Nonadherent Bone Marrow Cells Research Articles

Effects of 200cH medications on mice bone marrow cells and macrophages

Paracelsus once wrote: "All things are poison and nothing is without poison, only the dose permits something not to be poisonous." Latter Hahnemann formulated the law of similars, preparations which cause certain symptoms in healthy individuals if given in diluted form to patients exhibiting similar symptoms will cure it. Highly diluted natural complexes prepared according to Hahnemann’s ancient techniques may represent a new form of immunomodulatory therapy. The lack of scientific research with highly diluted products led us to investigate the in vivo and in vitro actions of commonly used medications. Here we describe the results of experimental studies aimed at verifying the effects of Mercurius solubilis, Atropa Belladonna, Lachesis muta and Bryonia alba. All medications were at 200cH dilution. Animals were maintained for 7 days and were allowed to drink the medications, which were prepared in a way that the final dilution and agitation (200cH) was performed in drinking water. The medication bottle was changed and sucussed every afternoon. Co-culture of non treated mice bone marrow cells and in vitro treated peritoneal macrophages were also performed. After animal treatment the bone marrow cells were immunophenotyped with hematopoietic lineage markers on a flow cytometer. We have determined CD11b levels on bone marrow cells after culture and co-culture with treated macrophages and these macrophages were processed to scanning electron microscopy. We have observed by morphological changes that macrophages were activated after all treatments. Mercurius solubilis treated mice showed an increase in CD3 expression and in CD11b on nonadherent bone marrow cells after co-culture with in vitro treatment. Atropa Belladonna increased CD45R and decreased Ly-6G expression on bone marrow cells after animal treatment. Lachesis muta increased CD3, CD45R and, CD11c expression and decreased CD11b ex vivo and in nonadherent cells from co-culture. Bryonia alba increased Ly-6G, CD11c and CD11b expression ex vivo and when in co-culture CD11b was increased in adherent cells as well as decreased in nonadherent cells. With these results we have demonstrated that highly diluted medications act on immune cells activating macrophages, and changing the expression profile of hematopoietic lineage markers. Highly diluted medications are less toxic and cheaper than other commonly used medications and based on our observations, it is therefore conceivable that this medications which are able to act on bone marrow and immune cells may have a potential therapeutic use in clinical applications in diseases were the immune system is affected and also as regenerative medicine as it may allow proliferation and differentiation of progenitor cells.

Lithium attenuates lead induced toxicity on mouse non-adherent bone marrow cells

Lead is a poisonous heavy metal that occurs in all parts of environment and causes serious health problems in humans. The aim of the present study was to investigate the possible protective effect of lithium against lead nitrate induced toxicity in non-adherent bone marrow stem cells. Trypan blue and MTT assays represented that exposure of the cells to different concentrations of lead nitrate decreased viability in a dose dependent manner, whereas, pretreatment of the cells with lithium protected the cells against lead toxicity. Lead reduced the number and differentiation status of bone marrow-derived precursors when cultured in the presence of colony stimulating factor (CSF), while the effect was attenuated by lithium. The cells treated with lead nitrate exhibited cell shrinkage, DNA fragmentation, anion superoxide production, but lithium prevented lead action. Moreover, apoptotic indexes such as PARP cleavage and release of HMGB1 induced by lead, were protected by lithium, suggesting anti-apoptotic effect of lithium. Immunoblot analysis of histone H3K9 acetylation indicated that lithium overcame lead effect on acetylation. In conclusion, lithium efficiently reduces lead toxicity suggesting new insight into lithium action which may contribute to increased cell survival. It also provides a potentially new therapeutic strategy for lithium and a cost-effective approach to minimize destructive effects of lead on bone marrow stem cells.

