Bone Marrow Nucleated Cells Research Articles

PS1349 UPDATED RISK STRATIFICATION MODEL FOR SMOLDERING MULTIPLE MYELOMA (SMM) INCORPORATING THE REVISED IMWG DIAGNOSTIC CRITERIA

Background: Deep understanding of the complexity and diversity of the TIME and its influence on response to therapy is needed to improve the ability to predict, monitor and guide immunotherapeutic responsiveness. Among cells composing the MM-TIME, granulocytic-MDSCs (G-MDSCs) have a prominent role in promoting tumor growth and inducing immunosuppression; however, their phenotype in MM is not well-established. Aims: To define the phenotype of G-MDSCs based on the integration of phenotypic data, immunosuppressive potential, gene regulatory networks and impact on patients’ outcome of different granulocytic subsets present in the MM-TIME. Methods: We used multidimensional flow cytometry (MFC) to evaluate the pre-established phenotype of G-MDSCs in bone marrow (BM) samples from healthy donors (HD) (n = 4) and MM patients (n = 5). We then used principal component analysis (PCA) to unbiasedly identify different granulocytic subsets in the MM-TIME, and FACSorted them for in vitro experiments to analyze their immunosuppressive potential (n = 9) and for RNAseq to analyze the molecular profile of G-MDSCs in MM (n = 5) vs HD (n = 5). The clinical significance of these subsets was determined comparing their numbers at diagnosis within the BM TIME of MM patients (n = 71) included in the GEM2012MENOS65 trial. Results: Human G-MDSCs have been defined as a cell population displaying a CD11b-CD14-CD15+CD33+HLADR- phenotype, representing 1% of total BM nucleated cells in HD and nearly 25% in MM patients. However, we found that the percentage of cells with this phenotype was similar in the BM of HD and MM patients (median of 8% in both, P>.99). Using MFC and according to PCA, 3 granulocyte subsets were unbiasedly identified in the BM of HD and MM, based on homogeneous CD14-CD15+CD33+HLADR- expression but differential reactivity against CD11b, CD13 and CD16: CD11b-CD13lo/-CD16-, CD11b+CD13lo/-CD16- and CD11b+CD13+CD16+. The percentage of all three subsets was similar (P>.5) between HD and MM. Afterwards, we used FACSorting to isolate (1) or deplete (2) individually each of the 3 subsets from MM-BM and determine its immunosuppressive potential in 2 assays. In (1), we noted a significant decrease in T cell proliferation when these were stimulated with CD3/CD28 and in presence of the CD11b+CD13+CD16+ neutrophil subset (0.5-fold, P = .01) but not of the CD11b-CD13lo/-CD16- and CD11b+CD13lo/-CD16- subsets. In (2), we noted that the cytotoxic potential of T cells significantly increased in presence of the BCMAxCD3 bispecific antibody (P = .04) reaching its maximum with the depletion of the CD11b+CD13+CD16+ subset (4-fold, P = .007). Additionally, RNAseq of the 3 subsets in HD and MM patients revealed that genes commonly associated with G-MDSCs (e.g. PTGS2, TGFβ1) were specifically upregulated in the CD11b+CD13+CD16+ granulocytic subset. Finally, we analyzed the distribution of the 3 subsets in the BM of MM patients. Interestingly, we observed that patients with a high number of CD11b+CD13+CD16+ neutrophils at baseline had lower count of T cells and that patients with low ratio between CD11b+CD13+CD16+ and T cells had inferior 3-year rates of progression-free survival as compared to the remaining patients (100% vs 75%, P = .03). Summary/Conclusion: In this comprehensive analysis that integrated molecular, phenotypic, functional and clinical data, we determined a correlation between three well-defined granulocytic subsets and their immunosuppressive potential, thereby providing for the first time, a set of optimal markers (CD11b/CD13/CD16) for monitoring G-MDSCs in MM.

