PTH (1-34) affects bone turnover governed by osteocytes exposed to fluoride.

Exposure to fluoride from environmental sources remains an overlooked, but serious public health risk. In this study, we looked into the role osteocytes play on the mechanism underlying fluoride induced osteopathology. We analyzed bone formation and resorption related genes generated by osteocytes that were exposed to varied doses of fluoride with and without PTH in vitro. Correspondingly, osteogenesis and osteoclastogenesis related genes were also investigated in rats exposed to fluoride for 8 weeks, and the PTH(1-34)was applied at the last 3 weeks to observe its role in regulating bone turnover upon fluoride treatment. The data in vitro indicated that fluoride treatment inhibited Sost expression of mRNA and protein and stimulated RANKL mRNA protein expression as well as the RANKL/OPG ratio in the primary osteocytes. Single PTH treatment played the similar role on expression of these genes and proteins. The PTH combined administration enhanced the action of fluoride treatment on RNAKL/OPG and SOST/Sclerostin. The up-regulation of RANKL and decreasing of Sost induced by fluoride and/or PTH treatment was validated in vivo and suggests that osteocytes are a major source of RANKL and Sost, both of which play essential roles in fluoride affecting osteogenesis and osteoclastogenesis. Expression of Wnt/ß-catenin was up-regulated in both in vitro osteocytes treated with high dose of fluoride and bone tissue of rats in the presence of fluoride and PTH. In vivo, fluoride and single PTH stimulated bone turnover respectively, furthermore, PTH combined with low dose of fluoride treatment reinforced the osteogenesis and osteoclastogenesis genes expression, however, co-treatment of PTH reversed the effect of high dose of fluoride on osteogenesis and osteoclastogenensis related factors. In conclusion, this study demonstrated that osteocytes play a key role in fluoride activated bone turnover, and PTH participates in the process of fluoride modulating SOST/Sclerostin and RANKL expression.

Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins.

Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins. Marked glycoproteins were labeled via bioorthogonal click chemistry and isolated by affinity purification. We assessed two labeling compounds conjugated with either biotin or desthiobiotin and the respective secretome fractions. 356 proteins were quantified using the biotin probe and 463 using desthiobiotin. 59 proteins were found differentially abundant (adjusted p-value ≤0.05, absolute fold change ≥1.5) between inactive and activated T cells using the biotin method and 86 using the desthiobiotin approach, with 31 mutual proteins cross-verified by independent experiments. Moreover, we analyzed the cellular proteome of the same model to demonstrate the benefit of secretome analyses and provide comprehensive data sets of both. 336 proteins (61.3%) were quantified exclusively in the secretome. Data are available via ProteomeXchange with identifier PXD004280.

Identification of Targets of a New Nutritional Mixture for Osteoarthritis Management Composed by Curcuminoids Extract, Hydrolyzed Collagen and Green Tea Extract.

OBJECTIVE: We have previously demonstrated that a mixture of curcuminoids extract, hydrolyzed collagen and green tea extract (COT) inhibited inflammatory and catabolic mediator's synthesis by osteoarthritic human chondrocytes. The objective of this study was to identify new targets of COT using genomic and proteomic approaches. DESIGN: Cartilage specimens were obtained from 12 patients with knee osteoarthritis. Primary human chondrocytes were cultured in monolayer until confluence and then incubated for 24 or 48 hours in the absence or in the presence of human interleukin(IL)-1ß (10-11M) and with or without COT, each compound at the concentration of 4 µg/ml. Microarray gene expression profiling between control, COT, IL-1ß and COT IL-1ß conditions was performed. Immunoassays were used to confirm the effect of COT at the protein level. RESULTS: More than 4000 genes were differentially expressed between conditions. The key regulated pathways were related to inflammation, cartilage metabolism and angiogenesis. The IL-1ß stimulated chemokine ligand 6, matrix metalloproteinase-13, bone morphogenetic protein-2 and stanniocalcin1 gene expressions and protein productions were down-regulated by COT. COT significantly decreased stanniocalcin1 production in basal condition. Serpin E1 gene expression and protein production were down-regulated by IL-1ß. COT reversed the inhibitory effect of IL-1ß. Serpin E1 gene expression was up-regulated by COT in control condition. CONCLUSION: The COT mixture has beneficial effect on osteoarthritis physiopathology by regulating the synthesis of key catabolic, inflammatory and angiogenesis factors. These findings give a scientific rationale for the use of these natural ingredients in the management of osteoarthritis.

