Search

Purpose: We investigated the in vitro and in vivo anti-multiple myeloma activity of monoclonal antibody (mAb) 1339, a high-affinity fully humanized anti-interleukin 6 mAb (immunoglobulin G1), alone and in combination with conventional and novel anti-multiple myeloma agents, as well as its effect on bone turnover. Experimental Design: We examined the growth inhibitory effect of 1339 against multiple myeloma cell lines in the absence and in the presence of bone marrow stromal cells, alone or in combination with dexamethasone, bortezomib, perifosine, and Revlimid. Using the severe combined immunodeficient (SCID)–hu murine model of multiple myeloma, we also examined the effect of 1339 on multiple myeloma cell growth and multiple myeloma bone disease. Results: mAb 1339 significantly inhibited growth of multiple myeloma cell in the presence of bone marrow stromal cell in vitro, associated with inhibition of phosphorylation of signal transducer and activator of transcription 3, extracellular signal-regulated kinase 1/2, and Akt. In addition, mAb 1339 enhanced cytotoxicity induced by dexamethasone, as well as bortezomib, lenalidomide, and perifosine, in a synergistic fashion. Importantly mAb 1339 significantly enhanced growth inhibitory effects of dexamethasone in vivo in SCID-hu mouse model of multiple myeloma. mAb 1339 treatment also resulted in inhibition of osteoclastogenesis in vitro and bone remodeling in SCID-hu model. Conclusions: Our data confirm in vitro and in vivo anti-multiple myeloma activity of, as well as inhibition of bone turnover by, fully humanized mAb 1339, as a single agent and in combination with conventional and novel agents, providing a rationale for its clinical evaluation in multiple myeloma. (Clin Cancer Res 2009;15(23):7144–52)

Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)–hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated β-catenin level while down-regulating nuclear factor-κB (NF-κB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.

As the only cells capable of efficiently resorbing bone, osteoclasts are central mediators of both normal bone remodeling and pathologies associates with excessive bone resorption. However, despite the clear evidence of interplay between osteoclasts and the bone surface in vivo, the role of the bone substrate in regulating osteoclast differentiation and activation at a molecular level has not been fully defined. Here, we present the first comprehensive expression profiles of osteoclasts differentiated on authentic resorbable bone substrates. This analysis has identified numerous critical pathways coordinately regulated by osteoclastogenic cytokines and bone substrate, including the transition from proliferation to differentiation, and sphingosine-1-phosphate signaling. Whilst, as expected, much of this program is dependent upon integrin beta 3, the pre-eminent mediator of osteoclast-bone interaction, a surprisingly significant portion of the bone substrate regulated expression signature is independent of this receptor. Together, these findings identify an important hitherto underappreciated role for bone substrate in osteoclastogenesis.

Analysis of tissues retrieved from the bone-pannus interface from patients with rheumatoid arthritis (RA) and studies in animal models of inflammatory arthritis provide strong evidence that osteoclasts, the cells that are essential for physiological bone resorption, are responsible for articular bone destruction in RA. However, current treatments that specifically target osteoclast-mediated bone resorption in RA have not been successful in preventing bone erosions, and new therapeutic strategies are needed. It has been noted that, although osteoclast precursors are present within the bone microenvironment at sites of pathological bone resorption, cells expressing the full morphological and functional properties of mature osteoclasts are restricted to the immediate bone surface and adjacent calcified cartilage. These findings provide evidence that, in addition to requirements for specific cytokines, interaction of osteoclast precursors with these mineralised matrices results in activation of specific signal pathways and the induction of unique gene products that are essential for terminal osteoclast differentiation and activation. These studies are designed to define the gene products and signalling pathways regulated by bone and calcified cartilage, to identify new molecular targets and novel therapeutic approaches for preventing osteoclast-mediated joint destruction in RA and related forms of pathological bone loss.