Systems Biology Analysis of the Effect and Mechanism of Qi-Jing-Sheng-Bai Granule on Leucopenia in Mice.

Qi-Jing-Sheng-Bai granule (QJSB) is a newly developed traditional Chinese medicine (TCM) formula. Clinically, it has been used for the treatment of leucopenia. However, its pharmacological mechanism needs more investigation. In this study, we firstly tested the effects of QJSB on leucopenia using mice induced by cyclophosphamide. Our results suggested that QJSB significantly raised the number of peripheral white blood cells, platelets and nucleated bone marrow cells. Additionally, it markedly enhanced the cell viability and promoted the colony formation of bone marrow mononuclear cells. Furthermore, it reversed the serum cytokines IL-6 and G-CSF disorders. Then, using transcriptomics datasets and metabonomic datasets, we integrated transcriptomics-based network pharmacology and metabolomics technologies to investigate the mechanism of action of QJSB. We found that QJSB regulated a series of biological processes such as hematopoietic cell lineage, homeostasis of number of cells, lymphocyte differentiation, metabolic processes (including lipid, amino acid, and nucleotide metabolism), B cell receptor signaling pathway, T cell activation and NOD-like receptor signaling pathway. In a summary, QJSB has protective effects to leucopenia in mice probably through accelerating cell proliferation and differentiation, regulating metabolism response pathways and modulating immunologic function at a system level.

Pharmacological and transcriptome profiling analyses of Fufang E'jiao Jiang during chemotherapy-induced myelosuppression in mice

Ethnopharmacological relevanceFufang E'jiao Jiang (FEJ), a famous traditional Chinese medicine formula from “Liangyi Ointment”, consists of five crude drugs, Colla corii asini, Radix Ginseng Rubra, Radix Rehmanniae Preparata, Codonopsis pilosula, and Crataegus pinnatifida Bge. It has pronounced functions of qi-nourishing and blood-activating. Recently, it has been widely used in China as a medication against myelosuppression in cancer treatment. Aim of the studyWe aimed to investigate the complex mode of action and underlying mechanisms of Fufang E'jiao Jiang (FEJ) regarding its hematopoietic effect. Main methodsMice were divided into 5 groups of control, model, high dose FEJ (HFEJ), medium dose FEJ (MFEJ) and low dose FEJ (LFEJ). After 10 days from the administration, bone marrow cells (BMCs) were extracted for nucleated cells counts, flow cytometry analysis of hematopoietic stem cells (HSCs) population, as well as hematopoietic progenitor cells (HPCs) colony-forming unit (CFU) assay. A portion of bone marrow nucleated cells (BMNCs) of MFEJ group were prepared for RNA sequencing (RNA-Seq). The transcriptome data were analyzed based on the differentially expressed genes (DEGs). The molecular mechanisms of FEJ were deducted based on the biological processes and protein-protein interaction (PPI) network. ResultsFEJ could significantly increase the percentage of HSCs and the quantities of BFU-E and CFU-GM in BMSCs. FEJ could stimulate the proliferation of HSC and the differentiation of HPC to all lineages, which may thereby accelerate the recovery of hematopoietic function in myelosuppressive mice. By providing transcriptome profile we highlighted several genes and biological processes that might be applicable for FEJ to treat chemotherapy-induced myelosuppression. GO analysis showed that the co-expressed DEGs in FEJ vs model and model vs control group were involved in biological processes including ossification, osteoblast differentiation, bone mineralization and bone development. The KEGG pathway analysis pointed out ECM-receptor interaction and PI3K-AKT signaling pathway as the most relevant pathways to the function of FEJ on myelosuppression. PPI network showed MMP2 and COL1A1 were the relatively large nodes. ConclusionFEJ has the hematopoietic effect in chemotherapy-induced myelosuppression mice. It might be achieved by improving the proliferative capacity of HSCs and the differentiation ability of HPCs. The molecular mode of action of FEJ might be the improvement of the bone marrow microenvironment via ECM-receptor interaction, the promoted proliferation of HSC through regulation of PI3K-AKT signaling pathway, and the involvement of osteoblasts and osteoclasts. MMP2 and COL1A1 appear to be the key relevant regulatory molecules. These results provide significant insight into the hematopoietic effects of FEJ in myelosuppression and point out novel targets for future validating analyses.

