Anti-Inflammatory Effect of Ascochlorin in LPS-Stimulated RAW 264.7 Macrophage Cells Is Accompanied With the Down-Regulation of iNOS, COX-2 and Proinflammatory Cytokines Through NF-κB, ERK1/2, and p38 Signaling Pathway.

A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 µM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

Assessment of a 50:50 mixture of two Bacillus subtilis strains as growth promoters for finishing pigs: productivity improvement and noxious gas reduction.

In this study, we aimed to assess the potential of a 50:50 mixture of two Bacillus subtilis strains in improving the productivity and health of finishing pigs and reducing noxious gases in their feces. These strains were found to abundantly secrete surfactin which has been shown to alleviate the effects of lipopolysaccharides in vitro. For the 10-wk experiment, 200 finishing pigs ([Landrace × Yorkshire] × Duroc) with an average body weight of 54.15 ±â€…1.70 kg were divided into four groups. Each group was fed with a basal diet supplemented with an equal amount of spores from the two B. subtilis strains at different levels: control group, no addition; treatment group 1, 0.5 × 109; treatment group 2, 1.0 × 109; treatment group 3, 1.5 × 109 cfu·kg-1 addition. During the 10-wk feeding period, dietary supplementation of 0.5 × 109, 1.0 × 109, and 1.5 × 109 cfu·kg-1 of the spore cells from these two strains resulted in a 0.9%, 1.9%, and 2.5% increase in body weight, respectively (linear P < 0.095). During the final 5 wk, the average daily gain (ADG) in weight was increased by the strains at amounts of 0.5 × 109, 1.0 × 109, and 1.5 × 109 cfu·kg-1 with a clear dosage effect (linear P < 0.05). However, neither the gain-to-feed ratio, the average daily feed intake, nor nutrient digestibility was affected by the supplementation. In blood, the endotoxin lipopolysaccharides, and two liver toxicity indicator enzymes; aspartate aminotransferase and lactate dehydrogenase were decreased (P < 0.05) in the 1.0 × 109 cfu·kg-1 spores-feeding group. Furthermore, four noxious gases were reduced by 8 to 20% in feces excreted by pigs fed with 1.5 × 109 cfu·kg-1 spores with a linear dosage effect (linear P < 0.001 to 0.05) during the final 5 wk. Our findings suggest that the mixture of B. subtilis strains may enhance the productivity of finishing pigs by reducing the risk of mild endotoxemia, rather than increasing digestibility or daily feed intake. Therefore, these Bacillus strains have the potential to act as growth promoters for pigs, leading to improved animal health and productivity. These results have significant implications for pig farmers seeking to optimize the health and growth of their animals.

In a previous study, we discovered two new strains of Bacillus subtilis that showed high surfactin secretion during growth in culture media. This surfactin proved effective in reducing endotoxin effects, particularly lipopolysaccharides, in vitro. To explore their potential as pig growth promoters, we administered 50:50 bacteria blend to 200 finishing pigs, dividing them into four groups for a 10-wk trial. Results showed that supplementing the pigs' diet with 0.5, 1.0, or 1.5 billion bacteria per kilogram led to weight gains of 0.9%, 1.9%, and 2.5%, respectively, with a dosage effect. The weight gain was notably higher during the final 5 wk. However, there were no significant differences in feed intake or nutrient digestibility. Blood analysis revealed reduced lipopolysaccharides and liver toxicity indicators, suggesting improved animal health. Moreover, the pigs that received the bacterial mixture showed reduced noxious gas levels in their feces with a dosage effect. These findings suggest that these new B. subtilis strains could serve as effective growth promoters for pigs by minimizing the risk of mild endotoxemia, leading to enhanced animal health and productivity. These results could have valuable implications for pig farmers seeking to optimize the health and growth of their animals.

Throughput screening of Bacillus subtilis strains that abundantly secrete surfactin in vitro identifies effective probiotic candidates.

