The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

Hydroxysafflor Yellow A-Induced Osteoblast Differentiation and Proliferation of BM-MSCs by Up-Regulating Nuclear Vitamin D Receptor.

BACKGROUND: Vitamin D receptor (VDR) is critical for mineral and bone homeostasis since it plays an essential role in the osteoblast differentiation of bone marrow mesenchymal stem cells (BM-MSCs). Hydroxysafflor yellow A (HSYA) has the potential to promote bone mineralization and inhibit bone resorption, while its detailed mechanism needs to be elaborated. OBJECTIVE: This study intends to explore the action of HSYA on the proliferation and differentiation of BM-MSC and the underlying mechanism. METHODS: Different concentrations of HSYA to BM-MSC and CCK-8, and EdU were used to detect cell viability and proliferation. The alkaline phosphatase (ALP) was used to observe the differentiation ability of BM-MSC osteoblasts. The calcium uptake and mineralization of osteoblast-like cells were observed by alizarin red staining. The level of calcium ion uptake in cells was detected by flow cytometry. AutoDock was performed for molecular docking of HSYA to VDR protein. Immunofluorescence and western blotting were performed to detect the expression of VDR expression levels. Finally, the effect of VDR was verified by a VDR inhibitor. RESULTS: After treatment with HSYA, the proliferation and calcium uptake of BM-MSC were increased. The level of ALP increased significantly and reached its peak on the 12th day. HSYA promoted calcium uptake and calcium deposition, and mineralization of osteoblasts. The western blotting and immunofluorescence showed that HSYA increased the expression of VDR in the osteoblast-like cell's nucleus and upregulated Osteocalcin, S100 calcium-binding protein G, and CYP24A1. In addition, HYSA treatment increased the expression of osteopontin and the synthesis of osteogenic proteins, such as Type 1 collagen. After the addition of the VDR inhibitor, the effect of HSYA was weakened. CONCLUSION: HSYA could significantly promote the activity and proliferation of osteoblasts and increase the expression level of VDR in osteoblasts. HSYA may also improve calcium absorption by osteoblasts by regulating the synthesis of calciumbinding protein and vitamin D metabolic pathway-related proteins.

Lyme Disease, Borrelia burgdorferi, and Lipid Immunogens.

The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete Borrelia burgdorferi, and a galactoglycerolipid (1) has been identified as a key trigger for the inflammatory immune response that characterizes LD. This report corrects the original structural assignment of 1 to 3, a change of an α-galactopyranose to an α-galactofuranose headgroup. The seemingly small change has important implications for the diagnosis, prevention, and treatment of LD.

Mechanisms involved in suppression of osteoclast supportive activity by transforming growth factor-ß1 via the ubiquitin-proteasome system.

Orthodontic treatment requires the regulation of bone remodeling in both compression and tension sides. Transforming growth factor-ß1 (TGF-ß1) is an important coupling factor for bone remodeling. However, the mechanism underlying the TGF-ß1-mediated regulation of the osteoclast-supporting activity of osteoblasts and stromal cells remain unclear. The current study investigated the effect of TGF-ß1 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in stromal cells induced by 1α,25(OH)2D3 (D3) and dexamethasone (Dex). TGF-ß1 downregulated the expression of RANKL induced by D3 and Dex in mouse bone marrow stromal lineage, ST2 cells. Co-culture system revealed that TGF-ß1 suppressed osteoclast differentiation from bone marrow cell induced by D3 and Dex-activated ST2 cells. The inhibitory effect of TGF-ß1 on RANKL expression was recovered by inhibiting the interaction between TGF-ß1 and the TGF-ß type I/activin receptor or by downregulating of smad2/3 expression. Interestingly, TGF-ß1 degraded the retinoid X receptor (RXR)-α protein which forms a complex with vitamin D receptor (VDR) and regulates transcriptional activity of RANKL without affecting nuclear translocation of VDR and phosphorylation of signal transducer and activator of transcription3 (STAT3). The degradation of RXR-α protein by TGF-ß1 was recovered by a ubiquitin-proteasome inhibitor. We also observed that poly-ubiquitination of RXR-α protein was induced by TGF-ß1 treatment. These results indicated that TGF-ß1 downregulates RANKL expression and the osteoclast-supporting activity of osteoblasts/stromal cells induced by D3 and Dex through the degradation of the RXR-α protein mediated by ubiquitin-proteasome system.