Direct differentiation of bone marrow mononucleated cells into insulin producing cells using pancreatic \u03b2-cell-derived components

Transplantation of stem cell-derived insulin producing cells (IPCs) has been proposed as an alternative to islet transplantation for the treatment of diabetes mellitus. However, current IPC differentiation protocols are focused on generating functional cells from the pluripotent stem cells and tend to rely on multistep, long-term exposure to various exogenous factors. In this study, we addressed the observation that under stress, pancreatic β-cells release essential components that direct the differentiation of the bone marrow nucleated cells (BMNCs) into IPCs. Without any supplementation with known differentiation-inducing factors, IPCs can be generated from BMNCs by in vitro priming for 6 days with conditioned media (CM) from the β-cells. In vitro primed BMNCs expressed the β-cell-specific transcription factors, as well as insulin, and improved hyperglycemia and glucose intolerance after transplantation into the streptozotocin-induced diabetic mice. Furthermore, we have found that components of the CM which trigger the differentiation were enclosed by or integrated into micro particles (MPs), rather than being secreted as soluble factors. Identification of these differentiation-directing factors might enable us to develop novel technologies required for the production of clinically applicable IPCs.

Alterations of Hematologic and Hematopoietic Parameters in Mice Exposed to Pulsed Electromagnetic Field.

Effects of pulsed electromagnetic field (PEMF) on hematology and hematopoiesis might vary with different PEMF parameters. The purpose of this study was to evaluate the possible effects of PEMF exposure at different pulses on hematologic and hematopoietic parameters in mice. Groups of male BALB/c mice were whole body exposed or were sham exposed (control) to PEMF at 100, 1000, and 10000 pulses. After PEMF exposure, blood samples and bone marrow cells of mice were collected for hematologic examinations, bone marrow nucleated cell counting, colony-forming units of granulocyte-macrophage (CFU-GM) colony assay, and serum granulocyte-macrophage colony-stimulating factor (GM-CSF) assay. Compared with the control group, white blood cells (WBC) and lymphocytes (LYM) in the 100 and 1000 pulses exposed groups were significantly increased but not changed in the 10000 pulses exposed group. Red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) were not changed in all exposed groups. There was no significant difference in mouse bone marrow nucleated cell number between the control group and each exposed group 7 days after PEMF exposure. The CFU-GM clone number of bone marrow cells and serum GM-CSF level were significantly increased in the 100 and 1000 pulses exposed group but not changed in the 10000 pulses exposed group. Our results indicated that the PEMF exposure at fewer pulses may induce statistically significant alterations in some hematologic and hematopoietic parameters of mice but no changes can be found in the more pulses PEMF-exposed groups.

Low-Intensity Pulsed Ultrasound Relieves Mild and Severe Myelosuppression Induced by Cyclophosphamide in Rabbits.