Osteoadherin is a cell binding keratan sulfate proteoglycan which was recently isolated from mineralized bovine bone and subsequently cloned and sequenced. For studies of osteoadherin expression in rat tissues we isolated and sequenced a 1.3-kbp partial cDNA covering most of the coding region using a rat calvaria cDNA library. The most 5' end of the cDNA was obtained by reverse transcription PCR from the bone total RNA preparation. The deduced, translated protein sequence containing 423 amino acid residues shows high sequence identity to mouse, bovine and human osteoadherin except in the very acidic C-terminal region. However, the rat counterpart showed a similarly high content of acidic amino acid residues. Ribonuclease protection assay showed osteoadherin mRNA to be expressed in femoral bone and calvaria tissues, while no expression was detected in cartilage, tendon or liver. Using very sensitive nested RT-PCR, however, message was detected in femoral head, rib, tendon and bone marrow total RNA preparations. An antiserum specific for the rat C-terminal region of osteoadherin was generated and used for studies of protein distribution by immunohistochemistry during femoral head development. Osteoadherin was primarily present in bone trabeculae and no staining was seen in cartilage. In situ hybridization showed the strongest expression in osteoblasts close to the cartilage/bone interface of the growth plate and lower expression in diaphyseal osteoblasts. On maturation of the femoral head on day 60 some expression was detected immediately below the forming articular cartilage. Our data indicated that osteoadherin is primarily expressed by osteoblasts and might have a role in regulation of mineralization.

To examine the cytokine profile of epithelial cells lining the human urinary tract with the aim of differentiating between the constitutive and disease-related cytokine production in these tissues.Sections from the renal pelvis, ureter, bladder or urethra were obtained from 22 patients undergoing urinary tract surgery and were stained with monoclonal antibodies to interleukin(IL)-1beta, IL-4, IL-6, IL-8, interferon gamma (IFNgamma) and transforming growth factor beta (TGFbeta). Sections were classified according to the presence or absence of disease in the tissue.Epithelial cells lining the renal pelvis, ureter, bladder or urethra all stained for IL-8 and TGFbeta (100%) in disease-free tissues and sections with cancer or interstitial cystitis (IC). In contrast, staining for IL-1beta, IL-4, IL-6 and IFNgamma varied with the disease state of the patient. Epithelial IL-1beta staining was absent (0%) in sections from healthy bladder, but positive in tissues with IC or cancer-associated pathology (50 to 100%). IL-6 staining was detected in the epithelial layer of several patients with IC or cancer related pathology, but only in cells with non-epithelial morphology and not in disease-free tissues. IFNgamma and IL-4 staining were only observed in patients with IC and only in cells with non-epithelial morphology.The results show that epithelial cells from all parts of the urinary tract constitutively produce IL-8 and TGFbeta and suggest that the production of other cytokines varies with the disease of the patient. Constitutive cytokine production provides the basis for a rapid host response, in the defense against mucosal attack by microbes or toxic agents.

While attachment to bone is required for optimal osteoclast function, the molecular events that underlie this fact are unclear, other than that the cell requires adhesion to mineralized matrix to assume a fully differentiated phenotype. To address this issue, we cultured murine bone marrow-derived osteoclasts on either cell culture plastic or devitalized mouse calvariae to identify the distinct genetic profile induced by interaction with bone. Among a number of genes previously unknown to be expressed in osteoclasts we found that Annexin A8 (AnxA8) mRNA was markedly up-regulated by bone. AnxA8 protein was present at high levels in osteoclasts present in human tissues recovered from sites of pathological bone loss. The presence of bone mineral was required for up-regulation of AnxA8 mRNA since osteoclasts plated on decalcified bone express AnxA8 at low levels as did osteoclasts plated on native or denatured type I collagen. Finally, AnxA8-regulated cytoskeletal reorganization in osteoclasts generated on a mineralized matrix. Thus, we used a novel approach to define a distinct bone-dependent genetic program associated with terminal osteoclast differentiation and identified Anxa8 as a gene strongly induced late in osteoclast differentiation and a protein that regulates formation of the cell's characteristic actin ring. J. Cell. Physiol. 226: 3413–3421, 2011. © 2011 Wiley Periodicals, Inc.