TNFα-induced M-MDSCs promote transplant immune tolerance via nitric oxide

Efficient induction of functional competent myeloid-derived suppressor cells (MDSCs) will be critical for the clinical application of MDSCs to treat autoimmune diseases and to induce transplantation immune tolerance. In the present study, we tried to establish the MDSC induction system with M-CSF and tumor necrosis factor α (TNFα) and investigated the immunosuppressive function of M-CSF + TNFα-induced MDSCs in transplant mouse models. Monocytic MDSCs (M-MDSCs) were induced by culture of the non-adherent mouse bone marrow cells with M-CSF or M-CSF + TNFα, respectively, for 7days. Phenotype analysis revealed that the majority of M-CSF- and M-CSF + TNFα-induced MDSCs express F4/80. The addition of TNFα in the induction period increased Gr-1, Ly6C, CD80, and CD274 expressions on these cells. M-CSF + TNFα-induced M-MDSCs showed poor TNFα, IL-12, and IL-6 expressions after lipopolysaccharide (LPS) stimulation and decreased arginase 1 (Arg-1) and Fizz expressions after IL-4 stimulation compared with M-CSF-induced M-MDSCs. M-CSF + TNFα-induced M-MDSCs showed enhanced ability to suppress T cell proliferation and cytokine production than M-CSF-induced M-MDSCs. M-CSF + TNFα-induced M-MDSCs express high levels of inducing nitric oxide synthase (iNOS) and blocking iNOS activity by a chemical inhibitor or gene deficiency significantly reversed the inhibitory effects of M-CSF + TNFα-induced M-MDSCs on T cells. Adoptive transfer of M-CSF + TNFα-induced M-MDSCs promoted immune tolerance in a male-to-female skin-grafted mice, but M-CSF + TNFα-induced iNOS-deficient M-MDSCs failed to do so. Thus, M-CSF + TNFα-induced M-MDSCs have powerful immunosuppressive activity, which is mediated by an iNOS-dependent pathway. M-CSF + TNFα-induced M-MDSCs can promote immune tolerance to donor antigens in a transplant mouse model. The combination of M-CSF and TNFα efficiently induces functional M-MDSCs in vitro. M-CSF + TNFα-induced M-MDSCs promote immune tolerance in a transplant mouse model. The immunosuppressive ability of M-CSF + TNFα-induced M-MDSCs is dependent on iNOS.

Abstract 4131: MEK inhibitor diminishes nasopharyngeal carcinoma (NPC) cell growth and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expression

Abstract Nasopharyngeal carcinoma (NPC) is a common malignant cancer in Guangxi, as well as in southern China and Southeast Asia. NPC preferentially metastasizes to bone in advanced patients, major factor resulting in high mortality. The molecular basis of NPC development and cancer-induced bone lesions are still unclear. We and others have reported that chemokine, like CCL2, CXCL16, CXCL12, and their corresponding receptors played important roles in cancer bone metastasis. In this study, we firstly investigated that CCR2 and CXCR6 expressions in clinical specimens. Then, we measured CCL2, CCR2, CXCL16 and CXCR6 expression levels by RT-PCR, ELISA and western blot in NPC cell lines. Further, we used MEK inhibitor, U0126, to treat the NPC cell lines and found CCL2 and CXCL16 expressions significantly decreased. The NPC cell lines proliferation were inhibited by U0126 in dose- and time-dependently manner. Finally, we collected conditioned medium (CM) from the NPC cell lines absent or present U0126 treatments, to treat mouse bone marrow non-adherent cells in order to detect NPC CM-induced multinucleated osteoclast-like formation. The CMs collected from U0126 treated NPC cells were dramatically diminished the numbers of multinucleated osteoclasts. These results indicate that U0126 inhibits NPC cell growth and diminishes cancer-induced osteoclast formation partially through modulating the expression of CCL2 and CXCL16. Thus this study may provide a novel therapeutic strategy for advanced NPC patients, especially for treatment of NPC bone metastasis. Citation Format: Yu Zhu, Qiong Song, Yi Lu. MEK inhibitor diminishes nasopharyngeal carcinoma (NPC) cell growth and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4131. doi:10.1158/1538-7445.AM2015-4131

MEK inhibitor diminishes nasopharyngeal carcinoma (NPC) cell growth and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions.

