Effect of recombinant TRAIL in a murine co-culture system of osteoclastogenesis

Although some experimental evidence has implicated the TRAIL/TRAIL-receptor system in the regulation of osteoclastogenesis, the only available studies performed so far have been performed on isolated pre-osteoclasts, induced to differentiate by the addition of recombinant RANKL and M-CSF. Using a more physiological co-culture system in the absence of exogenous cytokines, we have here demonstrated that recombinant TRAIL inhibits osteoclast formation, but only at relatively high concentrations (500 ng/mL)

Metabolic bone disorders: from bench to bedside

Bone is a complex tissue that have many function, when bone remodeling becomes perturbed we can assist at many pathologies. The key factor in these pathologies is a perturbation of RANKL/RANK/OPG pathways. We have tune up a 3D culture system that have the advantage to produce osteoclast cells more physiological than the administration of the recombinant RANKL protein. This can be useful when study new drugs to avoid the cross-reaction with the exogenous cytokines

Targeting RANKL in the management of bone loss in patient with breast cancer

The receptor activator of nuclear factor-κB ligand (RANKL), its signaling receptor RANK, and its natural decoy receptor OPG are members of the tumour necrosis factor (TNF) and TNF receptor superfamily and are best known for their essential role in controlling osteoclastogenesis. RANKL in bone has also been shown to serve as a chemoattractant for cancer cells, thus explaining the tropism of certain cancers such as breast and prostate cancer to preferentially metastasize to bone. Here, we will discuss the important role of RANKL and its possible role in the management of bone loss in patients with breast cancer

Chairside CAD/CAM Materials: Current Trends of Clinical Uses

Restorative materials are experiencing an extensive upgrade thanks to the use of chair- side Computer-aided design/computer-assisted manufacturing (CAD/CAM) restorations. There- fore, due to the variety offered in the market, choosing the best material could be puzzling for the practitioner. The clinical outcome of the restoration is influenced mainly by the material and its handling than by the fabrication process (i.e., CAD/CAM). Information on the restorative materials performances can be difficult to gather and compare. The aim of this article is to provide an over- view of chairside CAD/CAM materials, their classification, and clinically relevant aspects that en- able the reader to select the most appropriate material for predictable success

BAG3 localizes in axonal structures during neuronal differentiation and is expressed in cellular processes of migrating cells in mouse cerebral cortex

BAG3 protein belongs to the family of co-chaperones involved in protein quality control and in the clearance of misfolded proteins [1]. Few studies have addressed BAG3 distribution and function in the central nervous system (CNS) and little is known about the cellular localization of BAG3 during neuronal differentiation in vitro and migration in vivo. Therefore we analysed by immunofluorescence microscopy the cellular distribution of BAG3 in the PC12 cell model treated or not with NGF and in developing and adult cortex of mice brain. Our results shows that BAG3 localizes mainly in vesicle structures of the neuritic domain during cell differentiation, while in undifferentiated cells it appears confined to the cytoplasm near the nuclear membrane. These observations were corroborated by transmission electron microscopy (TEM) which revealed that in NGF-differentiated PC12 cells, BAG3 localizes into electron-dense vesicles clustered along the axon and showing the typical aspect of the large dense core vesicles (LDCVs). Interestingly, the change of BAG3 localization during neuronal differentiation was associated only to a slight increase in the total BAG3 immunoreactivity as shown by western blot analysis. In order to provide further insights on the role of BAG3 in neuronal differentiation and migration, we also analysed BAG3 localization in mice developing and adult cerebral cortex. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, while in adult brain a low expression was detected in neuronal cell bodies and glial cells. In conclusion, our findings suggest that the presence and differential expression of BAG3 might be required for the correct development of the nervous system as well as for the maintenance of protein homeostasis

Osteoimmunology represents a link between skeletal and immune system

There is a complex interplay between the cells of the immune system and bone. These interactions are not only mediated by the release of cytokines and chemokines but also by direct cell–cell contact. Studies of intracellular signaling mechanisms in osteoclasts have revealed that numerous immunomodulatory molecules are involved in the regulation of bone metabolism. Recently, it was proposed that immunoreceptors found in the immune cells are also an essential signal for osteoclasts activation, along with receptor activator of NF-κB (RANK) ligand (RANKL). Collectively, these and similar observations regarding cross-regulation between the immune and skeletal systems constitute the field of osteoimmunology. Here we briefly highlight core areas of interest and selected recent advances in this field

The endocannabinoid anandamide inhibits colon cancer cell growth by modulating different survival and proliferating pathways

The Endocannabinoid System (ECS) comprising the CB1 and CB2 receptors and their endogenous ligands is a central signalling system regulating food intake and energy balance. It is also present in peripheral tissues where it is involved in cell proliferation and survival. It has been shown that in colon cancer cells, the CB1 receptor antagonist SR171416 reduces colon cancer cell growth by acting as an inverse agonist rather than an antagonist [1]. Starting from this observation and from evidence indicating that some biological responses to cannabinoids depend on estrogen levels and some selective estrogen receptor modulators can bind the CB1 receptor [2], we aimed to study the effects of the CB1 receptor ligand anandamide (AEA) on colon cancer cell proliferation and its ability to modulate some survival and proliferating pathways including Akt, MAPK/ERK and estrogen receptor (ER) b signalling which is the predominant ER pathway in colonic epithelium. We used an AEA-analogue and a selective inhibitor of fatty acid amide hydrolase (FAAH) that enhances intracellular levels of AEA and studied proliferation and cell cycle progression on human adenocarcinoma cells DLD1 and SW620. Our results showed that increased levels of AEA significantly reduced cell proliferation in both cell lines at 24 and 48 h also inducing an S phase cell cycle accumulation. The AEA-induced inhibition of cell growth was mediated by a reduced expression of phoshoAkt and phosphoERK and, at the same time, by an induction of ERβ expression. These data suggest that AEA can reduces colon cancer cell proliferation by interfering with different signalling pathways