Quantification of age-related changes of α-tocopherol in lysosomal membranes in murine tissues and human fibroblasts.

Considering the biological function of α-tocopherol (α-Toc) as a potent protective factor against oxidative stress, this antioxidant is in the focus of aging research. To understand the role of α-Toc during aging we investigated α-Toc concentrations in young and aged primary human fibroblasts after supplementation with RRR-α-Toc. Additionally, α-Toc contents were determined in brain, kidney, and liver tissue of 10 week-, 18 month-, and 24 month-old mice, which were fed a standard diet containing 100 mg/kg dl-α-tocopheryl acetate. α-Toc concentrations in isolated lysosomes and the expression of the α-Toc transport proteins Niemann Pick C1 (NPC1), Niemann Pick C2 (NPC2), and lipoprotein lipase were also analyzed. Obtained data show a significant age-related increase of α-Toc in murine liver, kidney, and brain tissue as well as in human dermal fibroblasts. Also liver and kidney lysosomes are marked by elevated α-Toc contents with aging. NPC1 and NPC2 protein amounts are significantly decreased in adult and aged murine kidney tissue. Also aged human dermal fibroblasts show decreased NPC1 amounts. Supplementation of young and aged fibroblasts led also to decreased NPC1 amounts, suggesting a direct role of this protein in α-Toc distribution. Our results indicate an age-dependent increase of α-Toc in different murine tissues as well as in human fibroblasts. Furthermore saturation and intracellular distribution of α-Toc seem to be strongly dependent on the availability of this vitamin as well as on the presence of the lysosomal protein NPC1. © 2016 BioFactors, 42(3):307-315, 2016.

(D)-Ribose supplementation in the equine: lack of effect on glycated plasma proteins suggesting safety in humans.

BACKGROUND: d-Ribose is a popular dietary supplement for humans and the equine because of its crucial role in cellular bioenergetics. However, as a reducing sugar, it has been suggested that ingestion of d-ribose might promote the formation of glycated proteins in vivo with potential adverse consequences. OBJECTIVE: The aim of this study was to examine if d-Ribose would promote the formation of glycated proteins in vivo following exercise in training thoroughbred racehorses. METHODS: Two groups of horses received the supplement (30 and 50 g d-Ribose daily) for 17 weeks, during which period the horses were subjected to low-intensity exercises followed by high-intensity exercises. Blood samples were analyzed for glycated plasma proteins at baseline and following the 2 exercise regimens. RESULTS: This study shows that long-term ingestion of d-Ribose at 30-50 g a day does not promote the formation of glycated plasma proteins in thoroughbred racehorses. Ribose supplementation also protected the horses from cramping while enhancing muscle recovery at the same time. No adverse effects were reported. CONCLUSION: Ribose supplementation is safe and does not cause glycation in vivo. This investigation also establishes safety of d-Ribose in thoroughbred racehorses, suggesting similar implications in humans as well.

Influence of dietary tender cluster beans (Cyamopsis tetragonoloba) on biliary proteins, bile acid synthesis and cholesterol crystal growth in rat bile.