Nasopharyngeal carcinoma (NPC) is a common malignancy in southern China and Southeast Asia. NPC frequently metastasizes to the bone in advanced patients resulting in high mortality. The molecular mechanisms for NPC development and cancer-induced bone lesions are unclear. In this study, we firstly determined chemokine receptor CCR2 and CXCR6 expressions in clinical specimens and CNE2, SUNE1, CNE1, and HK1 cell lines. Then, we measured chemokine CCL2 and CXCL16 production in these NPC cell lines by ELISA. Expression levels of these chemokines and their receptors were observed to positively correlate with tumor aggressiveness. Furthermore, U0126 (MEK inhibitor) was used to treat these NPC cell lines. CCL2 and CXCL16 expression levels and cell proliferation were significantly inhibited by U0126 in a dose- and time-dependent manner. Finally, we collected conditioned medium (CM) from NPC cell cultures in the presence of U0126 treatment. When mouse bone marrow non-adherent cells were treated with the CM, the numbers of multinucleated osteoclast formation were dramatically diminished. These results indicate that MEK inhibitor diminishes NPC cell proliferation and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions. This study provides novel therapeutic targets such as CCL2/CCR2 and CXCL16/CXCR6 for advanced NPC patients.

An in vitro study on the effect of vinca alkaloid, vinorelbine, on chromatin histone, HMGB proteins and induction of apoptosis in mice non-adherent bone marrow cells

Context: Vinoreline is a vinca alkaloid anticancer drug widely used in cancer therapy. Drugs are not target specific, therefore might affect normal tissues/cells, in which bone marrow is the important one. Objective: To elucidate the cytotoxic and genotoxic effect of vinca alkaloid anti cancer drug, vinorelbine, on mice non-adherent bone marrow cells in vitro. Materials and methods: Non-adherent bone marrow cells were isolated and exposed to various concentrations (0–160 µg/ml) for 4 h at 23 °C. The chromatin proteins were analyzed by SDS PAGE and western blot. Fluorescent dye staining of the cells, anion superoxide and DNA fragmentations assays were also employed. Result: The results from MTT and trypan blue exclusion assays represented reduction of the cells viability. Extractability of histones and HMG proteins contrasted with difficulty as their content was decreased on SDS-gel upon increasing drug concentration as western blots confirmed it. The amount of degradation form of PARP (89 KD) increased significantly in a dose dependent manner. Increase in anion superoxide production and DNA fragmentation together with cytological detection of chromatin condensation and cellular damage upon exposure of the cells to vinorelbine were indicative of apoptosis induction in these normal cells. Conclusion: Vinorelbine is genotoxic in non-adherent bone marrow cells as affects chromatin components, DNA, histone and HMGB1 proteins and induces apoptosis.

High Bone Mass in Mice Lacking Cx37 Because of Defective Osteoclast Differentiation

Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37(-/-)) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37(-/-) mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37(-/-) mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37(+/+) littermates. sRANKL/M-CSF treatment of nonadherent Cx37(-/-) bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37(+/+) cell cultures. Further, Cx37(-/-) osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37(-/-) osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37(-/-) mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.

In Vitro Generation Of Osteoclasts From Interleukin-3 (IL-3)-Dependent Mouse Hematopoietic Cells

ObjectiveThis work aims to develop a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells Methods1. Here, we describe an alternative method for in vitro generation of osteoclasts, which involves the use of interleukin (IL)-3-dependent murine bone marrow cells. Bone marrow cells, isolated from 6- to 8-week old C57BL/6 were cultured in α-MEM containing 10% FBS in tissue culture dishes overnight to remove stromal cells. Then, non-adherent bone marrow cells were harvested and continued in α-MEM containing 10% FBS without (control) or with IL-3 (1 ng/ml) for 6 days. While no cells survived in the control culture after the 6-day culturing, the IL-3-treated culture gave rise to a significant number of surviving cells. These IL-3-depedent cells were capable of differentiating to osteoclasts in response to M-CSF and RANKL stimulation. Moreover, these IL-3-dependent cells can be further expanded by plating them in non-treated plastic dishes followed with M-CSF treatment; they continued to survive and proliferate in non-treated plastic dishes in the presence of M-CSF for up to 4 days. After 4-day M-CSF treatment, these cells can be lifted by EDTA, and they were still able to differentiate into osteoclasts upon subsequent stimulation of M-CSF and RANKL.2. We performed the in vitro bone resorption assay, Semiquantitative Reverse Transcription (RT)-PCR, Western Analysis, Infection of Murine Bone Marrow Cells (BMCs) to test whether the osteoclasts generated from IL-3-dependent murine bone marrow cells are different from the osteoclasts generated from traditional method. Results1. IL-3 can maintain the survival of murine bone marrow cells for up to 6 days and these cells still keep their capacity to generate osteoclasts. The capacity of IL-3-dependent cells to form osteoclasts decreases with time of IL-3 treatment and IL-3 dependent cells can be further expanded by M-CSF without significant loss of the osteoclastogenic potential.2. IL-3-dependent cells can form functional osteoclasts. RANKL induces the expression of osteoclast genes in IL-3-dependent cells. RANKL activates some of RANK signaling pathways in IL-3-dependent cells. Importantly, we found that IL-3 dependent murine bone marrow cells can be infected by retrovirus encoding GFP. Conclusions1) We have developed a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells.2) IL-3-dependent cells can be infected by retrovirus, permitting further experimental manipulations to express or knock down genes in IL-3-dependent cells for studying the molecular mechanism controlling differentiation and proliferation of osteoclast precursors or delineating molecular events in early osteoclastogenesis. Disclosures:No relevant conflicts of interest to declare.