Since the prohibition of antibiotics as animal growth promoters, demand for effective probiotic strains has steadily increased. The goal is to maintain productivity and mitigate environmental concerns in the livestock industry. There are many probiotic animal-diet supplements available, over 2,000 products in the Republic of Korea alone, with little explanation about the desirable properties of each probiotic strain. The purpose of this study was to describe the underlying logic and methods used to select two novel strains of probiotic candidates. To economically screen these candidates, the abundance of surfactin secreted was used as an in vitro marker. We used a modified oil-misting method to screen ~2,000 spore-forming bacteria for novel strains of Bacillus subtilis. Of these, 18 strains were initially selected based on the semiquantitative criterion that they secreted more surfactin than B. subtilis ATCC21322 on Luria-Berani (LB) agar plates. The whole genome sequence was determined for two of the 18 strains to verify their identity. A phylogeny of 1,162 orthologous genes, genome contents, and genome organization confirmed them as novel strains. The surfactin profiles produced by these two strains consisted of at least four isoforms similar to standard surfactin and enhanced cellulase activities up to 50%. Four fractionated individual isoforms of surfactin suppressed inflammation induced by lipopolysaccharides. The half-maximal inhibitory concentration (IC50) was about 20 µM for each isoform. Both selected strains were susceptible to seven important antibiotics. Our results implied that an abundant secretion of surfactin was a useful biomarker in vitro and could be utilized for mining probiotic candidates through high-throughput screening of environmental samples.

Effects of TGFbeta1 and extracts from Cervus korean TEMMINCK var. mantchuricus Swinhoe on acute and chronic arthritis in rats.

AIM OF THE STUDY: To elucidate the pharmacological activities of deer antler acupuncture and TGF61538;1 on the acute and chronic phases of rheumatoid arthritis diseases. MATERIALS AND METHODS: Polyarthritis rats were administered with TGF61538;1 and water extract of deer antler acupunture (DAA), prepared from the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe. TGF61538; (0.1 to 2 61549;g/animal) and DAA (5-100 61549;g/kg animal) were initiated 1 day before an arthritogenic dose of streptococcal cell wall fragments to see the effects on the joint swelling and distortion during the acute phase and the chronic phase of the disease. Arthritic index suppression of rat arthritis model was examined by TGF61538; and DAA administrations. RESULTS: TGF61538;1 and DAA diminished the polyarthritis development in rats. TGF61538; and DAA eliminated the joint swelling and distortion observed during the acute phase and the chronic phase of the disease. The TGF61538; and DAA suppressed the arthritis progress when administration was begun after acute phase of arthritis. DISCUSSION: Consistent with the inhibition of inflammatory cell recruitment into the synovium, TGF61538;1 and DAA reversed the leukocytosis associated with the chronic phase of the arthritis, respectively.

Herbal medicine Gamgungtang down-regulates autoimmunity through induction of TH2 cytokine production by lymphocytes in experimental thyroiditis model.

The crude herbal formulation, Gamgungtang (GGT), has been shown to protect animals against a wide range of spontaneously developing or induced autoimmune diseases. We have previously reported that GGT shows marked down-regulation of several experimental autoimmune diseases. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. In this study, in an effort to elucidate the mechanisms by which GGT suppresses EAT, and autoimmunity in general, we investigated the in vivo effects of this drug on the Th1/Th2 lymphocyte balance, which is important for the induction or inhibition of autoreactivity. Naive SJL/J mice were treated orally for 5 days with GGT (80 mg/(kg day)). Spleen cells were obtained at various time points during the treatment period and were stimulated in vitro with concanavalin A. Interleukins IL-4, IL-10 and IL-12, transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) cytokine production was evaluated at the protein levels of the cytokines in the medium and mRNA expressions. A significant upregulation of IL-4, IL-10 and TGF-beta was observed following treatment with GGT, which peaked at day 5 (IL-10) or day 10 (IL-4). On the other hand, IL-12 and IFN-gamma production were either unchanged or decreased. It seems therefore that GGT induces in vivo a shift towards Th2 lymphocytes which may be one of the mechanisms of down-regulation of the autoimmune reactivity in EAT. Our observations indicate that down-regulation of TH1 cytokines (especially IL-12) and enhancement of Th2 cytokine production may play an important role in the control of T-cell-mediated autoimmunity. These data may contribute to the design of new immunomodulating treatments for a group of autoimmune diseases.