Tender cluster beans (CBs; Cyamopsis tetragonoloba) are observed to possess anti-lithogenic potential in experimental mice. Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile in addition to supersaturation of cholesterol. This study aimed at evaluating the influence of CB on biliary glycoproteins, low molecular weight (LMW) and high molecular weight (HMW) proteins, cholesterol nucleation time, and cholesterol crystal growth in rat hepatic bile. Groups of rats were fed for 10 weeks with 0.5% cholesterol to render the bile lithogenic. Experimental dietary interventions were: 10% freeze-dried CB, 1% garlic powder or their combination. Incorporation of CB into HCD decreased the cholesterol saturation index in bile, increased bile flow and biliary glycoproteins. Dietary CB prolonged cholesterol nucleation time in bile. Electrophoresis of biliary proteins showed the presence of high concentration of 27 kDa protein which might be responsible for the prolongation of cholesterol nucleation time in the CB fed group. Proteins of 20 kDa and 18 kDa were higher in CB treated animals, while the same were less expressed in HCD group. Biliary proteins from CB fed animals reduced cholesterol crystal growth index which was elevated in the presence of proteins from HCD group. Cholesterol-7α-hydroxylase and cholesterol-27-hydroxylase mRNA expression was increased in CB treated animals contributing to the bile acid synthesis. Thus, the beneficial anti-lithogenic effect of dietary CB which primarily is due to reduced cholesterol saturation index was additionally affected through a modulation of the nucleating and anti-nucleating proteins that affect cholesterol crystallization.

Selenoglycoproteins attenuate adhesion of tumor cells to the brain microvascular endothelium via a process involving NF-κB activation.

Selenium-containing compounds and selenized yeast have anticancer properties. In order to address possible mechanisms involved in these effects, selenoglycoproteins (SGPs) were extracted from selenium-enriched yeast at pH 4.0 and 6.5 (the fractions are called SGP40 and SGP65, respectively), followed by evaluation of their impact on the interactions of lung and breast tumor cells with human brain microvascular endothelial cells (HBMECs). Extracted SGPs, especially SGP40, significantly inhibited adhesion of tumor cells to HBMECs and their transendothelial migration. Because the active components of SGPs are unknown, small selenium-containing compounds [leucyl-valyl-selenomethionyl-arginine (LVSe-MR) and methylselenoadenosine (M-Se-A)], which are normally present in selenized yeast, were introduced as additional treatment groups. Treatment of HBMECs with SGP40, LVSe-MR and M-Se-A induced changes in gene signatures, which suggested a central involvement of nuclear factor (NF)-κB-dependent pathway. These observations were confirmed in the subsequent analysis of NF-κB DNA binding activity, quantitative measurements of the expression of selected genes and proteins, and tumor cell adhesion assay with a specific NF-κB inhibitor as the additional treatment factor. These findings indicate that specific organic selenium-containing compounds have the ability to inhibit tumor cell adhesion to brain endothelial cells via down-regulation of NF-κB. SGPs appear to be more effective than small selenium-containing compounds, suggesting the role of not only selenium but also the glycoprotein component in the observed protective impact.

Pharmacologic induction of epidermal melanin and protection against sunburn in a humanized mouse model.

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection (1). Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis.

BACKGROUND AND AIMS: Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparative studies on structure, transcription regulation and responses to phosphate (Pi) deprivation of the soybean PAP gene family should facilitate further insights into the potential physiological roles of GmPAPs. METHODS: BLAST searches were performed to identify soybean PAP genes at the phytozome website. Bioinformatic analyses were carried out to investigate their gene structure, conserve motifs and phylogenetic relationships. Hydroponics and sand-culture experiments were carried out to obtain the plant materials. Quantitative real-time PCR was employed to analyse the expression patterns of PAP genes in response to P deficiency and symbiosis. KEY RESULTS: In total, 35 PAP genes were identified from soybean genomes, which can be classified into three distinct groups including six subgroups in the phylogenetic tree. The expression pattern analysis showed flowers possessed the largest number of tissue-specific GmPAP genes under normal P conditions. The expression of 23 GmPAPs was induced or enhanced by Pi starvation in different tissues. Among them, nine GmPAP genes were highly expressed in the Pi-deprived nodules, whereas only two GmPAP genes showed significantly increased expression in the arbuscular mycorrhizal roots under low-P conditions. CONCLUSIONS: Most GmPAP genes are probably involved in P acquisition and recycling in plants. Also we provide the first evidence that some members of the GmPAP gene family are possibly involved in the response of plants to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions.