This study aimed to investigate the effect of low-intensity pulsed ultrasound (LIPUS) on cyclophosphamide (CTX)-induced rabbit myelosuppression. Rabbits (n = 90) were randomly divided into a mild myelosuppression group (n = 40), a severe myelosuppression group (n = 40), and a normal control group (group Cu28 ; n = 10). The mild and severe myelosuppression models were established by daily ear vein injection of 15- and 40-mg/kg CTX for 4 continuous days, respectively. Then they were randomly divided into LIPUS groups (Au and Bu ) and control groups (Ac and Bc ). LIPUS was applied once per day for 20 minutes for 7 (Au7 and Bu7 ) and 28 (Au28 and Bu28 ) days. Physical conditions, mortality, blood cell counts, and bone marrow proliferation were calculated. Erythropoietin interleukin 3, and granulocyte-macrophage colony-stimulating factor levels were measured by an enzyme-linked immunosorbent assay. Flow cytometry was used to detect the granulocyte phagocytosis rate. Hematoxylin-eosin staining was performed to analyze changes of skin and muscle. Compared with the control group, LIPUS improved the number of peripheral blood cells (P < .05) and bone marrow nucleated cells and reduced the mortality of rabbits with myelosuppression of different degrees. Long-term treatment for 28 days had no effect on the levels of erythropoietin, interleukin 3, and granulocyte-macrophage colony-stimulating factor and granulocyte phagocytosis (P > .05). The parts of the skin where LIPUS was applied did not show any burning marks, and the muscle tissue in the path of LIPUS acoustic channels showed no obvious pathologic changes. Low-intensity pulsed ultrasound is a safe and effective method to relieve CTX-induced myelosuppression.

ACE Insertion/Deletion Polymorphism (rs4646994) Is Associated With the Increased Risk of Multiple Myeloma.

Introduction: The insertion (I allele) deletion (D allele) polymorphism of ACE gene (rs4646994) may influence the etiopathogenesis of multiple myeloma (MM). ACE gene is expressed in bone marrow cells and encodes angiotensin converting enzyme (ACE). It converts angiotensin I to active peptide angiotensin II, which stimulates proliferation of hematopoietic stem cells. This suggests possible association of ACE I/D gene polymorphism with MM. The aim of our study was to check possible impact of this polymorphism on risk of development and outcome of MM, as well as, sensitivity to bortezomib in cell cultures derived from MM patients.Objects and Methods: Genomic DNA from 98 newly diagnosed MM patients and 100 healthy blood donors were analyzed by PCR method. Chromosomal aberrations were detected by use of cIg-FISH. In a subgroup of 40 MM patients nucleated bone marrow cells were treated with bortezomib in vitro.Results: The Hardy-Weinberg equilibrium test showed that genotypic frequencies diverged significantly from the equilibrium. The differences between I and D allele frequencies in control and study population were significant (p = 0.046). We observed the association between DD genotype and more than 2-fold risk of MM - OR = 2.69; p < 0.0001. We did not detect any significant differences among studied genotypes regarding clinical and laboratory parameters. Moreover, we did not observe the association between survival of MM patients and I/D genotypes. Bortezomib increased number of apoptotic and necrotic cells, but the only statistically significant differences were observed in the number of viable cells at 1 nM between ID and DD genotypes (p = 0.026).Conclusion: Presented results confirmed the significant relationship between ACE (I/D) polymorphism and risk of MM development. We did not observe the association of ACE I/D polymorphism with disease outcome and bortezomib in vitro sensitivity.

Study of the Effects and Mechanisms of Ginsenoside Compound K on Myelosuppression.

Ginsenoside compound K (CK) is not a ginsenoside that naturally exists in Panax ginseng Meyer. However, CK is a major metabolite of ginsenoside Rb1, Rb2, or Rc in the intestine under the effects of bacteria. In this study, we first investigated the effects of CK on myelosuppression in mice induced by cyclophosphamide (CTX). The respective quantities of white blood cells, blood platelets, and bone marrow nucleated cells (BMNCs) were determined to be 8.54 ± 0.91 (109/L), 850.90 ± 44.11 (109/L), and 1.45 ± 0.22 (109/L) in the CK-H group by detecting peripheral blood cells and BMNCs. CK-H and CK-L both increased the thymus index by up to 0.62 ± 0.06 (mg/g) and 0.52 ± 0.09 (mg/g), respectively, and significantly increased the yields of colony formation units-granulocyte monocyte and colony formation units-megakaryocytic. According to our study, CK could control apoptosis and promote cells to enter the normal cell cycle by the bcl-2/bax signaling pathway and MEK/ERK signaling pathway. Therefore, the BMNCs could proliferate and differentiate normally after entering the normal cell cycle. So the peripheral blood cells could show a trend of returning to normal. The recovery of peripheral blood cells resulting in the level of cytokines tended to normal. This process may be the mechanisms of CK on myelosuppression. This study provides a reference for ginseng in the treatment of myelosuppression.