Enhancing the in vitro anticancer activity of albendazole incorporated into chitosan-coated PLGA nanoparticles.

To improve the solubility and anticancer activity of albendazole (ABZ), chitosan (CS)-coated poly-dl-lactic-co-glycolic acid (PLGA) nanoparticles were developed. CS was used to coat ABZ-loaded PLGA nanoparticles to enhance both mucoadhesiveness and colloidal stability. CS-coated PLGA nanoparticles were prepared by suspending the nanoparticles in CS solution after solvent diffusion. The CS-coated PLGA nanoparticles were characterized, and ABZ release was studied in vitro from various formulations. The mucoadhesive properties and in vitro anticancer activities of CS-coated PLGA nanoparticles were investigated by measurement of zeta potentials and the MTT assay, respectively. Spherical nanoparticles below 500nm in diameter were successfully prepared; the particle size distribution was narrow. Complete encapsulation of ABZ in CS-coated PLGA nanoparticles was confirmed by SEM, FTIR, DSC, and XRD. The particle sizes of CS-coated PLGA nanoparticles were in the range of 260-480nm; the encapsulation efficiency was 43.4-54.6%; and the yield 58.5-67.8%. The zeta potential of CS-coated nanoparticles was above +27mV and stability was maintained for 4 weeks. At pH 7.4, the in vitro release of ABZ from nanoparticles (P188-5) was 200-fold higher than that from untreated ABZ; this persisted for 12h. Moreover, ABZ release from CS-coated PLGA nanoparticles (P188-CS0.5) was 1.5-fold higher than that from untreated ABZ at pH 1.2. Additionally, the ABZ-loaded CS-coated nanoparticles exhibited superior mucoadhesion and improved cytotoxicity. The results show that CS coating of PLGA nanoparticles may improve the anticancer effect and the mucoadhesive properties of ABZ-loaded nanoparticles.

Exploring the Preparation of Albendazole-Loaded Chitosan-Tripolyphosphate Nanoparticles.

The objective of this study was to improve the solubility of albendazole and optimize the preparation of an oral nanoparticle formulation, using ß-cyclodextrin (ßCD) and chitosan-tripolyphosphate (TPP) nanoparticles. The solubility of albendazole in buffers, surfactants, and various concentrations of acetic acid solution was investigated. To determine drug loading, the cytotoxic effects of the albendazole concentration in human hepatocellular carcinoma cells (HepG2) were investigated. The formulations were prepared by mixing the drug solution in Tween 20 with the chitosan solution. TPP solution was added dropwise with sonication to produce a nanoparticle through ionic crosslinking. Then the particle size, polydispersity index, and zeta potential of the nanoparticles were investigated to obtain an optimal composition. The solubility of albendazole was greater in pH 2 buffer, Tween 20, and ßCD depending on the concentration of acetic acid. Drug loading was determined as 100 µg/mL based on the results of cell viability. The optimized ratio of Tween 20, chitosan/hydroxypropyl ßCD, and TPP was 2:5:1, which resulted in smaller particle size and proper zeta positive values of the zeta potential. The chitosan-TPP nanoparticles increased the drug solubility and had a small particle size with homogeneity in formulating albendazole as a potential anticancer agent.

Combined delivery of the adiponectin gene and rosiglitazone using cationic lipid emulsions.