UM171A-induced ROS promote antigen cross-presentation of immunogenic peptides by bone marrow-derived mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been extensively used in the clinic due to their exquisite tissue repair capacity. However, they also hold promise in the field of cellular vaccination as they can behave as conditional antigen presenting cells in response to interferon (IFN)-gamma treatment under a specific treatment regimen. This suggests that the immune function of MSCs can be pharmacologically modulated. Given the capacity of the agonist pyrimido-indole derivative UM171a to trigger the expression of various antigen presentation-related genes in human hematopoietic progenitor cells, we explored the potential use of UM171a as a means to pharmacologically instill and/or promote antigen presentation by MSCs. METHODS: Besides completing a series of flow-cytometry-based phenotypic analyses, several functional antigen presentation assays were conducted using the SIINFEKL-specific T-cell clone B3Z. Anti-oxidants and electron transport chain inhibitors were also used to decipher UM171a's mode of action in MSCs. Finally, the potency of UM171a-treated MSCs was evaluated in the context of therapeutic vaccination using immunocompetent C57BL/6 mice with pre-established syngeneic EG.7T-cell lymphoma. RESULTS: Treatment of MSCs with UM171a triggered potent increase in H2-Kb cell surface levels along with the acquisition of antigen cross-presentation abilities. Mechanistically, such effects occurred in response to UM171a-mediated production of mitochondrial-derived reactive oxygen species as their neutralization using anti-oxidants or Antimycin-A mitigated MSCs' ability to cross-present antigens. Processing and presentation of the immunogenic ovalbumin-derived SIINFEKL peptide was caused by de novo expression of the Psmb8 gene in response to UM171a-triggered oxidative stress. When evaluated for their anti-tumoral properties in the context of therapeutic vaccination, UM171a-treated MSC administration to immunocompetent mice with pre-established T-cell lymphoma controlled tumor growth resulting in 40% survival without the need of additional supportive therapy and/or standard-of-care. CONCLUSIONS: Altogether, our findings reveal a new immune-related function for UM171a and clearly allude to a direct link between UM171a-mediated ROS induction and antigen cross-presentation by MSCs. The fact that UM171a treatment modulates MSCs to become antigen-presenting cells without the use of IFN-gamma opens-up a new line of investigation to search for additional agents capable of converting immune-suppressive MSCs to a cellular tool easily adaptable to vaccination.

Synthesis and Evaluation of Structurally Diverse C-2-Substituted Thienopyrimidine-Based Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase.

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.

The effect of P2X7R- mediated Ca2+ and MAPK signaling in OPG-induced duck embryo osteoclasts differentiation and adhesive structure damage.

Various factors cause animal bone malnutrition disease during intensive culture. Osteoclasts play an important role in regulating bone metabolism disease. Osteoprotegerin (OPG) modulates osteoclast function; however, the mechanism underlying this effect is unknown. Therefore, the present study aimed to explore whether OPG affects duck embryo osteoclast function via purinergic receptor P2X7. OPG significantly inhibited duck embryo osteoclast differentiation and bone resorption, and suppressed F-actin formation. In addition, OPG remarkably impaired duck embryo osteoclasts' adhesive structure. After OPG treatment, the expression of P2X7R significantly reduced, the ATP level and Ca2+-ATPase activity decreased rapidly, and concomitantly suppressed calcium and MAPK signaling. A438079 (a selective P2X7R inhibitor) significantly inhibited duck embryo osteoclast differentiation and bone resorption, and the phosphorylation of Ca2+ regulated proteins (CAM, CAMKII, CAMKIV) and MAPKs (ERK, JNK, and P38) were markedly suppressed. Pretreatment of duck embryo osteoclasts with BzATP, a P2X7R agonist, activated Ca2+ and MAPK signaling. BzATP alleviated OPG-induced duck embryo osteoclast differentiation and adhesive structure damage, and recovered the distribution of adhesion-related proteins in mature duck embryo osteoclasts. Thus, P2RX7-mediated Ca2+ and MAPK signaling has a key function in OPG-induced duck embryo osteoclast differentiation and adhesive structure damage. P2X7R might be an ideal target to treat bone diseases through regulating bone cell activation.