Bone marrow nucleated cell concentrate autograft in temporomandibular joint degenerative disorders: 1-year results of a randomized clinical trial

Objectiveto assess the reliability of bone marrow nucleated cell (BMNc) intra-articular injection in patients with degenerative temporomandibular joint disorders (TMDs), and to compare its efficacy with that of hyaluronic acid (HA). Materials and methodsthis study was designed as a randomized, controlled trial of parallel groups. Patients affected by degenerative joint mandibular disorders were enrolled in this prospective clinical trial and randomly divided into two groups. The HA group underwent temporomandibular joint (TMJ) arthrocentesis and HA injection, whilst patients in the BMNc group were inoculated with BMNc inside the joint after lavage. Outcome measures were: assessing pain at rest and during motion, joint noises, chewing efficiency, and maximum interincisal opening. A postoperative MRI scan was performed and compared with the preoperative one, while examining for cartilage regeneration. Clinical and radiological data were collected from baseline to 12 months follow-up. ResultsThirty patients, 15 for each group, complaining of different degrees of unilateral TMD with internal derangement, were enrolled and treated. In both groups, significant clinical improvements were detected after the procedure up to 1 year postoperatively. The BMNc group presented significantly better pain relief than the HA group after 6 months (p = 0.028) and 12 months (p = 0.000). No significant differences were observed in terms of joint noises. In terms of chewing efficiency, the BMNc group showed positive significant differences after 12 months (p = 0.000). Maximum interincisal opening presented significantly better values in the BMNc group after 6 months (p = 0.001) and 12 months (p = 0.000). No MRI evidence of cartilage regeneration was reported. Conclusionintra-articular TMJ BMNc injection improved clinical outcomes in TMD treatment. The Results of this first human-model study are promising but further studies are needed to determine whether BMNc can represent the best treatment for TMDs.

Salvianolic acid B, an antioxidant derived from Salviamilitarize, protects mice against γ‑radiation‑induced damage through Nrf2/Bach1.

Salvianolic acid B (SB) is an antioxidant derived from Salvia militarize, and is one of the most widely used herbs in traditional Chinese medicine. SB is a potent antioxidant that has been well documented as a scavenger of oxygen free radicals, and has been used for the prevention and treatment of atherosclerosis-associated disorders. To explore its potential therapeutic effects in treating radiation damage, in this study, mice were treated with SB at different doses of 5, 12.5 and 20 mg/kg, subsequent to receiving γ-irradiation. The effects of SB on peripheral blood, bone marrow nucleated cells, spleen and thymus indices, and oxidation resistance were evaluated in both radiated mice and control groups. The results indicated that SB significantly increased the counts of peripheral white blood cells, red blood cells and platelets. The number of nucleated cells in the bone marrow and the level of protein increased as well. In addition, improved spleen and thymus indices in the bone marrow were observed. SB treatment additionally reversed the deterioration of both the thymus and spleen indices, which is associated with increased serum superoxide dismutase activity and decreasing malondialdehyde levels via nuclear factor (erythroid-derived 2)-like 2 protein/BTB and CNC homology 1 mediated antioxidant effect. Furthermore, ROS levels and Bax protein expression were also suppressed by SB. The data suggested that SB is effective in protecting mice from γ-radiation injury, and could potentially be applicable for clinical use. Notably, the present study identified a promising candidate drug for enhancing the hematopoietic and immune systems.

