of the population. OA is a multifactorial disorder, and no single etiological mechanism has been found to be common to all forms of the disease. Currently used therapies for control of the disease are mainly nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroid medications. The aim of this study was to investigate the extract from Crocus sativus as a biological disease-suppressing therapy agent. METHODS: Balb/c mice were injected intra-articularly with Clostridium histolyticum type IA for induction of osteoarthritis. The mice were randomized to five groups: control group, I group (CIOA untreated), II group (CIOA + 100 mg/kg/daily saffron), III group (CIOA + 50 mg/kg/daily saffron), IV group (CIOA + 25 mg/kg/daily saffron). Flow-cytometry analysis was used to study the splenocytes' phenotype isolated from the treated animals. The serum levels of inflammatory and anti-inflammatory cytokines were analyzed with ELISA. The histological assessment was used to analyze the saffron extract effect on histopathological alterations. RESULTS: Saffron treatment significantly decreased osteoarthritis-associated joint histological manifestations and decreased serum TNFα levels. The flow-cytometry analysis showed a decrease in pro-inflammatory immune cell subtypes in the spleen. CONCLUSIONS: The results obtained suggest that saffron affected the disease progression and could be a potential therapeutic approach in osteoarthritic patients' therapy.

Collagenase-Induced Mouse Model of Osteoarthritis-A Thorough Flow Cytometry Analysis.

OBJECTIVES: Osteoarthritis (OA) is a chronic degenerative disorder of the joint characterized by cartilage breakdown and synovial inflammation. A number of different cells of innate and adaptive immunity contribute to joint pathology during OA inflammation. The interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is still unknown. The objective of this study was to investigate the role of the different types of immune cells in the development of OA. METHODS: Collagenase-induced osteoarthritis was induced in Balb/c mice; flow cytometry analysis; and histopathological damages were assessed in histological sections stained with H&E, Toluidine blue, and Safranin O. RESULTS: Flow cytometry analysis showed B lymphocyte infiltration in the active phase of inflammation and an increase in the effector T cell population into the synovium. An increased activation state of cytotoxic T cells and of NK cell populations in the spleen and synovium was also found. The differentiation of NK cells from a cytotoxic phenotype in early OA to cells with an effector phenotype in the chronic phase of the disease followed. CONCLUSIONS: A number of different cells contribute to inflammatory processes in OA. The correlation between their phenotype and the inflammatory pathophysiology could result in the development of novel approaches to suppress destructive changes in the joint.

Antitumor Properties of Epitope-Specific Engineered Vaccine in Murine Model of Melanoma.

Finding new effective compounds of natural origin for composing anti-tumor vaccines is one of the main goals of antitumor research. Promising anti-cancer agents are the gastropodan hemocyanins-multimeric copper-containing glycoproteins used so far for therapy of different tumors. The properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) upon their use as carrier-proteins in conjugated vaccines, containing ganglioside mimotope GD3P4 peptide, were studied in the developed murine melanoma model. Murine melanoma cell line B16F10 was used for solid tumor establishment in C57BL/6 mice using various schemes of therapy. Protein engineering, flow cytometry, and cytotoxicity assays were also performed. The administration of the protein-engineered vaccines RtH-GD3P4 or HaH-GD3P4 under the three different regimens of therapy in the B16F10 murine melanoma model suppressed tumor growth, decreased tumor incidence, and prolonged the survival of treated animals. The immunization of experimental mice induced an infiltration of immunocompetent cells into the tumors and generated cytotoxic tumor-specific T cells in the spleen. The treatment also generates significantly higher levels of tumor-infiltrated M1 macrophages, compared to untreated tumor-bearing control mice. This study demonstrated a promising approach for cancer therapy having potential applications for cancer vaccine research.

Influence of estradiol treatment on bone marrow cell differentiation in collagenase-induced arthritis.

OBJECTIVE AND DESIGN: Estrogen is one of the important regulators of the balance between bone formation and bone resorption that can modulate the levels and activity of certain growth factors and cytokines. In this study, we investigated the effect of 17ß-estradiol (ED) on bone marrow (BM) cell differentiation in vivo and ex vivo in a mouse model of collagenase-induced osteoarthritis (CIOA). SUBJECT: ICR (CD-2) female mice were used in present experiments (total number = 75) and bone marrow cells were used for in vitro studies. TREATMENT: Mice were orally fed under different schemes with 17ß-estradiol at a dose of 2 µg or 4 µg for 30 days. METHODS: The effect of estradiol was estimated by histopathological, flow cytometry, and ELISA assays. Statistical differences were determined by one-way ANOVA. RESULTS: Estradiol treatment ameliorated cartilage destruction and osteophyte formation if started from day 0 of CIOA induction, attended with a decrease of uterine and ovarian weights. Long time treatment lowered the percentage of megakaryocyte/platelet (CD62P+) populations and osteoclast (RANK+) populations in BM. Cells obtained from estradiol-treated CIOA mice showed inhibited capacity to differentiate into RANK+ and mesenchymal cells under osteoclastogenic conditions in vitro. Estrogen decreased serum IL-6 levels. CONCLUSION: Results indicate a potential protective role for estrogen against the development of OA.

Functional complement activity is decisive for the development of chronic synovitis, osteophyte formation and processes of cell senescence in zymosan-induced arthritis.

Synovial inflammation plays a critical role in the symptoms and structural progression of arthritis which leads to irreversible damage of the adjacent cartilage and bone. Activation of complement system is strongly implicated as a factor in the pathogenesis of chronic synovitis in human rheumatoid arthritis (RA). In this study, we show that the depletion of functional complement activity at the time of the initiation of zymosan-induced arthritis, significantly reduced the expression of TGF-beta1/3, BMP2 and pSmad2 and decreased the number of Sudan Black B positive cells in the synovium. Also, the excessive synthesis of proteoglycans and glycosaminoglycans was diminished. The appearance of apoptotic and senescent cells among the adherent bone marrow cells cultivated in vitro was not observed in complement depleted mice. Therefore, the lack of functional complement prevented the development of chronic synovitis, osteophyte formation and the generation of pathologic senescent arthritic cells.