F-spondin (spondin-1) null mice exhibit increased bone formation, decreased osteoclast function and accelerated osteoarthritis

Purpose: We have previously reported that F-spondin (spondin-1), a neuroregulatory protein, is upregulated by chondrocytes in osteoarthritis. These studies showed that spondin-1, a member of the TSR (thrombospondin) type I class super family, activated latent TGF-b1, which appeared to account for selected in vitro effects, including induction of the hypertrophic chondrocyte phenotype. In this study we generated Spon1 knockout mice to investigate the effect of F-spondin in vivo in i) skeletal development and ii) OA progression following surgical destabilization of the medial meniscus (DMM). Methods: Knockout mice were generated in collaboration with the Texas Institute of Genomic Medicine by targeted deletion of exon 1 in C57BL/6 mice. Exon deletion was confirmed by Southern blot analysis and PCR using probes specific for wild type (WT) and mutant loci. Total TGFb-1 was detected using R D p<0.002). Similarly, cultured chondrocytes isolated from the rib cages of 5 day old Spon1-/mice also produced significantly less TGF-b1 (30%) compared to WT controls (p<0.01). To determine whether Spon1 deletion affected bone phenotype, we performed microCT of tibia and femurs in mutant and WT mice aged 1-6 months Relative to WT mice, Spon1-/exhibited increased bone formation at 6 months (Figure 1), evidenced by, a) increased trabecular and cortical bone volume fraction (Bone volume/Total volume: 0.26 0.03 versus 0.16 0.05, p<0.0002; Fig 1), b) decreased trabecular spacing (0.14 0.02 versus 0.19 0.03, p<0.0003); and c) increased trabecular number (7.7 1.2, versus 5.5 0.9, p<0.0005). Interestingly, no significant changes were observed at 1 or 3 months, suggesting that Spon1 effects are age-dependent. Histologically, tibia from Spon1-/mice displayed increased bone ingrowth in the bone marrow cavity, particularly within the spongy bone of the ephysiseal regions. This was accompanied by decreased TRAP staining compared to WT mice, suggesting decreased numbers of osteoclasts. Supporting this observation, osteoclast differentiation, performed by RANKL, M-CSF induction of nonadherent bone-marrow cells, revealed impaired differentiation in Spon1-/mice compared to WT. Preliminary analyses suggest that Spon1 deletion also accelerated cartilage degradation in the DMM model of osteoarthritis. The increased severity of osteoarthritis-like cartilage destruction 7 weeks post surgery was by accompanied by increased thickening of subchondral bone (Figure 2). Conclusions: Our studies indicate that spondin-1 (F-spondin), a latent TGF-b1 activating ECM protein, over-expressed in OA bone and cartilage, regulates bone metabolism in aging mice. Spon1-/mice exhibit increased trabecular and cortical bone formation, decreased osteoclast function and increased susceptibility to surgical induced OA. Together these data suggest that a primary function of spondin-1 in skeletal tissue is the regulation of bone mass via latent TGF-b1 activation. Further studies are in progress regarding the potential of spondin-1 as a drugable target in future therapy of osteoporosis or osteoarthritis. 103 GENE ARRAY PROFILING OF ARTICULAR CHONDROCYTES IN MICE WITH DIFFERENT SUSCEPTIBILITY TO NATURAL DISEASE REVEALS SPECIFIC GENE SIGNATURES LINKED TO HEALTHY AGEING AND SPONTANEOUS OA.