Retinoic acid as a vaccine adjuvant enhances CD8+ T cell response and mucosal protection from viral challenge.

Vaccine-induced memory T cells localized at mucosal sites can provide rapid protection from viral infection. All-trans-retinoic acid (ATRA) has been shown to act physiologically to induce the expression of gut-homing receptors on lymphocytes. We tested whether the administration of exogenous ATRA during a systemic vaccination of mice could enhance the generation of mucosal CD8(+) T cell immunity, which might represent a strategy for establishing better protection from viral infection via mucosal routes. ATRA induced the expression of CCR9 and α4ß7 on both mouse and human CD8(+) T cells activated in vitro. The administration of ATRA to mice during in vivo priming with a replication-defective recombinant adenovirus vector expressing the lymphocytic choriomeningitis virus glycoprotein (LCMVgp) (Ad5gp) increased numbers of both effector and memory T cells in intestinal mucosal tissues and showed higher frequencies of systemic central memory-like T cells that exhibited enhanced proliferation during boosting immunization with recombinant modified vaccinia virus Ankara expressing LCMVgp (MVAgp). Mice that received ATRA during Ad5gp vaccination were more resistant to intravaginal challenge by recombinant vaccinia virus expressing LCMVgp (VVgp), reflecting in part stronger T cell recall responses in situ. Thus, ATRA appears to be useful as an adjuvant during vaccination to increase memory T cell responses and protection from viral infection at mucosal sites and may facilitate the development of more effective vaccines against mucosally transmitted pathogens such as HIV.

Low AZGP1 expression predicts for recurrence in margin-positive, localized prostate cancer.

BACKGROUND: Men with positive margins after radical prostatectomy (RP) for localized prostate cancer (PC) have a 40-50% biochemical relapse rate at 5 years. Adjuvant radiotherapy improves biochemical progression-free and overall survival in men with positive margins, but is associated with increased toxicity. There is an urgent need to identify new prognostic markers to define the group of patients who would benefit from multimodality therapy. METHODS: Nuclear ß-catenin, membranous secreted frizzled-related protein 4 (sFRP4), zinc-alpha 2-glycoprotein (AZGP1), and macrophage inhibitory cytokine-1 (MIC-1) have previously been identified as molecular markers of outcome in localized PC. From these published studies, we identified a subset of patients with positive margins. The aim of this study was to assess the association between these four molecular markers and outcome in men with margin-positive, localized PC. RESULTS: We identified 186 men with positive margins from 330 men with localized PC; 53% had preoperative PSA >10 ng/ml, 72% extraprostatic extension (EPE), 24% seminal vesicles involvement (SVI), and 57% RP Gleason score ≥ 7. AZGP1 (P = 0.009), membranous sFRP4 (P = 0.03) and MIC-1 (P = 0.04) expression predicted for biochemical relapse on univariate analysis. Only absent/low AZGP1 expression (P = 0.01) was an independent predictor of recurrence in margin-positive, localized PC when modeled with preoperative PSA (P = 0.2), EPE (P = 0.2), SVI (P = 0.4), Gleason score ≥ 7 (P = 0.5) and adjuvant treatment (P = 0.4). Furthermore, there was an association between absent/low AZGP1 expression and clinical recurrence (P = 0.007). CONCLUSIONS: AZGP1 is a potential molecular marker for biochemical relapse in men with margin-positive, localized PC. Routine assessment of this biomarker may lead to better selection of patients who will benefit from post-RP radiotherapy.

Regulation of glioblastoma stem cells by retinoic acid: role for Notch pathway inhibition.

It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48 h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell-matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.

Phenolic content and in vitro inhibitory effects on oxidation and protein glycation of some Thai medicinal plants.

