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ABSTRACTGlycosphingolipids (GSLs) are key constituents of membrane bilayers playing a role in structural integrity, cell signalling in microdomains, endosomes and lysosomes, and cell death pathways. Conversion of ceramide into GSLs is controlled by GCS (glucosylceramide synthase) and inhibitors of this enzyme for the treatment of lipid storage disorders and specific cancers. With a diverse range of functions attributed to GSLs, the ability of the GSC inhibitor, eliglustat, to reduce myeloma bone disease was investigated. In pre-clinical models of multiple myeloma, osteoclast-driven bone loss was reduced by eliglustat in a mechanistically separate manner to zoledronic acid, a bisphosphonate that prevents osteoclast-mediated bone destruction. Autophagic degradation of TNF receptor-associated factor 3 (TRAF3), a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. By altering GSL composition, eliglustat prevented lysosomal degradation whilst exogenous addition of missing GSLs rescued TRAF3 degradation to restore osteoclast formation in bone marrow cells from myeloma patients. This work highlights the clinical potential of eliglustat as a therapy for myeloma bone disease. Furthermore, using eliglustat as a lysosomal inhibitor in osteoclasts may widen its therapeutic uses to other bone disorders such as bone metastasis, osteoporosis and inflammatory bone loss.

The use of endosseous implants is firmly established in skeletal reconstructive surgery, with rapid and permanent fixation of prostheses being a highly desirable feature. Implant coatings composed of hydroxyapatite (HA) have become the standard and have been used with some success in prolonging the time to revision surgery, but aseptic loosening remains a significant issue. The development of a new generation of more biologically active coatings is a promising approach for tackling this problem. Bioactive glasses are an ideal candidate material due to the osteostimulative properties of their dissolution products. However, to date, they have not been formulated with stability to devitrification or thermal expansion coefficients (TECs) that are suitable for stable coating onto metal implants while still retaining their bioactive properties. Here, we present a strontium-substituted bioactive glass (SrBG) implant coating which has been designed to encourage peri-implant bone formation and with a TEC similar to that of HA. The coating can be successfully applied to roughened Ti6Al4V and after implantation into the distal femur and proximal tibia of twenty-seven New Zealand White rabbits for 6, 12, or 24 weeks, it produced no adverse tissue reaction. The glass dissolved over a 6 week period, stimulating enhanced peri-implant bone formation compared with matched HA coated implants in the contralateral limb. Furthermore, superior mechanical fixation was evident in the SrBG group after 24 weeks of implantation. We propose that this coating has the potential to enhance implant fixation in a variety of orthopedic reconstructive surgery applications.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin−cKit+Sca1+ Flk2− (LKS Flk2−) and long-term culture–initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin−cKit+Sca1+Flk2−(LKS Flk2−) and long-term culture–initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

Chronic inflammation, as seen in conditions such as rheumatoid arthritis and Crohn disease, is in part driven by discordant production of inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6 (IL-6). Tyrosine kinase activity is essential to lipopolysaccharide-induced cytokine production in monocytes, and previous studies by us and others have implicated a role for the Tec kinase Bruton's tyrosine kinase (Btk) in inflammatory cytokine production. Here we show that knockdown of Btk using RNA interference results in decreased tumor necrosis factor-α, but not IL-6 production. Further investigations into the signaling mechanisms regulating IL-6 production led to the discovery that the Tec kinase bone marrow tyrosine kinase gene in chromosome X (Bmx) regulates Toll-like receptor-induced IL-6 production. Our data further showed that Bmx-dependent super-induction of IL-6 does not involve nuclear factor–κB activity. More detailed investigations of pathways downstream of Bmx signaling revealed that Bmx targets the IL-6 3′ untranslated region to increase mRNA stabilization via a novel, thus far undefined, p38 mitogen activated protein kinase-independent pathway. These data have important implications for the design of therapeutics targeted against specific cytokines and their regulators in inflammatory disease.