For the combined delivery of an insulin-sensitizing adipokine; i.e., the ADN gene, and the potent PPARγ agonist rosiglitazone, cationic lipid emulsions were formulated using the cationic lipid DOTAP, helper lipid DOPE, castor oil, Tween 20 and Tween 80. The effect of drug loading on the physicochemical characteristics of the cationic emulsion/DNA complexes was investigated. Complex formation between the cationic emulsion and negatively charged plasmid DNA was confirmed and protection from DNase was observed. The in vitro transfection efficiency and cytotoxicity were evaluated in HepG2 cells. The particle sizes of the cationic emulsion/DNA complex were in the range 230-540 nm and those of the rosiglitazone-loaded cationic emulsion/DNA complex were in the range 220-340 nm. Gel retardation of the complexes was observed when the complexation weight ratios of the cationic lipid to plasmid DNA exceeded 4:1 for both the drug-free and rosiglitazone-loaded complexes. Both complexes stabilized plasmid DNA against DNase. The ADN expression level increased dose-dependently when cells were transfected with the cationic emulsion/DNA complexes. The rosiglitazone-loaded cationic emulsion/DNA complexes showed higher cellular uptake in HepG2 cells depending on the rosiglitazone loading, but not depending on the type of plasmid DNA type such as pVAX/ADN, pCAG/ADN, or pVAX. The drug-loaded cationic emulsion/plasmid DNA complexes were less cytotoxic than free rosiglitazone. Therefore, a cationic emulsion could potentially serve as a co-delivery system for rosiglitazone and the adiponectin gene.

Heat-shock response is associated with enhanced contractility of vascular smooth muscle in isolated rat aorta.

Stress proteins have been implicated in pathological cardiovascular conditions. We hypothesized that a heat-shock response modulates contractility of vascular smooth muscles. Rat aortic ring preparations were mounted in organ baths, exposed to 42 degrees C for 45 min, and subjected to contractions. Expression of HSP70 and phosphorylation of myosin light chain were examined with immunoblots. Heat shock enhanced contractile response to KCl in parallel with HSP70 expression in rat aortic rings from 8 h but not 1 h after the end of heat shock. Heat shock also augmented vascular contractility to phenylephrine whether endothelium was intact or denuded. Treatment of heat shock-preconditioned aortic rings with Bay K8644, a calcium channel activator, but not treatment with phorbol dibutyrate (1 micromol/l), a protein kinase C activator, enhanced contractions of the rings as compared with those of the control. The levels of phosphorylation of myosin light chains after administration of phenylephrine in heat shock-preconditioned tissues were statistically significantly higher than those in control tissues. Pretreatment with wortmannin (300 nmol/l), an inhibitor of myosin light chain kinase, decreased both contractility and phosphorylation of myosin light chains in parallel. However, heat-shock response did not affect relaxation responses to either acetylcholine in endothelium-intact aortic rings or sodium nitroprusside in endothelium-denuded rings. These results suggest that the heat-shock response is associated with enhanced vascular smooth muscle contractility through a modulation of thick-filament regulation.

Thermosensitive and mucoadhesive sol-gel composites of paclitaxel/dimethyl-ß-cyclodextrin for buccal delivery.

The purpose of this study was to develop a buccal paclitaxel delivery system using the thermosensitive polymer Pluronic F127 (PF127) and the mucoadhesive polymer polyethylene oxide (PEO). The anticancer agent paclitaxel is usually used to treat ovarian, breast, and non-small-cell lung cancer. To improve its aqueous solubility, paclitaxel was incorporated into an inclusion complex with (2,6-di-O-methyl)-ß-cyclodextrin (DMßCD). The formation of the paclitaxel inclusion complex was evaluated using various techniques, including x-ray diffractometry (XRD), Fourier-transform infrared (FT-IR) spectrophotometry, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Hydrogels were prepared using a cold method. Concentrations of 18, 20, and 23% (w/v) PF127 were dissolved in distilled water including paclitaxel and stored overnight in a refrigerator at 4 °C. PEO was added at concentrations of 0.1, 0.2, 0.4, 0.8, and 1% (w/v). Each formulation included paclitaxel (0.5 mg/mL). The sol-gel transition temperature of the hydrogels was measured using the tube-inverting method. Drug release from the hydrogels was measured using a Franz diffusion cell containing pH 7.4 phosphate-buffered solution (PBS) buffer at 37 °C. The cytotoxicity of each formulation was measured using the MTT assay with a human oral cancer cell (KB cell). The sol-gel transition temperature of the hydrogel decreased when PF127 was present and varied according to the presence of mucoadhesive polymers. The in vitro release was sustained and the release rate was slowed by the addition of the mucoadhesive polymer. The cytotoxicity of the blank formulation was low, although the drug-loaded hydrogel showed acceptable cytotoxicity. The results of our study suggest that the combination of a PF 127-based mucoadhesive hydrogel formulation and inclusion complexes improves the in vitro release and cytotoxic effect of paclitaxel.