Fifteen medicinal plant extracts were investigated for: total phenolic content and free radical scavenging effect by DPPH and ABTS assays; anti-lipid peroxidation activity by TBARS; and for antiglycation activity. The results revealed that the total phenolic content showed good correlation with free radical scavenging by ABTS (r = 0.721) and anti-lipid peroxidation by TBARS (r = -0.659), but showed no correlation with antiglycation. Three extracts from Tamarindus indica, Camellia sinensis and Artocarpus lakoocha demonstrated a significant antioxidant effect, and also showed a promising antiglycation effect. The IC50 (mg/ml) were 0.9-0.16 for the DPPH method; TEAC values (mg Trolox/mg sample) of 1.72-2.83 for the ABTS method; IC50 (mg/ml) of 0.64-1.22 for the TBARS method; and IC50 ranging from 0.01 to 3.20 for the antiglycation method. These three herbs were found to possess effective antioxidant and antiglycation activities, and could be further developed for use in anti-aging cosmetics.

Monoclonal antibodies to rhamnogalacturonan I backbone.

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides--BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [-->2)-alpha-L-rhamnosep-(1-->4)-alpha-D-galacturonic acid p-(1-->](7) structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose-galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.

Optimization and validation of FePro cell labeling method.

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 microg/ml of Fe and 3 microg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 microg/ml of Fe and 3 microg/ml of Pro is effective in labeling cells for cellular MRI.

Chimeric pestiviruses: candidates for live-attenuated classical swine fever marker vaccines.

The use of attenuated classical swine fever virus (CSFV) strains as live vaccines is no longer allowed for the control of classical swine fever in Europe, due to the inability to differentiate between infected and vaccinated animals (Differentiating Infected from Vaccinated Animals; DIVA), except as emergency vaccines or as bait vaccines for wild boars. Thus, the establishment of a DIVA vaccine(s) is of pivotal importance for the control of this infectious disease. In this study, recombinant versions of the live-attenuated vaccine strain CSFV Riems were generated by replacing parts of the E2 gene with the corresponding sequence of border disease virus strain Gifhorn. Three cDNA clones were constructed: pRiems-ABC-Gif, pRiems-A-Gif and pRiems-BC-Gif. Infectious particles were obtained from clones pRiems-ABC-Gif and pRiems-BC-Gif only, whereas transfected RNA from clone pRiems-A-Gif behaved like a replicon. Based on its ability to be differentiated in vitro from wild-type CSFV by mAbs, vRiems-ABC-Gif was assessed for immunogenicity and protection against challenge infection in pigs. Before challenge, no CSFV-specific anti-E2 antibodies could be detected with commercial E2-blocking ELISAs in vRiems-ABC-Gif-vaccinated animals, whereas vRiems-vaccinated pigs developed high titres of anti-E2 antibodies, confirming the marker properties of this vaccine candidate. After oral vaccination, only partial protection against challenge infection was observed in the vRiems-ABC-Gif vaccinees, whereas all intramuscularly vaccinated animals and all vRiems-vaccinated animals were fully protected. These experiments suggest that the strategy of exchanging specific antigenic epitopes among pestiviruses is a promising tool for the development of new CSFV marker vaccines.

[Effects of soybean isoflavones on expression levels of osteoprotegerin and osteoprotegerin ligand mRNAs in bone tissues of ovariectomized rats].

OBJECTIVE: To investigate the effects of soybean isoflavones (SI) on expression levels of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) mRNAs in bone tissues of ovariectomized rats, and to discuss the mechanism of soybean isoflavaones in preventing and treating postmenopausal osteoporosis (PMO). METHODS: Thirty adult SD rats were randomly divided into 3 groups: sham-operation group, untreated group and SI-treated group. The rats in the last two groups were bilaterally ovariectomized. The bone density of L(3) to L(6) vertebrae was detected after 12-week intervention. Total RNA was extracted from femur head and the expression levels of OPG and OPGL mRNAs were examined by real-time quantitative polymerase chain reaction. RESULTS: SI could increase the bone density of lumbar vertebrae in ovariectomized rats, up-regulate the expression level of OPG mRNA and down-regulate the ratio of OPGL mRNA/OPG mRNA, but exert no significant effect on the expression of OPGL mRNA. CONCLUSION: The therapeutic effects of SI on PMO may be related to regulating the expression levels of OPG and OPGL mRNAs, and the ratio of OPGL mRNA/OPG mRNA.