Chronic inflammation, as seen in conditions such as rheumatoid arthritis and Crohn disease, is in part driven by discordant production of inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6 (IL-6). Tyrosine kinase activity is essential to lipopolysaccharide-induced cytokine production in monocytes, and previous studies by us and others have implicated a role for the Tec kinase Bruton's tyrosine kinase (Btk) in inflammatory cytokine production. Here we show that knockdown of Btk using RNA interference results in decreased tumor necrosis factor-α, but not IL-6 production. Further investigations into the signaling mechanisms regulating IL-6 production led to the discovery that the Tec kinase bone marrow tyrosine kinase gene in chromosome X (Bmx) regulates Toll-like receptor-induced IL-6 production. Our data further showed that Bmx-dependent super-induction of IL-6 does not involve nuclear factor–κB activity. More detailed investigations of pathways downstream of Bmx signaling revealed that Bmx targets the IL-63′ untranslated region to increase mRNA stabilization via a novel, thus far undefined, p38 mitogen activated protein kinase-independent pathway. These data have important implications for the design of therapeutics targeted against specific cytokines and their regulators in inflammatory disease.

Osteoblasts are a key component in the regulation of the hematopoietic stem cell (HSC) niche. Manipulating osteoblast numbers results in a parallel change in HSC numbers. We tested the activity of strontium (Sr), a bone anabolic agent that enhances osteoblast function and inhibits osteoclast activity, on hematopoiesis. In vitro treatment of primary murine osteoblasts with Sr increased their ability to form bone nodules, and in vivo it increased osteoblast number, bone volume, and trabecular thickness and decreased trabecular pattern factor. However, the administration of Sr had no influence on primitive HSCs, although the number of hematopoietic progenitors was higher than in control cells. When Sr-treated mice were used as donors for HSC transplantation, no difference in the engraftment ability was observed, whereas hematopoietic recovery was delayed when they were used as recipients. Despite the changes in osteoblast numbers, no increment in the number of N-cadherin+ osteoblasts and N-cadherin transcripts could be detected in Sr-treated mice. Therefore, increasing the overall number and function of osteoblasts without increasing N-cadherin+ cells is not sufficient to enhance HSC quantity and function. Our study further supports the notion that N-cadherin+ osteoblasts are fundamental in the hematopoietic niche.

Osteoblasts are a key component in the regulation of the hematopoietic stem cell (HSC) niche. Manipulating osteoblast numbers results in a parallel change in HSC numbers. We tested the activity of strontium (Sr), a bone anabolic agent that enhances osteoblast function and inhibits osteoclast activity, on hematopoiesis. In vitro treatment of primary murine osteoblasts with Sr increased their ability to form bone nodules, and in vivo it increased osteoblast number, bone volume, and trabecular thickness and decreased trabecular pattern factor. However, the administration of Sr had no influence on primitive HSCs, although the number of hematopoietic progenitors was higher than in control cells. When Sr-treated mice were used as donors for HSC transplantation, no difference in the engraftment ability was observed, whereas hematopoietic recovery was delayed when they were used as recipients. Despite the changes in osteoblast numbers, no increment in the number of N-cadherin+osteoblasts and N-cadherin transcripts could be detected in Sr-treated mice. Therefore, increasing the overall number and function of osteoblasts without increasing N-cadherin+cells is not sufficient to enhance HSC quantity and function. Our study further supports the notion that N-cadherin+osteoblasts are fundamental in the hematopoietic niche.

Mesenchymal stem cells have the capacity to differentiate into several mesenchymal tissues, including the components of the hematopoietic stem cell niche. Mesenchymal stem cells also exhibit a powerful immunosuppressive activity. Here we review the most recent data to identify the properties of therapeutic significance.

We determined that sFRP‐1 mRNA was differentially expressed by osteoblast/stromal cell lines and that sFRP‐1 neutralizing antibodies and siRNA complementary to sFRP‐1 coding sequence enhanced, while recombinant sFRP‐1 inhibited, osteoclast formation. In studying the mechanism of action for sFRP‐1, we found that sFRP‐1 could bind recombinant RANKL. These results suggest potential cross‐talk between Wnt and RANKL pathways.