Gd-complexes of 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-1,4,7,10-tetraacetic acid (DOTA) conjugates of tranexamates as a new class of blood-pool magnetic resonance imaging contrast agents.

Gd-complexes of the type [Gd(L)(H(2)O)]·xH(2)O (5a-c), where L is DOTA conjugates of tranexamic acid (4a) and tranexamic esters (4b,c), have been prepared as a new class of MRI blood-pool contrast agents (BPCAs). Thermodynamic stability (K(GdL)) and pharmacokinetic inertness of 5 compare well with or better than those of analogous MRI contrasting agents (CAs) such as Gd-DOTA and Gd-DTPA-BMA. Their R(1)-relaxivities are significantly higher than those of any of the clinically used MRI CAs. T(1)-weighted MR images of mice administered by 5c demonstrate high blood-pool effect with simultaneous contrast enhancement in liver. The structural uniqueness of 5c lies in the fact that it adopts macrocyclic DOTA instead of acyclic DTPA. In addition, 5c is nonionic and makes no resort to aromatic substituent(s) in the chelate backbone for the blood-pool enhancement. The nature of hepatobiliary uptake demonstrated by 5c may be explained in terms of lipophilicity of tranexamate in the chelate (4c). The cell cytotoxicity test shows no toxicity found with 5, suggesting their use as a practical MRI BPCAs.

Roles of MMP/TIMP in regulating matrix swelling and cell migration during chick corneal development.

Tissue remodeling is central to embryonic development. Here, we used immunohistochemistry, Western blotting, and RT-PCR analysis to investigate the roles of matrix metalloproteinases (MMPs) and the related "a disintegrin and metalloproteinase" (ADAM) family proteinases in chick corneal development. While MMP-13 was expressed in developing chick corneas from embryonic day (ED) 5 to ED 10, its inhibitor, tissue inhibitors of metalloproteinase-1 (TIMP-1), was expressed from ED 18 to 2 days post-hatching (P2). Early MMP-13 activity may be associated with degradation of type IX collagen from the primary stroma, which loosens the collagen fibrils and facilitates neural crest (NC) cell migration. The membrane-bound and secreted forms of ADAM10 were both detected throughout corneal development, and active ADAM10 formed a cleavage complex with CD44v6, a CD44 splice variant that is a major cell surface adhesion molecule for hyaluronic acid (HA) and has been implicated in cell migration. Both CD44v6 and its ectodomain cleavage products were detected from ED 5 to ED 14, and a broad-spectrum MMP inhibitor blocked ectodomain cleavage in cultured stromal cells. These findings suggest that ADAM10 mediates CD44v6 cleavage in the developing cornea, facilitating NC cell-derived mesenchymal cell migration. Finally, we identified high levels of active membrane-type 3-MMP (MT3-MMP) in developing corneas at ED 7, ED 14, and ED 18. MT3-MMP takes part in MMP-2 activation and possibly also CD44v6 shedding, suggesting that this pathway may be involved in cell migration. These findings collectively show for the first time that multiple MMPs, ADAMs, and TIMPs appear to functionally interact during corneal development.

Effect of the traditional Korean immunomodulating formulation, Gamguntang (GGT), on experimental thyroiditis model.

The crude herbal formulation, Gamgungtang (GGT), is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on different experimental models of thyroid disease was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the GGT. One surprising fact to emerge was that GGT-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with GGT. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.

Brief exposure to ethanol augments vascular contractility in human chorionic plate arteries.