Two birds with one stone: YQSSF regulates both proliferation and apoptosis of bone marrow cells to relieve chemotherapy-induced myelosuppression.

ETHNOPHARMACOLOGICAL RELEVANCE: Yiqi Shengsui formula (YQSSF) is a commonly used formula to treat chemotherapy-induced myelosuppression, but little is known about its therapeutic mechanisms. AIM OF THIS STUDY: This study aims to examine the effect of YQSSF in treating myelosuppression and explore its mechanism. MATERIALS AND METHODS: A myelosuppression BALB/c mouse model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CTX). The efficacy of YQSSF in alleviating chemotherapy-induced myelosuppression was evaluated by blood cell count, immune organ (thymus, spleen, liver) index, bone marrow nucleated cell (BMNC) count and histopathological analysis of bone marrow and spleen. Then, ultra-performance liquid chromatograph quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to analyze the ingredients of YQSSF extract. Key effects and potential mechanism of YQSSF extract in alleviating myelosuppression were predicted by network pharmacology method. Finally, cell cycle and TUNEL staining of bone marrow cells was detected to verify the key effects, and RT-qPCR or Western blotting were performed to measure the gene and protein expressions of the effect targets respectively to confirm the predicted mechanism of YQSSF for myelosuppression. RESULTS: YQSSF up-regulated the number of peripheral blood leukocytes and BMNC, reduced spleen index and liver index, improved the pathological morphology of bone marrow and spleen. A total of 40 ingredients were isolated from YQSSF extract using UPLC-Q/TOF-MS analysis. Network pharmacology revealed that YQSSF regulated both proliferation and apoptosis to alleviate myelosuppression. Finally, YQSSF decreased G0/G1 ratio, increased the proportion of bone marrow cells in S phase and proliferation index (PI), and reduced apoptotic cells in femur bone marrow. RT-qPCR and Western blotting showed that YQSSF up-regulated the expression levels of CDK4, CDK6, CyclinB1, c-Myc and Bcl-2, as well as down-regulated the expression levels of Cyt-c, Fas, Caspase-8/3 and p53. CONCLUSIONS: YQSSF promotes the proliferation and inhibits the apoptosis of bone marrow cells to relieve chemotherapy-induced myelosuppression.

Endosomal Sequestration of TLR4 Antibody Induces Myeloid-Derived Suppressor Cells and Reverses Acute Type 1 Diabetes.

We previously showed that treating NOD mice with an agonistic monoclonal anti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type 1 diabetes (T1D). Here, we show that TLR4-Ab reverses T1D by induction of myeloid-derived suppressor cells (MDSCs). Unbiased gene expression analysis after TLR4-Ab treatment demonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells and downregulation of T-cell genes. Further RNA sequencing of purified, TLR4-Ab-treated CD11b+ cells showed significant upregulation of genes associated with bone marrow-derived CD11b+ cells and innate immune system genes. TLR4-Ab significantly increased percentages and numbers of CD11b+ cells. TLR4-Ab-induced CD11b+ cells, derived ex vivo from TLR4-Ab-treated mice, suppress T cells, and TLR4-Ab-conditioned bone marrow cells suppress acute T1D when transferred into acutely diabetic mice. Thus, the TLR4-Ab-induced CD11b+ cells, by the currently accepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanism, we compared TLR4-Ab with TLR4 agonist lipopolysaccharide (LPS), which cannot reverse T1D. TLR4-Ab remains sequestered at least 48 times longer than LPS within early endosomes, alters TLR4 signaling, and downregulates inflammatory genes and proteins, including nuclear factor-κB. TLR4-Ab in the endosome, therefore, induces a sustained, attenuated inflammatory response, providing an ideal "second signal" for the activation/maturation of MDSCs that can reverse acute T1D.