Morphological and Molecular Analysis of Angiogenesis after Intramyocardial Transplantation of Autologous Bone Marrow Mononuclear Cells

We studied the peculiarities of angiogenesis in the postinfarction period after transmyocardial laser revascularization and intramyocardial implantation of mononuclear bone marrow cells into the pericicatrical zone of the left ventricular myocardium in dogs. Morphological manifestation of angiogenesis in the myocardium after application of laser and cell technologies are angiomatosis, formation of large thin-wall vessels and sinusoids. The angiogenic effect of implanted mononuclear bone marrow cells is determined by high content (43-47%) of CD31+ cells in both adherent and nonadherent fractions. More pronounced angiogenic potential of nonadherent cells is determined by intensive expression of cytokine VEGF-B and D mRNA essential for arterial vessels growth. Immunohistochemical studies showed that about 90% cells of the nonadherent fraction are endothelial precursors expressing endothelial cell markers isolectin B4 and VEGF-R2. It was found that the use of adherent mononuclear bone marrow cells during the postinfarction period induces ossification of the epicardium and subepicardial myocardium layer, formation of cartilage plates, and focal calcification. Implantation of nonadherent mononuclear bone marrow cells into transmyocardial laser channels did not induce ectopic ossification of the myocardium.

Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis.

Non-adherent bone marrow-derived cells (NA-BMCs) are a mixed cell population that can give rise to multiple mesenchymal phenotypes and that facilitates hematopoietic recovery. We characterized NA-BMCs by flow cytometry, fibroblast colony-forming units (CFU-f), real-time PCR, and in in vivo experiments. In comparison to adherent cells, NA-BMCs expressed high levels of CD11b(+) and CD90(+) within the CD45(+) cell fraction. CFU-f were significantly declining over the cultivation period, but NA-BMCs were still able to form CFU-f after 5days. Gene expression analysis of allogeneic NA-BMCs compared to bone marrow (BM) indicates that NA-BMCs contain stromal, mesenchymal, endothelial cells and monocytes, but less osteoid, lymphoid, and erythroid cells, and hematopoietic stem cells. Histopathological data and analysis of weight showed an excellent recovery and organ repair of lethally irradiated mice after NA-BMC transplantation with a normal composition of the BM.

Parathyroid Hormone Receptor Directly Interacts with Dishevelled to Regulate β-Catenin Signaling and Osteoclastogenesis

Bone growth and remodeling depend upon the opposing rates of bone formation and resorption. These functions are regulated by intrinsic seven transmembrane-spanning receptors, the parathyroid hormone receptor (PTH1R) and frizzled (FZD), through their respective ligands, parathyroid hormone (PTH) and Wnt. FZD activation of canonical beta-catenin signaling requires the adapter protein Dishevelled (Dvl). We identified a Dvl-binding motif in the PTH1R. Here, we report that the PTH1R activates the beta-catenin pathway by directly recruiting Dvl, independent of Wnt or LRP5/6. PTH1R coimmunoprecipitated with Dvl. Deleting the carboxyl-terminal PTH1R PDZ-recognition domain did not abrogate PTH1R-Dvl interactions; nor did truncating the receptor at position 480. However, further deletion eliminating the putative Dvl recognition domain abolished PTH1R interactions with Dvl. PTH activated beta-catenin in a time- and concentration-dependent manner and translocated beta-catenin to the nucleus. beta-Catenin activation was inhibited by Dvl2 dominant negatives and by short hairpin RNA sequences targeted against Dvl2. PTH-induced osteoclastogenesis was also inhibited by Dvl2 dominant negative mutants. These findings demonstrate that G protein-coupled receptors other than FZD directly activate beta-catenin signaling, thereby mimicking many of the functions of the canonical Wnt-FZD pathway. The distinct modes whereby FZD and PTH1R activate beta-catenin control convergent or divergent effects on osteoblast differentiation, and osteoclastogenesis may arise from PTH1R-induced second messenger phosphorylation.