We determined that sFRP‐1 mRNA was differentially expressed by osteoblast/stromal cell lines and that sFRP‐1 neutralizing antibodies and siRNA complementary to sFRP‐1 coding sequence enhanced, while recombinant sFRP‐1 inhibited, osteoclast formation. In studying the mechanism of action for sFRP‐1, we found that sFRP‐1 could bind recombinant RANKL. These results suggest potential cross‐talk between Wnt and RANKL pathways.Introduction:Osteoclast formation in normal bone remodeling requires the presence of osteoblast lineage cells that express RANKL and macrophage‐colony‐stimulating factor (M‐CSF), which interact with their cognate receptors on the osteoclast precursor. We identified secreted Frizzled‐related protein‐1 (sFRP‐1), which is known to bind to Wnt and inhibit the Wnt signaling pathway, as an osteoblast‐derived factor that impinges on osteoclast formation and activity.Materials and Methods:Differential display of mRNA from osteoblast lineage cell lines established sFRP‐1 to be highly expressed in an osteoclast supporting cell line. sFRP‐1 expression in bone was determined by in situ hybridization, and the effects of sFRP‐1 on osteoclast formation were determined using a neutralizing antibody, siRNA, for sFRP‐1 and recombinant protein.Results:In situ hybridization revealed sFRP‐1 mRNA expression in osteoblasts and chondrocytes in murine bone. sFRP‐1 mRNA expression could be elevated in calvarial primary osteoblasts in response to prostaglandin E2(PGE2) or interleukin (IL)‐11, whereas many other osteotropic agents (e.g., IL‐1, IL‐6, calcitrol, parathyroid hormone) were without any effect. In vitro assays of osteoclast formation established sFRP‐1 to be an inhibitor of osteoclast formation. Neutralizing antibodies against sFRP‐1 enhanced TRACP+mononuclear and multinuclear osteoclast formation (3‐ and 2‐fold, respectively) in co‐cultures of murine osteoblasts with spleen cells, whereas siRNA complementary to sFRP‐1 coding sequence significantly enhanced osteoclast formation in co‐cultures of KUSA O (osteoblast/stromal cell line) and bone marrow cells, cultured in the presence of PGE2and 1,25(OH)2vitamin D3. Recombinant sFRP‐1 dose‐dependently inhibited osteoclast formation in osteoblast/spleen co‐cultures, RANKL + M‐CSF‐treated splenic cultures, and RANKL‐treated RAW264.7 cell cultures, indicating a direct action of sFRP‐1 on hematopoietic cells. Consistent with this, sFRP‐1 was found to bind to RANKL in ELISAs.Conclusion:sFRP‐1 is expressed by osteoblasts and inhibits osteoclast formation. While sFRP‐1 activity might involve the blocking of endogenous Wnt signaling, our results suggest that, alternatively, it could be because of direct binding to RANKL. This study describes a new mechanism whereby osteoblasts regulate osteoclastogenesis through the expression and release of sFRP‐1.

Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)α production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNFα production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNFα production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNFα production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNFα production. We examined the role of Btk in TNFα production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNFα mRNA via the 3′ untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNFα production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNFα production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.

IL‐4 is an important cytokine that can influence bone. We identified two distinct actions of IL‐4 to inhibit osteoclast formation: one direct on osteoclast progenitors and the second through the production of a novel T‐cell surface‐associated molecule(s). These data show a new link between the immune system and bone.

