Ethical and scientific concerns regarding the use of human fetal bones in the SCID-hu model of primary human myeloma prompted us to develop a novel system that uses rabbit bones implanted subcutaneously in unconditioned SCID mice. Immunohistochemical analysis of the implanted bone revealed that the majority of bone marrow (BM) microenvironment cells such as blood vessels, osteoclasts and osteoblasts were of rabbit origin. The implanted bones were directly injected with myeloma cells from 28 patients. Successful engraftment of unseparated BM cells from 85% of patients and CD138-selected myeloma plasma cells from 81% of patients led to the production of patients' M-protein isotypes and typical myeloma manifestations (osteolytic bone lesions and angiogenesis of rabbit origin). Myeloma cells grew exclusively in the rabbit bone, but were able to metastasize into another bone at a remote site in the same mouse. Cells from patients with extramedullary disease also grew along the outer surface of the rabbit bones. This demonstrates the ability of SCID-rab model, marked by a nonmyelomatous, nonhuman, and nonfetal microenvironment, to support the growth of CD138-expressing myeloma cells. This system can now be widely used to study the biology of myeloma and its manifestations and to develop novel therapeutic approaches for this disease.
Bone marrow stromal cells (BMSCs) and osteoclasts (OCs) confer multiple myeloma (MM) cell survival through elaborating factors. We demonstrate herein that IL-6 and TNF family cytokines, TNFa, BAFF and APRIL, but not IGF-1 cooperatively enhance the expression of the serine/threonine kinase Pim-2 in MM cells. BMSCs and OCs upregulate Pim-2 expression in MM cells largely via the IL-6/STAT3 and NF-jB pathway, respectively. Pim-2 short interfering RNA reduces MM cell viability in cocultures with BMSCs or OCs. Thus, upregulation of Pim-2 appears to be a novel anti-apoptotic mechanism for MM cell survival. Interestingly, the mammalian target of rapamycin inhibitor rapamycin further suppresses the MM cell viability in combination with the Pim-2 silencing. The Pim inhibitor (Z)-5-(4-propoxybenzylidene) thiazolidine-2, 4-dione and the PI3K inhibitor LY294002 cooperatively enhance MM cell death. The Pim inhibitor suppresses 4E-BP1 phosphorylation along with the reduction of Mcl-1 and c-Myc. Pim-2 may therefore become a new target for MM treatment.
BackgroundMultiple myeloma (MM) expands almost exclusively in the bone marrow and generates devastating bone lesions, in which bone formation is impaired and osteoclastic bone resorption is enhanced. TGF-β, a potent inhibitor of terminal osteoblast (OB) differentiation, is abundantly deposited in the bone matrix, and released and activated by the enhanced bone resorption in MM. The present study was therefore undertaken to clarify the role of TGF-β and its inhibition in bone formation and tumor growth in MM.Methodology/Principal FindingsTGF-β suppressed OB differentiation from bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and also inhibited adipogenesis from C3H10T1/2 immature mesenchymal cells, suggesting differentiation arrest by TGF-β. Inhibitors for a TGF-β type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-β inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-β inhibition expedited OB differentiation in parallel with suppression of MM cell growth. The anti-MM activity was elaborated exclusively by terminally differentiated OBs, which potentiated the cytotoxic effects of melphalan and dexamethasone on MM cells. Furthermore, TGF-β inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models.Conclusions/SignificanceThe present study demonstrates that TGF-β inhibition releases stromal cells from their differentiation arrest by MM and facilitates the formation of terminally differentiated OBs, and that terminally differentiated OBs inhibit MM cell growth and survival and enhance the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. Therefore, TGF-β appears to be an important therapeutic target in MM bone lesions.
Pigmented neurons in the substantia nigra pars compacta (SNc) and locus coeruleus (LC) show decreased numbers differentially in Parkinson's disease (PD) and multiple system atrophy (MSA). Recent reports have described that fast spin-echo T1-weighted magnetic resonance imaging (MRI) by a 3-tesla machine can visualize neuromelanin-related contrast of the noradrenergic and dopaminergic neurons respectively in the LC and the SNc. Using neuromelanin MRI at 3 T, we investigated possible alterations of these catecholaminergic neurons in 32 PD and 9 MSA patients, and compared the results with those of 23 normal volunteers. The contrast ratio of the LC and SNc was decreased in MSA and PD patients, most prominently in the LC in MSA patients. The contrast ratio of the SNc was correlated with the Hoehn-Yahr stage of PD and the severity of neuroradiological abnormalities in MSA. These results indicate a potential diagnostic value of neuromelanin MRI to distinguish MSA patients from normal and PD patients.
Inflammation is crucially involved in the development of carotid plaques. We examined the relationship between plaque vulnerability and inflammatory biomarkers using intraoperative blood and tissue specimens. We examined 58 patients with carotid stenosis. Following carotid plaque magnetic resonance imaging, 41 patients underwent carotid artery stenting (CAS) and 17 underwent carotid endarterectomy (CEA). Blood samples were obtained from the femoral artery (systemic) and common carotid artery immediately before and after CAS (local). Seventeen resected CEA tissue samples were embedded in paraffin, and histopathological and immunohistochemical analyses for IL-6, IL-10, E-selectin, adiponectin, and pentraxin 3 (PTX3) were performed. Serum levels of IL-6, IL-1β, IL-10, TNFα, E-selectin, VCAM-1, adiponectin, hs-CRP, and PTX3 were measured by multiplex bead array system and ELISA. CAS-treated patients were classified as stable plaques (n = 21) and vulnerable plaques (n = 20). The vulnerable group showed upregulation of the proinflammatory cytokines (IL-6 and TNFα), endothelial activation markers (E-selectin and VCAM-1), and inflammation markers (hs-CRP and PTX3) and downregulation of the anti-inflammatory markers (adiponectin and IL-10). PTX3 levels in both systemic and intracarotid samples before and after CAS were higher in the vulnerable group than in the stable group. Immunohistochemical analysis demonstrated that IL-6 was localized to inflammatory cells in the vulnerable plaques, and PTX3 was observed in the endothelial and perivascular cells. Our findings reveal that carotid plaque vulnerability is modulated by the upregulation and downregulation of proinflammatory and anti-inflammatory factors, respectively. PTX3 may thus be a potential predictive marker of plaque vulnerability.
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