Tetracycline antibiotics are widely used in human and veterinary medicine; however, residual amounts of these antibiotics in the environment are of concern since they could contribute to selection of resistant bacteria. In this study, tetracycline (TC), chlortetracycline (CTC), doxycycline (DC) and oxytetracycline (OTC) were treated with laccase from the white rot fungus Trametes versicolor in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). High performance liquid chromatography demonstrated that DC and CTC were completely eliminated after 15 min, while TC and CTC were eliminated after 1 h. This system also resulted in a complete loss of inhibition of growth of Escherichia coli and Bacillus subtilis and the green alga Pseudokirchneriella subcapitata with decreasing tetracycline antibiotic concentration. These results suggest that the laccase-HBT system is effective in eliminating tetracycline antibiotics and removing their ecotoxicity.

Activated T cells secrete the cytokine IL-13, which regulates inflammatory and immune responses. To explore the role of IL-13 in bone metabolism, we examined the effects of the cytokine on bone resorption and PG synthesis in osteoblasts. IL-13 suppressed the bone-resorbing activity stimulated by IL-1 alpha, which was determined by the release of 45Ca from prelabeled mouse long bones. Histologic examinations revealed that IL-1 alpha markedly stimulated bone resorption with increased osteoclast recruitment, and that the simultaneous addition of IL-13 considerably inhibited it. The gamma-chain of IL-2 receptors may be functionally involved in the signal transduction of not only IL-2, but also IL-4, IL-7, and IL-13. Of these cytokines, IL-4 similarly suppressed IL-1 alpha-induced bone resorption, but IL-2 and IL-7 did not. Both IL-13 and IL-4 inhibited PGE2 production stimulated by IL-1 alpha in long bone cultures. Suppression of IL-1 alpha-induced bone resorption by IL-13 and IL-4 was recovered by adding exogenous PGE2 to the long bone cultures. Neither IL-4 nor IL-13 further inhibited IL-1 alpha-induced bone resorption in the presence of indomethacin. To examine the effects of IL-13 on PG synthesis, we measured the mRNA levels of cytosolic phospholipase A2 (cPLA2), constitutively expressed cyclooxygenase (COX-1) and inducible COX (COX-2) in mouse osteoblast-like cells. IL-1 alpha markedly stimulated the mRNA expression of COX-2, but not that of COX-1. Both IL-13 and IL-4 dose-dependently suppressed the IL-1 alpha-induced stimulation of both COX-2 mRNA expression and PGE2 synthesis. A small increase (1.7-fold) in cPLA2 mRNA levels was detected in the cultures with IL-1 alpha, but the expression was not affected by IL-13 or IL-4. These results indicated that IL-13 and IL-4 inhibit bone resorption by suppressing COX-2-dependent PG synthesis in osteoblasts.

A variety of studies have shown that osteoclasts originate from bone marrow, but their exact progenitors and differentiation pathway remain unclear. The treatment of mice with a high dose of 5-fluorouracil (5- FU) results in an enrichment for primitive hematopoietic progenitors; using this procedure, we prepared a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro. When spleen cells from mice pretreated in vivo with 5-FU were cultured in the presence of methylcellulose medium containing recombinant interleukin-3 (rIL-3), small colonies consisting of blast cells with little sign of differentiation developed on day 7 of culture. We lifted these blast colonies, pooled them, and replated them as secondary methylcellulose cultures in the presence of rIL-3 and erythropoietin. Approximately 60% of the cells formed colonies comprising various combinations of neutrophils, macrophages, eosinophils, mast cells, megakaryocytes, and erythroblasts. We replated such blast cells into microtiter wells and cultured them in the presence of rIL-3 (100 U/mL) or recombinant granulocyte-macrophage colony stimulating factor (GM- CSF) (100 U/mL) plus 1.25(OH)2D3 (10(-7) mol/L). Multinucleated cells appeared from day 14 of culture and approximately 100 giant cells per well were scored on day 21 of culture. Parathyroid hormone (1 U/mL) also induced the multinucleated cell formation. May-Grunwald-Giemsa staining revealed the large cells containing many nuclei in their cytoplasm, which is characteristic of bone-resorbing cells or osteoclasts. These cells showed a tartrate-resistant acid phosphatase (TRAP) activity. Calcitonin caused a striking shape change in these cells and suppressed the formation of multinucleated cells. Moreover, electron microscopy shows that these cells were able to resorb fetal calvariae. In the presence of r granulocyte-colony stimulating factor, r macrophage-colony stimulating factor, or r interleukin-6 plus 1.25(OH)2D3, formation of TRAP-positive multinucleated cells was lower compared with the support of rIL-3 or rGM-CSF. Mature macrophages collected from colonies did not form the multinucleated cells as described above, even in the presence of rIL-3 and 1.25(OH)2D3. Moreover, to exclude the possibility that osteoclasts generated from non-blast cells, we performed a cloning experiment from one isolated blast cell and demonstrated that single cells differentiate into osteoclasts or macrophages in the presence of rIL-3 with or without 1.25(OH)2D3. This system will provide a useful model for further analysis of osteoclast formation in vitro.

Platelet-rich plasma (PRP) is an autologous source of platelet-derived growth factors, and has been used successfully in oral surgery repair and in the placement of osseointegrated implants. However, little is known about the underlying release mechanisms of these factors or the manner in which inhibition of osteoclastic cells is regulated. The present study investigated the efficacy of PRP as an inhibitor of receptor activator of NF-kappaB ligand (RANKL)-induced TRAP-positive multinucleated cell formation in RAW264.7 cells. RANKLinduced formation of osteoclasts was significantly inhibited by treatment with PRP, while washed platelet treatment was more effective in inhibiting osteoclast formation. These results suggest that PRP and washed platelets are potent inhibitors of osteoclast differentiation.