KR-31831 improves survival and protects hematopoietic cells and radiosensitive tissues against radiation-induced injuries in mice.

This study explored the radioprotective effects and possible underlying mechanisms of KR-31831 against radiation-induced injury in a mouse model. KR-31831 (30 and 50 mg/kg) was administered to mice 24 h and 30 min before exposure to a single lethal or sublethal dose of whole-body irradiation (WBI) (7 or 4 Gy, respectively). These animals were then evaluated for changes in mortality, various hematological and biochemical parameters, and histological features in response to these treatments. In addition, RNA sequencing was used to profile the radiation-induced transcriptomic response in the bone marrow cells. The results showed that KR-31831 dose-dependently prolonged the 30-day survival period and prevented damage to radiation-sensitive organs, such as the intestine and testis, in response to WBI. Damage to the hematopoietic system was also notably improved in the KR-31831-treated mice, as evidenced by an increase in bone marrow and peripheral blood cells, as well as recovery of the histopathological characteristics of the bone marrow. These protective effects were achieved, at least in part, via the suppression of radiation-induced increases in apoptotic cell death and erythropoietin levels in the plasma. Furthermore, the gene expression profiles of the bone marrow cells of the WBI-treated mice suggested that KR-31831 upregulates the expression of the genes involved in regulating apoptosis and modulating the immune response, both of which are required for protecting the bone marrow. These results suggest the potential therapeutic efficacy of KR-31831 for protection against radiation-induced injury.

Dihydroartemisinin attenuates osteoclast formation and bone resorption via inhibiting the NF­κB, MAPK and NFATc1 signaling pathways and alleviates osteoarthritis.

Osteoarthritis (OA) is a chronic, progressive and degenerative disease, and its incidence is increasing on a yearly basis. However, the pathological mechanism of OA at each stage is still unclear. The present study aimed to explore the underlying mechanism of dihydroartemisinin (DHA) in terms of its ability to inhibit osteoclast activation, and to determine its effects on OA in rats. Bone marrow­derived macrophages were isolated as osteoclast precursors. In the presence or absence of DHA, osteoclast formation was assessed by tartrate­resistant acid phosphatase (TRAP) staining, cell viability was assessed by Cell Counting Kit­8 assay, the presence of F­actin rings was assessed by immunofluorescence, bone resorption was determined by bone slices, luciferase activities of NF­κB and nuclear factor of activated T cell cytoplasmic 1 (NFATc1) were determined using luciferase assay kits, the protein levels of biomolecules associated with the NF­κB, MAPK and NFATc1 signaling pathways were determined using western blotting, and the expression of genes involved in osteoclastogenesis were measured using reverse transcription­quantitative PCR. A knee OA rat model was designed by destabilizing the medial meniscus (DMM). A total of 36 rats were assigned to three groups, namely the sham­operated, DMM + vehicle and DMM + DHA groups, and the rats were administered DHA or DMSO. At 4 and 8 weeks postoperatively, the microarchitecture of the subchondral bone was analyzed using micro­CT, the thickness of the cartilage layers was calculated using H&E staining, the extent of cartilage degeneration was scored using Safranin O­Fast Green staining, TRAP­stained osteoclasts were counted, and the levels of receptor activator of NF­κB ligand (RANKL), C­X­C­motif chemokine ligand 12 (CXCL12) and NFATc1 were measured using immunohistochemistry. DHA was found to inhibit osteoclast formation without cytotoxicity, and furthermore, it did not affect bone formation. In addition, DHA suppressed the expression levels of NF­κB, MAPK, NFATc1 and genes involved in osteoclastogenesis. Progressive cartilage loss was observed at 8 weeks postoperatively. Subchondral bone remodeling was found to be dominated by bone resorption accompanied by increases in the levels of RANKL, CXCL12 and NFATc1 during the first 4 weeks. DHA was found to delay OA progression by inhibiting osteoclast formation and bone resorption during the early phase of OA. Taken together, the results of the present study demonstrated that the mechanism through which DHA could inhibit osteoclast activation may be associated with the NF­κB, MAPK and NFATc1 signaling pathways, thereby indicating a potential novel strategy for OA treatment.

