Targeting Regulatory T Cells by Addressing Tumor Necrosis Factor and Its Receptors in Allogeneic Hematopoietic Cell Transplantation and Cancer.

An intricate network of molecular and cellular actors orchestrates the delicate balance between effector immune responses and immune tolerance. The pleiotropic cytokine tumor necrosis factor-alpha (TNF) proves as a pivotal protagonist promoting but also suppressing immune responses. These opposite actions are accomplished through specialist cell types responding to TNF via TNF receptors TNFR1 and TNFR2. Recent findings highlight the importance of TNFR2 as a key regulator of activated natural FoxP3+ regulatory T cells (Tregs) in inflammatory conditions, such as acute graft-vs.-host disease (GvHD) and the tumor microenvironment. Here we review recent advances in our understanding of TNFR2 signaling in T cells and discuss how these can reconcile seemingly conflicting observations when manipulating TNF and TNFRs. As TNFR2 emerges as a new and attractive target we furthermore pinpoint strategies and potential pitfalls for therapeutic targeting of TNFR2 for cancer treatment and immune tolerance after allogeneic hematopoietic cell transplantation.

Genetic analysis of a novel antioxidant multi-target iron chelator, M30 protecting against chemotherapy-induced alopecia in mice.

BACKGROUND: Chemotherapy-induced alopecia has been well documented as a cause of distress to patients undergoing cancer treatment. Almost all traditional chemotherapeutic agents cause severe alopecia. Despite advances in the treatment of chemotherapy-induced alopecia, there is no effective treatment for preventing chemotherapy-induced alopecia. METHODS: In the present study, we investigated the potential role of a multi-target iron chelator, M30 in protecting against cyclophosphamide-induced alopecia in C57BL/6 mice implanted with an osmotic pump. M30 enhanced hair growth and prevented cyclophosphamide-induced abnormal hair in the mice. Furthermore, we examined the gene expression profiles derived from skin biopsy specimens of normal mice, cyclophosphamide-treated mice, and cyclophosphamide treated mice with M30 supplement. RESULTS: The top genes namely Tnfrsf19, Ercc2, Lama5, Ctsl, and Per1 were identified by microarray analysis. These genes were found to be involved in the biological processes of hair cycle, hair cycle phase, hair cycle process, hair follicle development, hair follicle maturation, hair follicle morphogenesis, regulation of hair cycle. CONCLUSION: Our study demonstrates that M30 treatment is a promising therapy for cyclophosphamide-induced alopecia and suggests that the top five genes have unique preventive effects in cyclophosphamide-induced transformation.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.

Effect of puerarin on expression of Fas/FasL mRNA in pulmonary injury induced by ischemia-reperfusion in rabbits.

The effect of puerarin (Pur) on expressions of Fas/FasL mRNAs in pulmonary ischemia and reperfusion injury (PIRI) in rabbit was investigated. The sole side lung ischemia and reperfusion model was used. Rabbits were randomly divided into three groups, a sham operated group (sham, n = 10), PIR group (IR, n = 30) and PIR + Pur group (Pur, n = 30). Changes of several parameters including apoptotic index (AI), wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA) were measured after 60, 180 and 300 minutes of reperfusion. Meanwhile, the location and expression of Fas/FasL mRNA were investigated. Lung tissue was prepared for light microscopic and electron microscopic observation after 60, 180 and 300 minutes of reperfusion. Compared with group IR, Fas/FasL mRNAs were slightly expressed in intima and extima of small pulmonary artery, alveoli, and bronchiole epithelia in group Pur. The values of AI, W/D and IQA were significantly lower than those in group IR after 60, 180, and 300 minutes of reperfusion in lung tissue (P <0.01 or P <0.05). Meanwhile, the abnormal changes in lung tissue morphology were markedly less in group Pur. Puerarin notably protects lung from PIRI by inhibiting Fas/FasL mRNA expression and decreasing lung cell apoptosis in rabbits.

Chronic activation of FXR in transgenic mice caused perinatal toxicity and sensitized mice to cholesterol toxicity.