The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment induces proliferation and survival of MM cells, as well as osteoclastogenesis. This study investigated the therapeutic potential of novel p38 mitogen-activated protein kinase (p38MAPK) inhibitor LY2228820 (LY) in MM. Although cytotoxicity against MM cell lines was modest, LY significantly enhanced the toxicity of bortezomib by down-regulating bortezomib-induced heat shock protein 27 phosphorylation. LY inhibited interleukin-6 secretion from long term cultured-BM stromal cells and BM mononuclear cells (BMMNCs) derived from MM patients in remission. LY also inhibited macrophage inflammatory protein-1alpha secretion from patient MM cells and BMMNCs as well as normal CD14 positive osteoclast precursor cells. Moreover, LY significantly inhibited in vitro osteoclastogenesis from CD14 positive cells induced by macrophage-colony stimulating factor and soluble receptor activator of nuclear factor-kappaB ligand. Finally, LY also inhibited in vivo osteoclatogenesis in a severe combined immunodeficiency mouse model of human MM. These results suggest that LY represents a promising novel targeted approach to improve MM patient outcome both by enhancing the effect of bortezomib and by reducing osteoskeletal events.

Osteoclast and their mononuclear cell precursors are present within the bone microenvironment at sites of physiologic and pathologic bone resorption. Analysis of tissues from sites of bone resorption reveal that cells expressing the full morphological and functional properties of mature osteoclasts are restricted to the immediate bone surface. We hypothesize that in addition to cytokines, components of the bone matrix and specific cell surface receptors on osteoclasts and their precursors play an essential role in determining the genetic profile and functional properties of fully differentiated resorbing osteoclasts. We have employed expression profiling, with an in vitro model of matrix-dependent osteoclast differentiation, to identify the molecular pathways by which bone matrix-interactions induce terminal osteoclast differentiation and activation. In preliminary studies, we have identified unique genes and transcriptional pathways that are induced by interaction of osteoclast precursors with specific components of the mineralized bone matrix. The authenticity of the gene profiles, as markers of osteoclast differentiation and activation, have been provisionally validated using an in vivo animal bone implantation model and by examination of tissues from patients with specific forms of pathologic osteoclast-mediated bone resorption. The ultimate goal of our studies is to identify new molecular targets for inhibiting osteoclast-mediated bone loss in disorders of pathologic bone loss. The early work of Walker et al. (Walker 1972) in parabiotic animals, and the subsequent studies of Burger et al. (Burger, Van der Meer, van de Gevel, et al. 1982) using a co-culture model with fetal bone rudiments and bone marrow-derived cells, have helped to establish that osteoclasts are derived from macrophage precursors of colony forming unit-macrophage (CFU-M lineage). As such, they share a common hematopoietic origin with other CFU-M lineage cells, including tissue macrophages that populate the lung (alveolar macrophages), liver (Kupfer cells), synovium (synovial macrophages) and other organs. They also share a common lineage

Chondroadherin is a cartilage protein with cell binding properties. The expression of chondroadherin was studied in rat tissues and during postnatal femoral head development. For design of oligonucleotide probes and primers a 1664 bp, full length, rat chondroadherin cDNA was isolated from a rat chondrosarcoma library and sequenced. Northern blot analysis showed chondroadherin mRNA to be present in femoral head and rib cartilage, as well as in tendon. More sensitive reverse-transcriptase PCR additionally identified the mRNA in calvaria, long bone and bone marrow. Localization of chondroadherin by immunocytochemistry in the developing femoral head from postnatal day 14 to day 60 showed presence of the protein in cartilaginous regions. With increasing age a very distinct localization of chondroadherin was seen in the territorial matrix around late proliferative cells in the growth plate as well as in the developing articular cartilage in the maturing femoral head. Localization of chondroadherin mRNA by in situ hybridization was in agreement with immunocytochemistry with strong hybridization signals in late proliferative cells in the growth plate. In the articular cartilage the expression was restricted to cells in the lower regions. A three-fold increase of cartilage chondroadherin content in the growing femoral head was demonstrated by Western blot analysis. The high expression of this cell binding protein in a dynamic region of cartilage suggests an important role for chondroadherin in the regulation of chondrocyte growth and proliferation.