Stromal cell independent B cell development in vitro: Generation and recovery of autoreactive clones

We describe and characterize a stromal cell independent culture system that efficiently supports pro-B cell to IgM+ B cell development with near normal levels of IgH and Igκ diversity. Pro-B cells present in non-adherent bone marrow cells proliferate in the presence of IL-7 and subsequent to the removal of IL-7 and addition of BAFF, differentiate normally into IgM+ B cells. B cell development in vitro closely follows the patterns of development in vivo with culture-derived (CD) B cells demonstrating characteristic patterns of surface antigen expression and gene activation. IgM+ CD B cells respond to TLR stimulation by proliferation and differentiation into antibody-secreting cells. Self-reactive IgM+ B cell development is blocked in 3H9 IgH knockin mice; however, cultures of 3H9 IgH knockin pro-B cells yields high frequencies of “forbidden”, autoreactive IgM+ B cells. Furthermore, serum IgG autoantibody exceeded that present in autoimmune, C4−/− animals following the reconstitution of RAG1−/− mice with IgM+ CD cells derived from BL/6 mice.

Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment

BackgroundNon adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4+, murine CD4−/−, HLA-DR3+ mice.Methodology/Principal FindingsBone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45+ showed depletion of CD4+, CD8+, CD19+, and CD117+ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4+ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death.Conclusions/SignificanceNA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.

Inhibition of Cdk4/6 by PD 0332991 Results in Abrogation of Osteoclast Formation

Multiple myeloma (MM) is characterized by increased osteoclast activity resulting in bone destruction and development of lytic lesions. Despite overall survival achieved by new treatment modalities, new drugs are required to specifically and effectively inhibit bone destruction. PD 0332991 is a selective small molecule inhibitor of cyclin-dependent kinase (Cdk)4 and Cdk6 with oral bioavailability. We have demonstrated recently that inhibition of Cdk4/6 by PD 0332991 effectively controls MM tumor expansion in animal models and sensitizes chemoresistant MM tumor cells to bortezomib killing despite protection by bone marrow stromal cells. Currently clinical phase I/II trials are ongoing to test the efficacy of the combination of PD 0332991 and bortezomib.Selective inhibition of Cdk4/6 by PD 0332991 leads to sustained G1 arrest but not apoptosis, and this appears to be cell type-specific. Consistent with these findings, PD 0332991 is well tolerated in MM patients. On this basis, we address the possibility that PD 0332991, alone and in combination with bortezomib, may inhibit osteoclastogenesis, by culturing non-adherent mononuclear bone marrow cells from MM patients cultured in the presence of M-CSF and RANKL for three weeks. Treatment of human osteoclast cultures with PD 0332991 for 3 weeks decreased osteoclast formation in a dose-dependent manner with an IC50 50 nM. The combination of PD 0332991 and bortezomib led to synergistic inhibition of osteoclast formation and completely abrogated osteoclastogenesis with low dose PD 0332991 (25 nM) and bortezomib (2 nM). Furthermore, we show in a time course study that treatment with PD 0332991 for the first week, but not the second or third week, was sufficient to inhibit osteoclast formation. PD 0332991 is the only known selective inhibitor of Cdk4 and Cdk6, which at concentration below 5 uM does not cross react with at least 38 kinases or induce apoptosis. Taken together, our data suggest that by inducing G1 arrest and inhibiting progenitor expansion, PD 0332991 is a powerful and selective inhibitor for osteoclastogenesis. We propose that targeting Cdk4/6 with PD 0332291 in combination therapy is a promising therapeutic strategy to improve bone integrity in MM.

Dietary isoflavones act on bone marrow osteoprogenitor cells and stimulate ovary development before influencing bone mass in pre‐pubertal piglets