Heavy alcohol consumption has been known as a risk factor for hypertension, although the mechanism by which alcohol intake causes hypertension remains elusive. We tested the hypothesis that brief exposure to ethanol augments vascular contractility through the stress response in human chorionic plate arteries. Human chorionic plate arteries were mounted in organ baths and exposed to 5% ethanol for 15, 30 or 45 min. Brief exposure for 45 min, but not 15 min, not only augmented contractility to KCl and 5-hydroxytryptamine 5 h after the end of exposure, but also increased the expression of heat shock protein (HSP) 70 in the tissues. Reverse transcription-polymerase chain reaction showed gradual increases of hsp70 mRNA expression, but not heat shock cognate 70 (hsc70), hsp90alpha or glucose regulatory protein 78 (grp78) mRNA expression, in an exposure time-dependent manner 3 h after the end of exposure. These results indicate that ethanol augments vascular contractility through the stress response.

IL-1beta regulates cellular proliferation, prostaglandin E2 synthesis, plasminogen activator activity, osteocalcin production, and bone resorptive activity of the mouse calvarial bone cells.

Interleukin-1beta (IL-1beta) regulates several activities of the osteoblast cells derived from mouse calvarial bone explants in vitro. IL-1beta stimulated cellular proliferation and the synthesis of prostaglandin E2 in the cultured cells in a dose-dependent manner. Furthermore, plasminogen activator activity of the mouse osteoblast was positively affected by IL-1beta in a dose-dependent manner over the dosage range of 0.01 ng-2 ng/mL with a maximal effect being observed at 2 ng/mL. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to vitamin D, two characteristics of the osteoblast phenotype, were significantly antagonized by IL-1beta over a similar dose range. Treatment of mouse calvarial bone cells with IL-1beta resulted in a dose dependent stimulation of bone resorption and the bone resorption induced by IL-1beta was strongly inhibited by calcitonin treatment, indicating osteoclast-mediated bone resorption, suggesting that the bone resorption induced by IL-1beta appears to be osteoclast-mediated. This study supports the role of IL-1beta in the pathological modulation of bone cell metabolism, with regard to implication of the pathogenesis of osteoporosis by IL-1beta.

Spinal NF-kB activation induces COX-2 upregulation and contributes to inflammatory pain hypersensitivity.

Cyclooxygenase-2 (COX-2) is a major contributor to the elevation of spinal prostaglandin E2, which augments the processing of nociceptive stimuli following peripheral inflammation, and dynorphin has been shown to have an important role in acute and chronic pain states. Moreover, the transcription factor, nuclear factor-kappa B (NF-kB), regulates the expressions of both COX-2 and dynorphin. To elucidate the role of spinal NF-kB in the induction of inflammatory pain hypersensitivity, we examined whether activated NF-kB affects pain behavior and the expressions of the mRNAs of COX-2 and prodynorphin following peripheral inflammation. Intrathecal pretreatment with different NF-kB inhibitors, namely, NF-kB decoy or pyrrolidine dithiocarbamate, significantly reduced mechanical allodynia and thermal hyperalgesia following unilateral hindpaw inflammation evoked by complete Freund's adjuvant (CFA). These NF-kB inhibitors also suppressed the activation of spinal NF-kB and the subsequent remarkable elevation of spinal COX-2 mRNA, but not that of prodynorphin mRNA. In addition, the activation of spinal NF-kB following CFA injection was inhibited by intrathecal pretreatments with interleukin-1 beta receptor antagonist or caspase-1 inhibitor. In view of the fact that interleukin-1 beta (IL-1 beta) is the major inducer of spinal COX-2 upregulation following CFA injection, our results suggest that IL-1 beta-induced spinal COX-2 upregulation and pain hypersensitivity following peripheral inflammation are mediated through the activation of the NF-kB-associated pathways.

IL-1beta induces and TGF-beta reduces vitamin D3-induced bone resorption in mouse calvarial bone cells.