Structural Characterization of Polysaccharides Isolated from Panax notoginseng Medicinal Residue and Its Protective Effect on Myelosuppression Induced by Cyclophosphamide.

This study aims to establish the isolation and purification method of polysaccharides from medicinal residue of Panax notoginseng (PPN). The structure and protective effect of PPN on myelosuppression mice were investigated. One neutral polysaccharide (NPPN) and five acidic polysaccharides (APPN I, APPN II-A, APPN II-B, APPN III-A, and APPN III-B) were obtained. The results confirmed that NPPN, APPN I and APPN II-A are glycan with 1, 4 main chains. APPN III-A is a glycan. APPN II-B and APPN III-B are homogalacturonan pectin with 1, 4 main chains. This study demonstrated that NPPN played a bone marrow protective role in myelosuppression mice induced by cyclophosphamide. NPPN could relieve cell cycle arrest, reduce the apoptosis rate of marrow cells, and improve granulocyte-macrophage colony-stimulating (GM-CSF), thermoplastic polyolefin (TPO) and erythropoietin (EPO) serum level, which contributes to promoting the proliferation of hematopoietic cells.

Sunscreen filter octocrylene is a potential obesogen by acting as a PPARγ partial agonist.

Octocrylene (OC) is an extensively prescribed organic ultraviolet B filter used in sunscreen products. Due to its extensive use, a significant level of OC is detected in marine and freshwater environments. Notably, the bioaccumulation of OC in aquatic biota may affect human health. In this study, the effect of OC on metabolism was investigated using the adipogenesis model of human bone marrow mesenchymal stem cells (hBM-MSCs). OC promoted adiponectin production during adipogenesis in hBM-MSCs compared to the vehicle-treated control (EC50, 29.6 µM). In target identification, OC directly bound to peroxisome proliferator-activated receptor (PPAR) γ (Ki, 37.8 µM). OC-bound PPARγ also significantly recruited nuclear receptor coactivator proteins SRC-1 (EC50, 54.1 µM) and SRC-2 (EC50, 58.6 µM). In the molecular docking simulation study, the optimal ligand-binding mode of OC suggested that OC is a PPARγ partial agonist. A competitive analysis with a PPARγ full agonist pioglitazone revealed that OC acted as a PPARγ partial agonist. OC altered the gene transcription profile of lipid-metabolism associated enzymes in normal human keratinocytes, primarily exposed human cells after the application of sunscreens. In conclusion, OC is a potential metabolic disrupting obesogen.

Metformin attenuates osteoclast-mediated abnormal subchondral bone remodeling and alleviates osteoarthritis via AMPK/NF-κB/ERK signaling pathway.