IL‐4 is an important cytokine that can influence bone. We identified two distinct actions of IL‐4 to inhibit osteoclast formation: one direct on osteoclast progenitors and the second through the production of a novel T‐cell surface‐associated molecule(s). These data show a new link between the immune system and bone.The Th2 cytokine interleukin (IL)‐4 inhibits osteoclast formation in vitro but also acts on other cell types found in bone, including T‐cells and macrophages. Because some osteoclastogenesis inhibitors (e.g., IL‐12) act indirectly through T‐cells, we investigated IL‐4 action on osteoclastogenesis in the presence of T‐cells. Osteoclast formation from murine spleen cells treated with RANKL and macrophage colony‐stimulating factor (M‐CSF) was blocked by IL‐4 even when spleen cells were depleted of T‐cells (Thy 1.2+) and/or B‐cells (B220+). Also, IL‐4 inhibited osteoclastogenesis in RANKL/M‐CSF‐stimulated adherent spleen cells, Rag1−/−(lymphocyte‐deficient) spleen cells, and bone marrow macrophages, indicating an action on myelomonocytic cells to block osteoclastogenesis. In contrast, IL‐4 did not inhibit osteoclastogenesis in cells from IL‐4 receptor null mice (IL‐4R−/−). However, when wildtype T‐cells were added to IL‐4R−/−spleen cell cultures, IL‐4 inhibited osteoclast formation, indicating a T‐cell‐dependent action. Osteoclast formation in RANKL‐stimulated RAW 264.7 cells was not inhibited by IL‐4 unless T‐cells were added to the culture. Separation of RAW 264.7 cells and T‐cells by semipermeable membrane ablated this action of IL‐4, suggesting the induction of a membrane‐associated osteoclastogenesis inhibitor. However, membrane‐bound inhibitors thymic shared antigen‐1 (TSA‐1) and osteoclast inhibitory lectin (OCIL) were not regulated by IL‐4. In summary, at least two mechanisms of IL‐4‐mediated osteoclastogenesis inhibition exist, including a direct action on myelomonocytic progenitors (from which osteoclasts derive) and an indirect action through T‐cells that may involve novel anti‐osteoclastic factors.

The elucidation of the signalling pathways involved in inflammatory diseases, such as rheumatoid arthritis, could provide long sought after targets for therapeutic intervention. Gene regulation is complex and varies depending on the cell type, as well as the signal eliciting gene activation. However, cells from certain lineages, such as macrophages, are specialised to degrade exogenous material and consequently do not easily transfect. Methods for high-efficiency gene transfer into primary cells of various lineages and disease states are desirable, as they remove the uncertainties associated with using transformed cell lines. Significant research has been undertaken into the development of nonviral and viral vectors for basic research, and as vehicles for gene therapy. We briefly review the current methods of gene delivery and the difficulties associated with each system. Adenoviruses have been used extensively to examine the role of various cytokines and signal transduction molecules in the pathogenesis of rheumatoid arthritis. This review will focus on the involvement of different signalling molecules in the production of tumour necrosis factor alpha by macrophages and in rheumatoid synovium. While the NF-κB pathway has proven to be a major mediator of tumour necrosis factor alpha production, it is not exclusive and work evaluating the involvement of other pathways is ongoing.

Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only is RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M‐CSF, and OPG messenger (mRNA). Stimulation by 1α,25 dihydroxyvitamin D3[1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate‐resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2(PGE2), interleukin‐11 (IL‐11), IL‐1, tumor necrosis factor‐α (TNF‐α), and parathyroid hormone‐related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte‐macrophage‐CSF (GM‐CSF), transforming growth factor‐β (TGF‐β), and OPG inhibited osteoclast formation in skin fibroblastic cell‐hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL‐positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.

Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only is RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M‐CSF, and OPG messenger (mRNA). Stimulation by 1α,25 dihydroxyvitamin D3[1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate‐resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2(PGE2), interleukin‐11 (IL‐11), IL‐1, tumor necrosis factor‐α (TNF‐α), and parathyroid hormone‐related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte‐macrophage‐CSF (GM‐CSF), transforming growth factor‐β (TGF‐β), and OPG inhibited osteoclast formation in skin fibroblastic cell‐hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL‐positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.