Food containing soybeans provide isoflavone phytoestrogens that can preserve bone mass in postmenopausal women, and prevent bone loss in ovariectomized rats. But their effects on bone remain unclear, particularly on bone formation during growth. Two groups of eight pre-pubertal piglets were fed a basal or an isoflavone-enriched (S800) diet for 6 weeks. The S800 diet contained 800 mg SoyLifetrade mark/kg, providing 2.8 mg isoflavones/kg body weight/day. Several bones were collected and tested for bone strength and density. Bone marrow was collected from humeri together with blood samples and genital tracts. The plasma concentrations of isoflavones were increased in the pigs fed S800, but growth rate, body weight, plasma bone markers, bone mineral density, and strength were all unaffected. In contrast, cultured stromal cells from S800 pigs had more alkaline phosphatase-rich cells and mineralized nodules, secreted more osteocalcin, osteoprotegerin and RANK-L, synthesized more osteoprotegerin, and RANK-L. Cultured mononucleated nonadherent bone marrow cells from S800 pigs developed fewer tartrate-resistant acid phosphatase mononucleated cells (osteoclast progenitors) when cultured with 1,25(OH)(2)D(3), and resorbed a smaller area of dentine slices. Freshly isolated bone marrow osteoclast progenitors from S800 pigs had more caspase-3 cleavage activity, and synthesized less RANK. Both osteoclast and osteoblast progenitors had ERalpha and ERbeta, whose syntheses were stimulated by the S800 diet. The S800 piglets had heavier ovaries with more follicles, but their uterus weight was unaffected. We conclude that dietary isoflavones have no detectable effect on the bone mass of growing female piglets, but act on bone marrow osteoprogenitors via ERs--mainly ERbeta, and stimulate ovary development.

Vaccination with Lymphoma and Dendritic Cell Hybrids Intended as an Adjuvant Therapy for Lymphoma Patients - A Feasibility Study.

A feasibility of hybrid cell vaccination performed in remission following a standard chemotherapy (CHT) is currently being studied in patients with indolent B cell lymphomas at our institution under approval of the Ethics Committee. Adequate samples of fresh tumor tissue were collected from 16 patients. Seven patients were vaccinated with irradiated lymphoma/dendritic cell (DC) hybrids, and whenever the amount of collected viable lymphoma cells was not sufficient to complete vaccination with the use of cell hybrids, booster injections were alternatively performed with the use autologous DCs pulsed with tumor lysate. Autologous lymphoma cells were electrofused with autologous and/or allogeneic DCs, depending on accessibility of autologous DCs. Monocyte-derived DCs were basically generated from the peripheral blood adherent cells stimulated with GM-CSF and IL-4, and if available, from the bone marrow (BM) adherent cells. If only possible, autologous DCs were also generated from the BM non-adherent cells or from a sample of leukapheresis product collected for transplantation cultured with GM-CSF, TNF-α, SCF and FLT3-L to induce differentiation of DC progenitors. DC hybrids and/or DCs preincubated with tumor lysate were administered to uninvolved lymph nodes under USG guidance. Four patients failed to achieve a complete remission after initial therapy and the vaccination was discontinued due to disease progression (after 2, 2, 6, and 5 cycles). Three patients were vaccinated during remission following CHT. At present - 16, 18, and 10 months after starting the immunization, and after 8, 9, and after 7 cycles of vaccination, one patient with mantle cell lymphoma (MCL) is symptomless with persistent bone marrow (BM) involvement, one patient with MCL presenting with IgM paraproteinemia is disease-free with residual BM involvement (Fig 1), and one patient with follicular lymphoma is disease-free. Cutaneous delayed type hypersensitivity (DTH) response to DC/lymphoma hybrids exceeded the response to DCs alone in 3 of 7 vaccinated patients, and interferon-γ producing CD8+ cells specific to autologous lymphoma cells were demonstrated in one patient. In another patient, cutaneous DTH reaction was accompanied by transient pruritus and swelling of the injected lymph node two days after the sixth booster injection. No other adverse reactions to vaccinations were observed. Vaccination using the DC/lymphoma hybrids appears feasible, safe, and capable of inducing lymphoma cell specific immune responses. Further evaluation of efficacy in terms of disease control is mandatory. [Display omitted]

Comparative analysis of hypermethylation of cell cycle control and DNA-mismatch repair genes in low-density and CD34+ bone marrow cells from patients with myelodysplastic syndrome