This study explored the mechanism by which metformin (Met) inhibits osteoclast activation and determined its effects on osteoarthritis (OA) mice. Bone marrow-derived macrophages were isolated. Osteoclastogenesis was detected using tartrate-resistant acid phosphatase (TRAP) staining. Cell proliferation was evaluated using CCK-8, F-actin rings were detected by immunofluorescence staining, and bone resorption was detected using bone slices. Nuclear factor kappa-B (NF-κB) and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) were detected using luciferase assays, and the adenosine monophosphate-activated protein kinase (AMPK), NF-κB, and mitogen-activated protein kinase (MAPK) signaling pathways were detected using western blotting. Finally, expression of genes involved in osteoclastogenesis was measured using quantitative polymerase chain reaction. A knee OA mouse model was established by destabilization of the medial meniscus (DMM). Male C57BL/6J mice were assigned to sham-operated, DMM+vehicle, and DMM+Met groups. Met (100 mg/kg/d) or vehicle was administered from the first day postoperative until sacrifice. At 4- and 8-week post OA induction, micro-computed tomography was performed to analyze microstructural changes in the subchondral bone, hematoxylin and eosin staining and Safranin-O/Fast Green staining were performed to evaluate the degenerated cartilage, TRAP-stained osteoclasts were enumerated, and receptor activator of nuclear factor κB ligand (RANKL), AMPK, and NF-κB were detected using immunohistochemistry. BMM proliferation was not affected by Met treatment below 2 mM. Met inhibited osteoclast formation and bone resorption in a dose-dependent manner in vitro. Met suppressed RANKL-induced activation of p-AMPK, NF-κB, phosphorylated extracellular regulated protein kinases (p-ERK) and up-regulation of genes involved in osteoclastogenesis. Met reversed decreases in BV/TV, Tb.Th, Tb.N, and CD, and an increase in Tb.Sp at 4 weeks postoperatively. The number of osteoclasts and OARSI score were decreased by Met without effect on body weight or blood glucose levels. Met inhibited RANKL, p-AMPK, and NF-κB expression in early OA. The mechanism by which Met inhibits osteoclast activation may be associated with AMPK/NF-κB/ERK signaling pathway, indicating a novel strategy for OA treatment.

Cyclodextrin metal-organic framework as vaccine adjuvants enhances immune responses.

It is urgently needed to develop novel adjuvants for improving the safety and efficacy of vaccines. Metal-organic frameworks (MOFs), with high surface area, play an important role in drug delivery. With perfect biocompatibility and green preparation process, the γ-cyclodextrin metal-organic framework (γ-CD-MOF) fabricated with cyclodextrin and potassium suitable for antigen delivery. In this study, we modified γ-CD-MOF with span-85 to fabricate the SP-γ-CD-MOF as animal vaccine adjuvants. The ovalbumin (OVA) as the model antigen was encapsulated into particles to investigate the immune response. SP-γ-CD-MOF displayed excellent biocompatibility in vitro and in vivo. After immunization, SP-γ-CD-MOF loaded with OVA could induce high antigen-specific IgG titers and cytokine secretion. Meanwhile, SP-γ-CD-MOF also significantly improved the proliferation of spleen cells and activated and matured the bone marrow dendritic cells (BMDCs). The study showed the potential of SP-γ-CD-MOF in vaccine adjuvants and provided a novel idea for the development of vaccine adjuvants.

Simultaneous Administration of NOD-2 (MDP) and TLP-4 (LPS) Ligands to Bone Marrow Donors 24 h before Transplantation Increases the Content of Multipotent Stromal Cells (MSCs) in Bone Marrow Grafts in CBA Mice Compared to the Total Result of Their Isolated Administration.

In 3-month bone marrow transplants of CBA mice from bone marrow donors receiving single injections of TLR-4 ligand (LPS) or NOD-2 ligand (muramyl dipeptide, MDP) 24 h before transplantation, an increase in the total number of MSCs (by 2.6 and 1.9 times, respectively), as well as a slight increase in the number of nuclear cells and the mass of bone capsules (by 1.3 and 1.2 times) were observed. After combined administration of MDР and LPS to donors, the total content of MSCs in the grafts was higher by 1.6 times in comparison with the total result of their isolated administration (and by 7.2 times in comparison with the control). At the same time, the concentration of osteogenic MSCs in the grafts of all groups was almost the same and corresponded to the control level. The number of nuclear cells and the mass of bone capsules of the grafts after combined administration of LPS and MDP were close (~80%) to the sum of the results of their isolated administration. These findings suggest that activation of the stromal tissue and the success of bone marrow transplantation depend on the intensity of innate immune responses. These data can be useful for the development of optimal methods of tissue transplantation.