Distribution and sites of synthesis of a cartilage extracellular matrix protein, cartilage oligomeric matrix protein (COMP), and of a bone extracellular matrix protein, bone sialoprotein (BSP), were studied in the femoral head of growing Wistar rats from day 14 to day 60 by immunocytochemistry and in situ hybridization. This period includes formation of the secondary ossification center and differentiation of articular cartilage. At early stages, immunoreactivity for COMP was pronounced throughout the cartilage. The localization of COMP was predominantly territorial in the center of the immature femoral head and in the growth plate at all ages studied. In the superficial parts, a shift from a uniform extracellular matrix staining at day 14 to an interterritorial localization at day 33 to day 60 was seen, apparently concurrent with formation of articular cartilage. COMP staining, representing cartilage remnants, also extended into the center of the trabecular bone in the primary spongiosa. In the secondary ossification center, the staining for COMP decreased at the onset of calcification. The protein was only synthesized by chondrocytes, as shown by in situ hybridization. The highest level of COMP mRNA was detected in chondrocytes in the central region of the growth plate. In the layer corresponding to the articular cartilage of the femoral head, mRNA levels for COMP were low from day 14 to day 33 but were increased on day 60. This shows substantial synthesis in the developing articular cartilage. Immunoreactivity for BSP was detected in bone trabeculae of primary spongiosa. In situ hybridization showed the highest levels of BSP mRNA in regions of newly formed bone. BSP mRNA was detected in hypertrophic chondrocytes in the secondary ossification center as early as day 18, well before the appearance of immunochemically detectable BSP. Interestingly, simultaneous expression of COMP and BSP mRNA was seen after day 18 in hypertrophic chondrocytes of the growth plate and later also in hypertrophic chondrocytes close to the mineralization zone of the articular cartilage.

Multiple Myeloma (MM) is associated with significant skeletal changes including ospeoporosis and bone lesions in more than 80% of patients. Bone resorption is caused by an enhanced osteoclast number and activation. However, molecular and cellular basis for these effects are not completely understood. Here we describe a model that allows us to evaluate in vivo, the relationship and interactions between MM cells and cellular elements of bone formation and resorption. In this model, the BM stroma and IL-6-dependent human MM cell line (INA-6) was injected in a human bone chip implanted into SCID mice. Bone chip in the control mice were injected with media alone. At different time points (days 1, 7 and 30) following injection, myelomatous and control bone chips were retrieved, fixed in paraformaldehyde and evaluated by micro-computed tomography (μCT) and histological examinations for effects of MM cells on bone compartment. The μCT is a non-invasive quantitative imaging method that can asses the degree and extent of bone disease as well as quantify changes in bone structure, that may reflect the effect of interaction between MM cells and bone elements. Additionally, we have measured the levels of human osteocalcin in murine serum to reflect the remodelling of human bone in mice. We have observed that only at day 30 after the MM cell injections, lesions in myelomatous bone is observed in mice; while no significant changes in the matched control bones are seen. By 3-D histomorphometric parameters, calculated from μCT (bone volume fraction= bone volume/total volume), the quantitative measurement confirmed that at day 30, a decreased bone volume fraction (314) was observed in bones with MM compared with normal bones (394), due to reduction of bone density in the trabecular bone. Moreover histological examination of the same bones revealed a significant increase in number of multinucleated TRAP-expressing osteoclasts in mice engrafted with MM versus control (52 versus 8 per field, p= 0.011). Also compared to serum from control mice, animals with bone containing myeloma cells had significantly elevated markers of bone remodelling. To confirm the validity of this model to study effect of MM cells on bone as well as to show its utility for evaluation of novel agents active in MM bone disease, we treated mice with Zoledronic acid at 0,6 mg/kg, weekly for 2 months. As evaluated by μCT, X-rays and TRAP staining Zoledronic acid was able to significantly inhibit the development of bone lesions in treated mice compared to untreated mice. Additionally, , we also observed that Zoledronic acid was also able to retard the MM tumor growth as measured by human IL-6sR released by INA-6 cells in mice sera corroborating its reported anti-MM activity. This model therefore provides a reproducible and predictable in vivo system to study biology of bone disease in MM and to identify and evaluate therapeutic targets and agents targeting MM bone disease.