Hypermethylation of CpG islands within the promoter region is one of the mechanisms by which genes are inactivated and may be one of the reason for silencing of cell cycle control or DNA-mismatch repair genes in myelodysplastic syndrome (MDS). Since the function of cell cycle control genes including the cyclin-dependent kinase inhibitors known as p15 INK4b and p16 INK4a, as well as p14 ARF which blocks MDM-2 (an inhibitor of p53), the retinoblastoma (RB1) protein and the mismatch repair gene MGMT is critical for hematopoietic proliferation and differentiation, we performed methylation specific polymerase chain reaction (MSP) in low-density, non-adherent bone marrow cells from 49 patients with MDS. In addition, expression of p15 INK4b and RB1 was analysed by quantitative real-time PCR. From selected patients, we analyzed the methylation pattern of cell cycle control genes in CD34+ bone marrow cells. Thirty-nine of 49 cases (80%) had at least one of five genes methylated in our MDS samples by analysing low-density non-adherent bone marrow cells. The frequency of p15 INK4b methylation was 34 of 49 samples (69%). The incidence of methylation of both p14 ARF and p16 INK4a was four of 49 (8%). RB1 gene was methylated in seven samples (14%) and each patient had RA. Interestingly, none of these genes were methylated in the purified CD34+ hematopoietic stem cells from the MDS patients. Furthermore, all our RARS patients had a methylated p15 INK4b promoter correlating with non-detectable expression of this gene in bone marrow cells from those patients. These results indicate that hypermethylation of cell cycle control genes in MDS may occur late during the differentiation of myelodysplastic stem cells.

Circulating Cells with Osteogenic Potential

While osteoclast lineage cells are clearly present in the peripheral circulation, whether there is a comparable pool of circulating osteoblast lineage cells has remained controversial. Using assays requiring adherence to plastic (as originally described by Friedenstein and colleagues for bone marrow stromal cells over four decades ago), several studies have shown that plastic adherent cells with osteogenic potential are, indeed, present in the circulation of a number of species, but at extremely low concentrations. Work from a number of independent groups over the past decade has also identified a population of nonadherent bone marrow cells with osteogenic potential. Since these nonadherent cells may be much more likely to access the peripheral circulation than plastic adherent cells, we tested for the presence of circulating osteoblast lineage cells in humans using flow cytometry to identify cells in the peripheral blood expressing bone-related proteins. Our findings indicate that these cells are present in the circulation in significant numbers, are markedly increased in the peripheral blood of adolescent boys going through the growth spurt, and may also increase following fractures. These circulating osteogenic cells express bone-related proteins, can mineralize in vitro, and form bone in vivo. The identification of these osteogenic cells in peripheral blood opens up new questions regarding the possible role of these cells in bone remodeling, in fracture repair, and possibly in vascular calcification.

Tumor Necrosis Factor-α Increases Circulating Osteoclast Precursor Numbers by Promoting Their Proliferation and Differentiation in the Bone Marrow through Up-regulation of c-Fms Expression

Osteoclasts are essential cells for bone erosion in inflammatory arthritis and are derived from cells in the myeloid lineage. Recently, we reported that tumor necrosis factor-alpha (TNFalpha) increases the blood osteoclast precursor (OCP) numbers in arthritic patients and animals, which are reduced by anti-TNF therapy, implying that circulating OCPs may have an important role in the pathogenesis of erosive arthritis. The aim of this study is to investigate the mechanism by which TNFalpha induces this increase in OCP frequency. We found that TNFalpha stimulated cell division and conversion of CD11b+/Gr-1-/lo/c-Fms- to CD11b+/Gr-1-/lo/c-Fms+ cells, which was not blocked by neutralizing macrophage colony-stimulating factor (M-CSF) antibody. Ex vivo analysis of monocytes demonstrated the following: (i) blood CD11b+/Gr-1-/lo but not CD11b-/Gr-1- cells give rise to osteoclasts when they were cultured with receptor activator NF-kappaB ligand and M-CSF; and (ii) TNF-transgenic mice have a significant increase in blood CD11b+/Gr-1-/lo cells and bone marrow proliferating CD11b+/Gr-1-/lo cells. Administration of TNFalpha to wild type mice induced bone marrow CD11b+/Gr-1-/lo cell proliferation, which was associated with an increase in CD11b+/Gr-1-/lo OCPs in the circulation. Thus, TNFalpha directly stimulates bone marrow OCP genesis by enhancing c-Fms expression. This results in progenitor cell proliferation and differentiation in response to M-CSF, leading to an enlargement of the marrow OCP pool. Increased marrow OCPs subsequently egress to the circulation, forming a basis for elevated OCP frequency. Therefore, the first step of TNF-induced osteoclastogenesis is at the level of OCP genesis in the bone marrow, which represents another layer of regulation to control erosive disease.