Physiology and pathophysiology of the RANKL/RANK system

The bone resorption activity of osteoclasts is regulated at many levels, including differentiation of their monocytes precursors, fusion into multi-nucleated cells, migration to the resorption site, polarization of the mature osteoclasts and assembly of a podosome-based sealing zone. Another function of osteoclasts is relative to the integrity of the actin cytoskeleton, depending on the substratum upon which the osteoclasts are spread. There are two different structures of actin known as podosomes and sealing zone, actived in specialized matrix contacts and delimiting the membrane domain, where the ruffled border is formed. When a dual coculture of murine osteoblasts and murine mononuclear monocytes, in absolute absence of exogenous cytokines and other growth factors, was cultured on glass, the basic architecture of podosomes units and ruffled border was maintained regularly (1). We studied the osteoclast morphology and its behaviour in adhesion and in vesicle traffic by combination of light microscopy immunohistochemistry and transmission electron microscopy (TEM) immunolabeling. The adhesion and the fusion of preosteoclasts were observed by scanner electron microscopy (SEM). The osteoclasts produced by our physiological dual co-culture (without interaction of specific cytokines) are functionally and biologically active TRAP + and multinucleated cells. In fact the role of RANK, expressed by osteoblasts, controls the modulation of OPG bioavailability in the extracellular compartment. The fusion of monocytes is influenced by the presence of osteoblasts, that is based on RANK-RANKL interaction and communication between osteoblasts and preosteoclasts mediated by several molecules (2). Osteoclasts and osteoblasts can make direct contact, allowing membrane-bound ligands and receptors to interact and initiate intracellular signalling. RANKL-RANK complexes are likely internalized via rafts and then degraded in lysosomes. A recent study has shown that membrane-bound RANKL complexes to OPG is internalized by endocytosis process. A potential interaction of RANKL with clathrin components prior to the OPG binding, as shown by the kinetic results, OPG is intracellularly degraded after being internalized: our observation of osteoclast membrane at TEM has shown that immunogold labelled RANKL colocalizes with immunoglod labelled clathrin via the clathrin-coated-pit-mediated (3) pathway and both proteins are degraded by lysosome and proteasome pathway. 1) Nicolin V. et al.,. 2006 J.Mol.Histol.37 :171-177 2) Narducci P., et al.,. Acta Histochem.113(2):73-81. 3) Narducci P., et al., 2010, Eur J Histochem 54(1):e6

Flavonoids and flavonoid-rich natural extracts inhibit cytokine release in cystic fibrosis bronchial epithelial cells by regulating NF-kB pathway

Cystic fibrosis (CF) is a genetic life-shortening condition in Caucasians. Despite being a multi-organ disease, CF is classically diagnosed by symptoms of acute/chronic respiratory disease, with persistent pulmonary infections (Amaral, 2015). CF is caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) protein. Inheritance of mutant CFTR alleles results in surface liquid depletion and defective mucociliary clearance leading to pulmonary failure. Defects in CFTR perturb the regulation of many intracellular signaling pathways including the NF-kB pathway causing excessive production of pro-inflammatory mediators. Current CF therapies are directed to delay CF lung damage by restoring CFTR function and controlling abnormal inflammation. However, only few anti-inflammatory drugs are effective for CF treatments (mainly oral corticosteroids and ibuprofen), these drugs have limited beneficial effects in presence of considerable side effects. Flavonoids have been reported as promising anti-inflammatory drugs and some of them seem to act as CFTR direct activators (Amaral, 2015). From this respect, herbal remedies or plant bioactive molecules may be of great interest. To this aim, we tested the anti-inflammatory activity of apolar extracts from the roots of three Peonia species (Paeoniaceae family), namely P. rockii P. ostii and P. lactiflora, on CFTR ΔF508/ΔF508 CuFi1 cells and normal counterpart. The effects of the extracts on intrinsic as well as TNFα-induced inflammation were evaluated by determining IL-8, IL-6 and RANTES production. Further- more, to study the direct effect of the extracts on NF-kB activation, Human Embryonic Kidney cells were used in transient transfection of NF-kB reporter plasmid and NF-kB activity and cytokine productions were also evaluated. Results showed a significant anti-inflammatory activity of all three Peonia extracts with the P. lactiflora being the most effective. Furthermore, we also tested the anti-inflammatory potential of the pure flavonoid naringin in the same model systems. We found that naringin was able to reduce cytokine release through inhibiting the key enzymes of the NF-kB and MAPK/ ERK pathways. Interestingly, preliminary results on spray dried pharmaceutical formulations of this molecule, show that naringin co-sprayed with leucine improves pharmacological activity of the flavonoid neat raw drug