Experimental Study of the Efficacy of Sodium Deoxyribonucleate Used in Combination with Co-Transplantation of Mesenchymal and Hematopoietic Stem Cells after Exposure to γ-Radiation.

We studied the possibility of using sodium deoxyribonucleate (Derinat) for improving the efficiency of co-transplantation of mesenchymal (MSC) and hematopoietic stem cells (HSC) to female F1(CBA×C57BL/6) mice with bone marrow aplasia caused by exposure to γ-radiation. It was found that immunomodulator Derinat enhanced the effect of co-transplantation, in particular, triple post-irradiation administration of Derinat accelerated hematopoiesis recovery judging from the parameters of peripheral blood, total cellularity of the bone marrow and spleen, and animal survival. Single or double administration of Derinat prior to irradiation was ineffective. The optimal result was obtained when the following scheme was applied: MSC→HSC with an interval of 48 h starting during the first hours after irradiation and triple administration of Derinat (in 10-15 min, 3 and 7 days after irradiation) in a dose of 3 mg/mouse.

Low Mg content on Ti-Nb-Sn alloy when in contact with eBMMSCs promotes improvement of its biological functions.

Magnesium is a metal used in the composition of titanium alloys and imparts porosity. Due to its osteoconductive, biocompatible and biodegradable characteristics, its application in the development of biomedical materials has become attractive. This study aimed to evaluate the influence of magnesium present in porous Ti-Nb-Sn alloys, which have a low elastic modulus in adhesive, osteogenic properties and the amount of reactive intracellular oxygen species released in mesenchymal stem cells derived from bone marrow equine bone (eBMMSCs). Mechanical properties of the alloy, such as hardness, compressive strength and elastic modulus, were analyzed, as well as surface morphological characteristics through scanning electron microscopy. The evaluation of magnesium ion release was performed by atomic force spectroscopy. The biological characteristics of the alloy, when in contact with the alloy surface and with the culture medium conditioned with the alloy, were studied by SEM and optical microscopy. Confirmation of osteogenic differentiation by alizarin red and detection of ROS using a Muse® Oxidative Stress Kit based on dihydroetide (DHE). The alloy showed an elastic modulus close to cortical bone values. The hardness was close to commercial Ti grade 2, and the compressive strength was greater than the value of cortical bone. The eBMMSCs adhered to the surface of the alloy during the experimental time. Osteogenic differentiation was observed with the treatment of eBMMMSCs with conditioned medium. The eBMMSCs treated with conditioned medium decreased ROS production, indicating a possible antioxidant defense potential of magnesium release.

Calycosin-7-O-ß-Glucoside Isolated from Astragalus membranaceus Promotes Osteogenesis and Mineralization in Human Mesenchymal Stem Cells.

Stem cells have received attention in various diseases, such as inflammatory, cancer, and bone diseases. Mesenchymal stem cells (MSCs) are multipotent stem cells that are critical for forming and repairing bone tissues. Herein, we isolated calycosin-7-O-ß-glucoside (Caly) from the roots of Astragalus membranaceus, which is one of the most famous medicinal herbs, and investigated the osteogenic activities of Caly in MSCs. Caly did not affect cytotoxicity against MSCs, whereas Caly enhanced cell migration during the osteogenesis of MSCs. Caly increased the expression and enzymatic activities of ALP and the formation of mineralized nodules during the osteogenesis of MSCs. The osteogenesis and bone-forming activities of Caly are mediated by bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, Wnt3a, phospho-GSK3ß, and phospho-AKT, inducing the expression of runt-related transcription factor 2 (RUNX2). In addition, Caly-mediated osteogenesis and RUNX2 expression were attenuated by noggin and wortmannin. Moreover, the effects were validated in pre-osteoblasts committed to the osteoblast lineages from MSCs. Overall, our results provide novel evidence that Caly stimulates osteoblast lineage commitment of MSCs by triggering RUNX2 expression, suggesting Caly as a potential anabolic drug to prevent bone diseases.

BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.