The nuclear receptor farnesoid X receptor (FXR) (nuclear receptor subfamily 1, group H, member 4, or NR1H4) is highly expressed in the liver and intestine. Previous reports have suggested beneficial functions of FXR in the homeostasis of bile acids, lipids, and glucose, as well as in promoting liver regeneration and inhibiting carcinogenesis. To investigate the effect of chronic FXR activation in vivo, we generated transgenic mice that conditionally and tissue specifically express the activated form of FXR in the liver and intestine. Unexpectedly, the transgenic mice showed several intriguing phenotypes, including partial neonatal lethality, growth retardation, and spontaneous liver toxicity. The transgenic mice also displayed heightened sensitivity to a high-cholesterol diet-induced hepatotoxicity but resistance to the gallstone formation. The phenotypes were transgene specific, because they were abolished upon treatment with doxycycline to silence the transgene expression. The perinatal toxicity, which can be rescued by a maternal vitamin supplement, may have resulted from vitamin deficiency due to low biliary bile acid output as a consequence of inhibition of bile acid formation. Our results also suggested that the fibroblast growth factor-inducible immediate-early response protein 14 (Fn14), a member of the proinflammatory TNF family, is a FXR-responsive gene. However, the contribution of Fn14 induction in the perinatal toxic phenotype of the transgenic mice remains to be defined. Because FXR is being explored as a therapeutic target, our results suggested that a chronic activation of this nuclear receptor may have an unintended side effect especially during the perinatal stage.

Inflammasome-dependent and -independent IL-18 production mediates immunity to the ISCOMATRIX adjuvant.

Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1ß and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.

Expression of TweakR in breast cancer and preclinical activity of enavatuzumab, a humanized anti-TweakR mAb.

BACKGROUND: The receptor for the cytokine TWEAK (TweakR) is a cell surface member of the tumor necrosis factor receptor superfamily with diverse biological roles. TNFRSF family members are appealing therapeutic targets in oncology due to their aberrant expression and function in tumor cells. The goal of the current study was to examine the potential of TweakR as a therapeutic target in breast cancer. METHODS: Expression of TweakR in primary breast cancer tissues and metastases was characterized using immunohistochemistry. To determine the functional relevance of TweakR, breast cancer cell lines were treated in vitro and in vivo with enavatuzumab, a humanized mAb against TweakR. RESULTS: Overexpression of TweakR was observed in infiltrating tumors compared to normal adjacent breast tissues, and strong staining of TweakR was observed in all subtypes of invasive ductal breast cancer. In addition, a positive correlation of TweakR and HER2 expression and co-localization were observed, irrespective of ER status. TweakR expression was also observed in bone metastasis samples from primary breast cancer but rarely in benign tumors. Enavatuzumab inhibited the in vitro growth of TweakR-expressing breast cancer cell lines, and this activity was augmented by cross-linking the mAb. In addition, enavatuzumab significantly inhibited the in vivo growth of multiple breast cancer xenograft models including a model of metastasis. CONCLUSIONS: TweakR is highly expressed in all subtypes of invasive ductal breast cancer, and enavatuzumab administration exhibited a dose-dependent inhibition of primary tumor growth and lung metastasis and enhanced the antitumor activity of several chemotherapy agents currently used to treat breast cancer. These data provide the rationale to evaluate enavatuzumab as a potential therapy for the treatment of breast cancer.

[Research on the changes of IL-1 receptor and TNF-alpha receptor in rats with cerebral ischemia reperfusion and the chronergy of acupuncture intervention].

OBJECTIVE: To explore the intervention timing of acupuncture in treatment of cerebral infarction and the relationship of cerebral ischemia reperfusion injury with inflammatory cytokine receptor. METHODS: One hundred and ten male healthy Wistar rats were randomly divided into a normal group (n=10), a sham operation group (n=10), a model group (n=10), an acupuncture at non-acupoint group (non-acupoint group, n=40), an acupuncture with regaining consciousness method group (regaining consciousness group, n=40). Four subgroups were set up 1 h ischemia reperfusion in 1 h group, 3 h group, 6 h group, 12 h group in the two acupuncture groups, 10 rats in each subgroup. Two acupuncture groups were treated with acupuncture at four time points (1 h, 3 h, 6 h and 12 h after ischemia reperfusion), and "Shuigou" (GV 26) and "Neiguan" (PC 6) were selected in regaining consciousness group, and the non-acupoints below the bilateral costal region were selected in non-acupoint group. At the corresponding time point, the tissues of the brain were removed and interleukin1 receptor (IL-1RI) and tumor necrosis factor receptor (TNFR-I) mRNA and protein changes were detected by using real-time quantitative polymerase chain reaction and immunoblot assay. RESULTS: The expression of IL-1RI and TNFR-I mRNA and protein in the model group were significantly higher than that in normal group, sham operation group, regaining consciousness group and non-acupoint group (P<0.01, P<0.05). The expression of IL-1RI and TNFR-I mRNA and protein in regaining consciousness group was weakest at 3 h after reperfusion followed successively by 6 h, 1 h, 12 h, and there was no significantly change of IL-1RI and TNFR-I mRNA and protein expression in non-acupoint group among different timing points, but which was decreased as compared with those in the model group at the same time point (all P<0.05). CONCLUSION: Acupuncture can reduce the expression of IL-1RI and TNFR-I mRNA and protein in rats with cerebral ischemia reperfusion, inhibit the excessive expression of proinflammatory cytokine receptor, block apoptosis signal transduction and extend time window for treatment of cerebral ischemia, so as to play the protective effect for brain. Within 3 h of ischemia is the best time for intervention of acupuncture treatment.