The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a crucial role not only in proliferation and survival of MM cells, but also in osteoclastogenesis. In this study, we examined diverse potential of novel p38MAPK inhibitor LSN2322600 (LSN) for MM therapy in vitro and in vivo. The cytotoxic activity of LSN against MM cell lines was modest; however, LSN significantly enhances the cytotoxicity of Bortezomib by down-regulating Bortezomib-induced heat shock protein (HSP) 27 phosphorylation. We next examined the effects of LSN on cytokine secretion in MM cells, bone marrow stromal cells and osteoclast precursor cells. LSN inhibited IL-6 secretion from long-term cultured-bone marrow stromal cells (LT-BMSCs) and bone marrow mononuclear cells (BMMNCs) from MM patients in remission. LSN also inhibited MIP-1 α secretion by fresh tumor cells, BMMNCs and CD14 positive cells. Since these cytokines mediate osteoclastogenesis, we further examined whether LSN could inhibit osteoclastogenesis. Importantly, LSN inhibited in vitro osteoclastogenesis induced by macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of nuclear factor- κ B ligand (sRANKL), as well as osteoclastogenesis in the severe combined immunodeficiency (SCID)-Hu mouse model of human MM. These results suggest that LSN represents a promising novel targeted strategy to reduce skeletal complications as well as to sensitize or overcome resistance to Bortezomib.

Prosthetic wear debris-induced peri-implant osteolysis is a major cause of aseptic loosening after total joint replacement. In this condition, wear particles released from the implant components induce a granulomatous inflammatory reaction at the interface between implant and adjacent bone, leading to progressive bone resorption and loss of fixation. The present study was undertaken to characterize definitively the phenotype of osteoclast-like cells associated with regions of peri-implant focal bone resorption and to compare the phenotypic features of these cells with those of mononucleated and multinucleated cells associated with polyethylene wear particles. Peri-implant tissues were obtained from patients undergoing hip revision surgery for aseptic loosening after total joint replacement. Cells were examined for the expression of several markers associated with the osteoclast phenotype using immunohistochemistry, histochemistry, and/or in situ hybridization. CD68 protein, a marker expressed by multiple macrophage lineage cell types, was detected in mononucleated and multinucleated cells associated with polyethylene particles and the bone surface. Cathepsin K and tartrate-resistant acid phosphatase were expressed highly in both mononucleated and multinucleated cells associated with the bone surface. Levels of expression were much lower in cells associated with polyethylene particles. High levels of β3 integrin protein were detected in cells in contact with bone. Multinucleated cells associated with polyethylene particles exhibited faint positive staining. Calcitonin receptor mRNA expression was detected solely in multinucleated cells present in resorption lacunae on the bone surface and was absent in cells associated with polyethylene particles. Our findings provide further evidence that cells expressing the full repertoire of osteoclast phenotypic markers are involved in the pathogenesis of peri-implant osteolysis after total joint replacement. They also demonstrate that foreign body giant cells, although believed to be phenotypically and functionally distinct from osteoclasts, express many osteoclast-associated genes and gene products. However, the levels and patterns of expression of these genes in the two cell types differ. We speculate that, in addition to the role of cytokines and growth factors, the substrate with which these cells interact plays a critical role in their differential phenotypic and functional properties.

Sixteen of 24 infants with vomiting were examined by real-time sonography, which established the diagnosis of hypertrophic pyloric stenosis (HPS); 12 underwent surgery. Of the three parameters measured, pyloric muscle wall thickness was the most reliable and accurate for the diagnosis of HPS. We suggest that a muscle wall thickness of 4 mm or more and stenosis index greater than 50% are standard findings in patients with HPS. Serial real-time sonography following pyloromyotomy showed that the pyloric tumor changed significantly during the 1st postoperative week and returned to normal size by the end of the 4th week.