Bone cells produce multiple growth factors and cytokines that have effects on bone metabolism and can be incorporated into the bone matrix. The present study was designed to extend these observations by examining the interactions between transforming growth factor-beta (TGF-beta) or interleukin-1beta (IL-1beta) and bone cells in a rat long bone culture model. IL-1beta regulates several activities of the osteoblast cells derived from rat long bone explants in vitro. IL-1beta stimulated cellular proliferation and the synthesis of prostaglandin E2 and plasminogen activator activity in the cultured cells in a dose-dependent manner. TGF-B is present in the bone matrix and potentially can be released during bone resorption. TGF-beta reduced basal bone resorption and inhibited vitamin D3 [1,25(OH)2D3]-induced bone resorption in rat long bone cells. These studies support the role of IL-1beta in the pathological modulation of bone cell metabolism, with regard to implication in the pathogenesis of osteoporosis by IL-1beta, and that TGF-beta is positively inhibiting the bone resorption.

Induction of a TC1-mediated experimental autoimmune thyroiditis by deglycosylated porcine thyroglobulin.

Contribution of cytotoxic T lymphocytes (CTL) to experimental autoimmune thyroiditis (EAT) was well defined (Speidel et al., Eur. J. Immunol. 1997, 27, 2391-2399, Ref. 7). The native porcine thyroglobulin (pTg) showed high sensitivity to endo-o-N-acetylglucosaminidase F (Endo F) and its molecular weights, corresponding to about 330 kDa as a monomer and 660 kDa as a dimer, were reduced to smaller molecular weight forms by Endo F and trifluoromethanesulfonic acid (TMSF). Deglycosylated porcine Tg (dgpTg) and native pTg were injected i.v. into CBA/J mice, without the aid of adjuvants. Both lymphocytic infiltrations of the thyroid glands and levels of Tg-specific CTL were similar to those found in conventional EAT induced by Tg and adjuvants. In contrast, proliferative responses in native pTg and dgpTg-injected mice could not be detected, and titers of antibodies to pTg and dgpTg were 20 times and 30 times lower than that of pTg and adjuvants, respectively. The EAT-inducer CTL belonged to the CD8+ cell subset and exerted their thyroiditogenic potential through release of IFN-gamma. It was concluded that dgpTg-induced EAT is mediated by type 1 cytotoxic T cells (Tcl). Also, results that EAT induction of the glycosylated pTg (gpTg) was much lower than that of dgpTg, suggested that the abberant and incomplete glycosylation of the thyroglobulin is responsible for the induction of autoimmune thyroiditis.

Interleukin-1 and tumor necrosis factor-alpha induce collagenolysis and bone resorption by regulation of matrix metalloproteinase-2 in mouse calvarial bone cells.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. We examined the effects of the two cytokines on the collagenolysis and bone resorption by induction of matrix metalloproteinases (MMPs). The cells were analyzed using zymographic analysis. It was shown that the mouse calvarial osteoblasts constitutively synthesize progelatinase-A (MMP-2). Interleukin-1beta markedly enhanced the messenger RNAs (mRNAs) expression of MMP-2 (gelatinase A), but slightly MMP-9 (gelatinase B), which associated with increases in bone matrix degradation. Both pro- and active-forms of MMP-2 were detected in the conditioned medium collected from calvarial cultures, and IL-1beta markedly stimulated both pro- and active-forms of the MMP-2. The expression of MMP-2 mRNAs could be detected, and they were markedly enhanced by IL-1beta on days 1 and 2. These results demonstrate that the potency of induction of MMP-2 by IL-1beta and TNF-alpha is closely linked to the respective bone-resorbing activity, suggesting that MMP-2-dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines. On the other hand, when the mouse osteoblasts were stimulated with parathyroid hormone, 1,25(OH)2D3, mononuclear cell conditioned medium (MCM) and IL-1 as bone resorption agents, collagenolysis was increased by producing the active gelatinase. Interleukin-1 in stimulating bone resorption was examined using fetal mouse long bone organ culture. Interleukin-1 stimulated bone resorption and produced marked resorption when present simultaneously. Furthermore, treatment of indomethacin and dexamethasone clearly abolished the responses of IL-1alpha and IL-1beta.