Phenotypic and functional maturation of murine dendritic cells (DCs) induced by purified Glycyrrhizin (GL).

The aim of this study is to investigate phenotypic and functional modulation of murine dendritic cells (DCs) with use of purified Glycyrrhizin (GL). These impacts of GL on DCs both from bone marrow derived DCs and established DC cell 2.4 were assessed with conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that the purified GL induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD80, CD83 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs2.4 cells were down- regulated after treatment with GL (which occurs when phagocytosis of DCs2.4 cells were decreased). Finally, we proved that GL increased the production of IL-12, IL-10 and decreased the production of tumor necrosis factor alpha (TNF-α). These data indicated that GL could promote maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that GL could exert positive modulation on murine DCs.

Purified cranberry proanthocyanidines (PAC-1A) cause pro-apoptotic signaling, ROS generation, cyclophosphamide retention and cytotoxicity in high-risk neuroblastoma cells.

Optimized purification of oligomeric proanthocyanidines (PAC) from cranberry generated PAC-1A which selectively affected the viability of various neuroblastoma (NB) cell lines representing a spectrum of high-risk NB features. PAC-1A caused a loss of mitochondrial transmembrane depolarization potential (∆Ψm) and increased generation of reactive oxygen species (ROS) which was directly correlated to the modulation of apoptotic marker proteins in SMS-KCNR cells. PAC-1A reduced the expression of pro-survival (Bcl-2, MCL-1, Bcl-xL) and increased levels of pro-apoptotic (Bax, Bad, Bid) Bcl family proteins, upregulated the activity of SAPK/JNK MAPK and downregulated expression or activity of PI3K/AKT/mTOR pathway components. PAC-1A increased the cellular uptake/retention of cyclophosphamide (CP). PAC-1A and CP synergistically increased cytotoxicity and expression of pro-apoptotic markers, reduced cellular glutathione (GSH) and superoxide dismutase (SOD) levels. Additional features of PAC-1A as an anticancer drug as shown in SMS-KCNR NB cells include delay of cell cycle progression and induction of cell death via TNF-family death receptor activity, thus, targeting both the extrinsic and intrinsic pathway of apoptosis. PAC-1A partially blocked the cell cycle in G2/M phase which correlated with a decrease of the G0/G1 subpopulation, upregulation of cyclin D1 and downregulation of CDK6 and p27 expression. In summary, PAC-1A has demonstrated chemotherapeutic potential to treat a broad spectrum of NBs including highly malignant tumors that show resistance to standard chemotherapeutics and apoptotic stimuli.

A non-innovator version of etanercept for treatment of arthritis.

Etanercept is a soluble tumor necrosis factor (TNF) receptor originally approved for treatment of moderate-to-severe rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis. We have developed a non-innovator version of the recombinant protein etanercept, with the investigational name AVG01 (trade name AVENT™), using a novel expression vector-based technology. Here we show, by extensive analytical characterization, that AVG01 is highly similar to the reference product Enbrel® and demonstrates similar efficacy in pre-clinical studies.

Constituents of Amoora cucullata with TRAIL resistance-overcoming activity.

In search of bioactive natural products for overcoming TRAIL resistance from natural resources, we previously reported a number of active compounds. Bioassay-guided fractionation of mangrove, Amoora cucullata, collected from Sundarbans Mangrove Forest, Bangladesh, led to the isolation of four new compounds (1-4), along with seven known compounds (5-11). Of the isolates, compounds 1, 5, 8, and 9 showed TRAIL resistance-overcoming activity, among which 8 showed the most potent activity and enhanced TRAIL-induced apoptosis in TRAIL-resistant human gastric adenocarcinoma (AGS) cells through the activation of caspase-3/7, enhancing the expression of DR4 and DR5 mRNA in AGS cells. Cell death caused by the combined treatment of 8 and TRAIL was inhibited by human recombinant DR5/Fc and DR4/Fc chimera proteins, indicating that 8 sensitizes TRAIL-resistant AGS cells to TRAIL through the induction of DR4 and DR5.

Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells.

UNLABELLED: Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte-mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our murine choline-deficient, ethionine-supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor-inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE-induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2-week-CDE-treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin-positive cells. Stimulation of an Fn14-positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NFkappaB) activation and dose-dependent proliferation, which was diminished after targeting of the p50 NFkappaB subunit by RNA interference. CONCLUSION: TWEAK acts directly and stimulates LPC mitosis in an Fn14-dependent and NFkappaB-dependent fashion, and signaling via this pathway mediates the LPC response to CDE-induced injury and regeneration.

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells.

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X(L) and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.

[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.

Influence of baicalin on the expression of receptor activator of nuclear factor-kappaB ligand in cultured human periodontal ligament cells.

BACKGROUND: Baicalin is a flavonoid purified from the medicinal plant Scutellaria baicalensis Georgi. It has been reported that baicalin exhibits antibacterial, anti-inflammatory and analgesic effects and can inhibit nuclear factor-kappaB activation. Periodontal disease is a chronic infective disease of the periodontium caused by bacteria present in dental plaque inducing alveolar bone resorption until teeth are lost. Human periodontal ligament (HPDL) is the connective tissue between alveolar bone and tooth. Receptor activator of nuclear factor-kappaB ligand (RANKL), a member of the tumor necrosis factor (TNF) ligand family, plays an important role in osteoclastogenesis from osteoclast precursors to mature osteoclasts. In this study we investigate the effects of baicalin on RANKL protein production and messenger RNA (mRNA) expression induced by IL-1beta in cultured HPDL cells. METHODS: To induce RANKL expression, IL-1beta was added to serum-free medium HPDL cells and incubated. Various concentrations of baicalin (0, 0.001, 0.01 and 0.1 microg/ml) were added to the medium and the cells were treated for 0, 12, 24, 48 and 72 h, respectively. RANKL in the cells was detected using immunocytochemistry. The mRNA of RANKL, osteoprotegerin (OPG) and cyclooxygenase-2 (COX-2) were measured by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: The expression of RANKL at mRNA and protein levels in HPDL cells was stimulated by IL-1beta. Baicalin suppressed IL-1beta-induced RANKL and COX-2 production at a concentration of 0.01 microg/ml. The most prominent effect was observed with 48 h of baicalin treatment. The inhibition of baicalin on the rhIL-1beta-stimulated OPG expression was first apparent at 24 h after the start of treatment, however it did not reach significant differences. CONCLUSIONS: The data suggest that baicalin may inhibit RANKL mRNA expression via the suppression of COX-2 expression induced by IL-1beta. In addition to its antibacterial and anti-inflammatory properties, baicalin was shown to be effective in periodontitis and alveolar bone resorption.

Regulation of osteoclastogenesis by gap junction communication.

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.

Resorption of auditory ossicles and hearing loss in mice lacking osteoprotegerin.

Bones conduct sound in the middle ear. The three ossicles-the malleus, incus, and stapes-form a chain that transmits vibrations from the tympanic membrane to the oval window of the inner ear. Little is known about bone remodeling events in these ossicles and about potential effects of osteoporosis on hearing loss. Osteoclastic bone resorption is enhanced in Opg(-/-) mice lacking osteoprotegerin, which is a soluble decoy receptor for the osteoclastogenic cytokine RANKL. We asked whether auditory ossicles are resorbed in Opg(-/-) mice, and whether these mice suffer from impaired auditory function. All three ossicles in Opg(-/-) mice showed thinning, especially at the malleal manubrium and incus body. Most notably, unlike in the case in wild-type mice, the junction between the stapes and the otic capsule was fixed in Opg(-/-) mice, and the stapedial footplate was thinner and broader. Radiological analyses revealed that malleal cortical thickness was positively correlated with tibial bone mineral density in Opg(-/-) and control littermate mice. Furthermore, progressive hearing loss was detected in Opg(-/-) mice starting at 6 to 15 weeks of age. These data suggest that osteoprotegerin plays a crucial role in hearing by protecting the auditory ossicles and otic capsule from osteoclastic bone resorption.