[Effects of kangfengshi granules on expressions of osteoprotegerin, RANKL and M-CSF in bone tissues of rats with collagen-induced arthritis].

OBJECTIVE: To observe the effects of Kangfengshi Granules (KFSG) on expressions of the mRNAs of osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) in bone tissues of rats with collagen-induced arthritis. METHODS: Forty SD rats were randomly divided into four groups: normal control group, untreated group, cyclosporine A (CsA)-treated group and KFSG-treated group. Except the rats in the normal control group, all the other rats received subcutaneous injection of collagen II to establish collagen-induced arthritis (CIA) models. Then the rats in each group were fed normal saline or corresponding drugs for four weeks. Total RNA was extracted from carpal and digital bones. The expressions of OPG, RANKL and M-CSF mRNAs were examined by real-time PCR. RESULTS: The total incidence of arthritis induced by collagen II in the rats was approximately 90%. The expression levels of RANKL and M-CSF mRNAs and the RANKL mRNA/OPG mRNA ratio in the untreated group, KFSG-treated group and CsA-treated group were all significantly higher than those in the normal control group, while the expression levels of OPG mRNA in those three groups were significantly lower than that in the normal control group. The expression level of OPG mRNA in the KFSG-treated group was obviously higher while the expression level of M-CSF mRNA and the RANKL mRNA/OPG mRNA ratio in the same group were both lower as compared with those in the untreated group. CONCLUSION: The molecular mechanism of effects of KFSG on bone erosion and destruction induced by rheumatoid arthritis is closely correlated with up-regulating the expression of OPG mRNA, down-regulating the expression of M-CSF mRNA and RANKL mRNA/OPG mRNA ratio.

[Effect of Herba Epimedii flavone on the osteoblasts metabolism in vitro].

OBJECTIVE: To explore the effect of Herba Epimedii flavone (HEF) on the osteoblast metabolism in vitro. METHOD: Osteoblast were obtained from new born rat calvaria by digestive enzymes. MTF, PNPP and RT-PCR were used to observe the proliferation, activity of ALP and mRNA expression of OPG and RANKL of cultured osteoblasts in vitro. RESULT: It was found that HEF had the effect on stimulating cell proliferation, activity of ALP and the mRNA expression of OPG of cultured osteoblasts (P < 0.01, P < 0.05). CONCLUSION: HEF can promote the proliferation, the differentiation and the expression of OPG mRNA of the osteoblasts cultured in vitro.

Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis.

OBJECTIVE: Intraarticular corticosteroids are frequently used as successful adjuvant therapy for inflammatory arthritides, but little is known about their effects on molecules that regulate bone biology. We undertook this study to investigate the effect of intraarticular corticosteroids on the synovial expression of RANKL and osteoprotegerin (OPG). METHODS: We evaluated RANKL, OPG, and surface marker expression by immunohistochemical methods in synovial knee biopsy samples obtained from 13 patients with inflammatory arthritis before and 2 weeks following intraarticular injection of triamcinolone hexacetonide. We further investigated the effect of dexamethasone (DEX) on RANKL expression by lymphocytes from rheumatoid arthritis synovial fluids (RA SF), using flow cytometric analysis. Finally, we evaluated the in vitro effect of DEX on RANKL and OPG expression in osteoblast-like cells, by Western blotting. RESULTS: Intraarticular corticosteroids induced a decrease in the number of synovial T cells without influencing the number of macrophages, evaluated as both CD68+ and CD163+ cells. This change was paralleled by a decrease of synovial RANKL expression with a concomitant reduction of the RANKL:OPG ratio. DEX down-regulated RANKL expression on lymphocytes derived from RA SF. Moreover, in vitro pretreatment of osteoblast-like cells with tumor necrosis factor favored an antiresorptive effect of DEX treatment through a similar down-regulation of RANKL expression. CONCLUSION: The decrease in inflammation attributed to intraarticular corticosteroids is accompanied by down-modulation of bone destruction markers. These findings offer a rationale for the beneficial effect of corticosteroids on joint erosion in arthritis.