Osteoblastic stromal cells are capable of supporting osteoclast formation from hematopoietic precursors in the presence of osteotropic factors such as 1α,25(OH)2D3, PTH, and IL-11. Osteoblastic stromal cells produce receptor activator of NF-κB ligand (RANKL), a type II membrane protein of the TNF ligand family, in response to these agents. Activated T lymphocytes also produce RANKL; however, the ability of this cell type to support osteoclast formation in vitrois unknown. Human PBMC-derived T cells, extracted using αCD3-coated magnetic beads, were cocultured with adherent murine spleen cells in the presence of Con A and a panel of cytokines. In the presence of Con A, bona fideosteoclasts were formed in vitrowith activated T cells: IL-1α and TGFβ further enhanced osteoclast numbers. PBMC-derived lymphocytes showed an increase in the mRNA expression of RANKL within 24 h of treatment with the same agents that were used to induce osteoclast formation. In synovial tissue sections with lymphoid infiltrates from RA patients, the expression of RANKL was demonstrated in CD3+T cells. The ability of activated T lymphocytes to support osteoclast formation may provide a mechanism for the potentiation of osteoclast formation and bone resorption in disease states such as rheumatoid arthritis.

We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1α,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11. The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-γ (IFN-γ) and granulocyte/macrophage colony-stimulating factor (GM–CSF) production in T cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM–CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-γ did not. In cocultures with osteoblasts and spleen cells from IFN-γ receptor type II–deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-γ production: IFN-γ had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were from wild-type mice and vice versa, we identified that the target cells for IFN-γ inhibition of OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM–CSF production and not via IFN-γ production.

The breadth of bone lesion types seen in spondyloarthritis is unprecedented in medicine and includes increased bone turnover, bone loss and fragility, osteitis, osteolysis and erosion, osteosclerosis, osteoproliferation of soft tissues adjacent to bone and spinal skeletal structure weakness. Remarkably, these effects can be present simultaneously in the same patient. The search for a potential unifying cause of effects on the skeleton necessarily focuses on inflammation arising from the dysregulation of immune response to microorganisms, particularly dysregulation of TH17 lymphocytes, and the dysbiosis of established gut and other microbiota. The compelling notion that a common antecedent pathological mechanism affects existing bone and tissues with bone-forming potential (entheses), simultaneously with variable effect in the former but bone-forming in the latter, drives basic research forward and focuses our awareness on the effects on these bone mechanisms of the increasing portfolio of targeted immunotherapies used in the clinic.

Horwood: Genzyme: Research Funding.

Multiple myeloma (MM) is an incurable cancer of plasma cells (PC), with a median survival of 5-7 years. Osteolytic bone disease and skeletal complications occur in more than 80% of MM patients and significantly contribute to the morbidity and mortality of these patients. Glycosphingolipid (GSL), an essential constituent of the outer leaflet of the cellular membrane, is altered in MM and other hematological cancers. We previously reported that GM3, a subtype of GSL promotes osteoclastogenesis. On the other hand, the GSL synthase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) reduces myeloma bone disease in the 5TGM1 mouse model of MM. Mechanistically, NB-DNJ prevents osteoclast (OC) development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of the transcriptional activator NFATc1.

Chemotherapy induced leucopoenia and neutropaenia results in an increased risk of infection, with febrile neutropaenia being associated with bacteraemia, sepsis and death. Haematopoietic regeneration is reliant on endogenous stem cells and the acceleration of this process would reduce the length of time (nadir) of clinical neutropaenia, thereby reducing the risk of infection and the treatment associated morbidity and mortality associated with chemotherapy. The haematopoietic stem cell injury-niche has not been extensively explored for potential novel in vivoregenerative targets. Our objective was to assess if alarmins, endogenous molecules which are released upon tissue damage and widely considered to only be triggers of the immune response, may potentiate stem-cell mediated repair and regeneration. Here we show that the alarmin HMGB1 transitions endogenous quiescent G0stem cells to the recently described GAlertphase, thereby enhancing regeneration in multiple tissues, including blood, bone, and muscle.