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Poly(DL-lactic acid) (PLA)/poly(ethylene glycol)-block-poly (propylene glycol)-block-poly(ethylene glycol) copolymer (PEG-PPG-PEG) nanoparticles loaded with camptothecin (CPT), called CPT-NP, were prepared and examined for particle size change and drug release in phosphate-buffered saline, pH 7.4, (PBS), and drug biodistribution profiles in mice bearing sarcoma 180 solid tumor. CPT-NP kept an almost constant mean size and exhibited an initial rapid release of approximately 20%, following by very slow release. As compared with CPT solution, CPT-NP showed higher tissue accumulation and better tumor localization, which were considered essentially associated with the better efficacy of CPT-NP reported in the previous study.

Camptothecin (CPT)-loaded nanoparticles were prepared using poly( dl -lactic acid) (PLA) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) copolymer (PEG-PPG-PEG), and examined for particle characteristics, in vitro release, pharmacokinetics and efficacy. The preparative condition, in which the ratio of PLA/PEG-PPG-PEG/CPT was 35/35/4 (w/w/w) and organic solvent (dichloromethane) was evaporated from the emulsion at 18 °C, gave the nanoparticles with the diameter of approximately 230 nm, fairly high drug content (ca. 1.6% (w/w)) and stable entrapment of the drug, which were used for in vivo studies. After i.v. administration to normal rats, the nanoparticles showed slightly smaller AUC but much larger MRT as compared with CPT solution, and delivered the drug greatly to the surrounding tissues, in particular to the liver. When antitumor effect was examined by i.v. administration to mice bearing sarcoma 180 (S-180) solid tumor, the nanoparticles showed a significant suppression of tumor growth without body weight loss, and their effect was better than that of CPT solution. The PLA/PEG-PPG-PEG nanoparticles were considered potentially useful to enhance the efficacy of CPT, to which the high drug retention in the body and gradual drug release appeared to be importantly related.

Background and objectives: Aminopeptidase N (APN)/CD13 is a multifunctional ectoenzyme that is involved in anti-inflammatory reactions, control of immune reactions and differentiation of many cellular systems. Here, we hypothesized that CD13/APN would be expressed on human gingival fibroblasts (hGF) and would contribute to the regulation of immune responses in periodontal tissue. Methods and results: CD13/APN was expressed on hGF at the mRNA and protein levels as determined by reverse transcriptase–polymerase chain reaction (RT–PCR) and flow cytometry, respectively. Enzymatic activities accompanying the expression were assessed by colorimetrical analysis using the synthetic substrate Leu-p-nitroanilide. We examined the possible regulation of CD13/APN expression on hGF in response to T cell-derived cytokines. T helper (Th) 2 cell type cytokines such as interleukin-4 and interleukin-13, but not interleukin-2 or interleukin-15, preferentially increased the expression of proteins as well as the enzymatic activities of CD13/APN in a dose-dependent manner. Receptors for these cytokines, the interleukin-4 receptor α chain, interleukin-13 receptor α1 chain, and interleukin-2R common γ chain, were expressed on hGF assessed by RT–PCR or flow cytometry. hGF exhibited inhibitory effects for formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte-activation that was evaluated by Mac-1 expression, and this inhibitory effect was partially recovered by pre-treatment with the APN-specific inhibitor bestatin. Conclusions: These findings suggested that CD13/APN expressed by hGF could contribute to the anti-inflammatory response in periodontal tissue, and may be involved in disease processes mediated by Th2 cells.

BACKGROUND AND OBJECTIVES CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. METHODS AND RESULTS We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. CONCLUSIONS These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.

BACKGROUND AND OBJECTIVES Retinoic acid (RA) exerts a wide variety of effects on development, cellular differentiation and homeostasis in various tissues. However, little is known about the effects of RA on the differentiation of periodontal ligament cells. In this study, we investigated whether RA can affect the dexamethasone-induced differentiation of periodontal ligament cells. METHODS AND RESULTS Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate for mineralized nodule formation, as characterized by von Kossa staining. Continuous treatment with all-trans-RA inhibited the mineralization in a dose-dependent manner, with complete inhibition over 1 microm RA. Other RA analogs, 9-cis-RA and 13-cis-RA, were also effective. Furthermore, addition of RA for just the first 4 days completely inhibited the mineralization; however, as RA was added at later stages of culture, the inhibitory effect was diminished, suggesting that RA had a phase-dependent inhibition of mineralization. RA receptor (RAR)-alpha agonist (AM-580), but not retinoid X receptor agonist (methoprene acid), inhibited the mineralization, and reverse transcription-polymerase chain reaction analysis revealed that RAR-alpha was expressed on the cells, suggesting that RAR-alpha was involved in the inhibitory mechanism. This inhibition was accompanied by inhibition of alkaline phosphatase activity; however, neither expression of platelet-derived growth factor (PDGF) receptor-alpha, PDGF receptor-beta, or epidermal growth factor (EGF) receptor, nor phosphorylation of extracellular signal-regulated kinases triggered by PDGF-ascorbic acid or PDGF-BB was changed, as assessed by flow cytometry or western blot analyses. CONCLUSIONS These findings suggest that RA is a potential negative regulator for differentiation of human periodontal ligament cells.

CD13/aminopeptidase N (APN) is a multifunctional ectoenzyme that is involved in anti-inflammatory reactions, control of immune reactions and differentiation of many cellular systems. In this study, we first demonstrated that CD13/APN was expressed on human gingival fibroblast (hGF). T helper (Th) 2 cell type cytokines such as IL-4 and IL-13, but not IL-2 or IL-15, preferentially increased the expression of CD13/APN, resulting in the inhibition of FMLP-mediated neutrophil activation through the degradation by CD13/APN on HGF. Our data suggested that CD13/APN expressed by hGF could contribute to the anti-inflammatory response in periodontal tissue, and may be involved in disease processes mediated by Th2 cells.