Detailed Phenotypic, Molecular and Functional Profiling of Myeloid Derived Suppressor Cells (MDSCs) in the Tumor Immune Microenvironment (TIME) of Multiple Myeloma (MM)

Detailed Phenotypic, Molecular and Functional Profiling of Myeloid Derived Suppressor Cells (MDSCs) in the Tumor Immune Microenvironment (TIME) of Multiple Myeloma (MM)

Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of Mitochondrial Vulnerability in Acute Myeloid Leukemia

BACKGROUND60% to 70% of Acute Myeloid Leukemia (AML) patients enter complete remission after induction regimen, but the majority relapse within 3 years due to the outgrowth of therapy resistant Leukemia Stem Cells (LSCs). Identification of novel treatment strategies effective against these cells thus represents an outstanding medical need. We developed a cell culture method, which transiently maintains LSC activity ex vivo (Pabst et al., Nature Methods, 2014) and enables chemical interrogation of cell types relevant for the progression of the disease. Overall, HSCs and LSCs share numerous biological traits, making specific LSC eradication challenging. However, striking differences in energy metabolism between normal and leukemic stem cells have recently been suggested. While HSCs appear to rely primarily on anaerobic glycolysis for energy production, LSCs seem to depend on mitochondrial oxidative phosphorylation for their survival. Targeting mitochondrial respiration could therefore represent an effective approach for the specific eradication of LSCs. AIMWe aimed to identify novel therapeutic targets for AMLs with poor treatment outcome. The study relied on the Leucegene approach that integrates results generated by RNA sequencing analysis of primary human AML specimens, detailed clinical and cytogenetic annotations provided by the Quebec leukemia cell bank and ex vivo responses of primary AML samples to various chemical compounds. Our study specifically focused on specimens originating from patients with poor (overall survival < 3 years) and good (overall survival ≥ 3 years) response to standard chemotherapy, and did not include cases of Acute Promyelocytic Leukemia (APL). RESULTSWe identified Mubritinib, previously described as an ERBB2 inhibitor, as a novel anti-leukemic agent, which selectively inhibits the viability of leukemic cells from therapy-resistant AML patients, but does not affect normal CD34+ cord blood cells. Exposure to Mubritinib triggered apoptotic cell death in a subset of AML samples with high mitochondrial function-related gene expression, high relapse rates, and short overall survival. Sensitivity to Mubritinib also strongly associated with the intermediate cytogenetic risk category, normal karyotype (NK), and NPM1, FLT3 (ITD) and DNMT3A mutations. Conversely, resistance to Mubritinib associated with favorable cytogenetic risk AMLs, Core Binging Factor (CBF) leukemias and KIT mutations. Mubritinib has been developed as an ERBB2 kinase inhibitor. Intriguingly, we found that ERBB2 is not expressed in Mubritinib-sensitive AML specimens, suggesting that the anti-leukemic activity of this compound is likely not mediated by ERBB2 inhibition. Using a combination of functional genomics and biochemical analyses, we demonstrated that Mubritinib directly inhibits the mitochondrial Electron Transport Chain (ETC) complex I, which leads to a decrease in oxidative phosphorylation activity and to induction of oxidative stress. The impact of Mubritinib on AML progression was explored using a syngeneic mouse model (MLL-AF9 tdTomato-positive leukemia). Recipients of MLL-AF9 cells treated with Mubritinib exhibited a 19-fold decrease in the number of tdTomato-positive cells in the bone marrow and a 42-fold decrease in the spleens compared to control mice. Short-term treatment also led to a 37% increase in the median overall survival of Mubritinib exposed recipients compared to vehicle treated mice. Importantly, and in agreement with our observation that Mubritinib treatment does not impede proliferation of normal hematopoietic CD34+ cells in vitro, Mubritinib treatment had no impact on the number of non-transduced (tdTomato negative) nucleated bone marrow cells of recipients. CONCLUSIONSWe uncovered the clinical, mutational, and transcriptional landscape of mitochondrial vulnerability in AML and identified Mubritinib as a novel ETC complex I inhibitor with therapeutic potential for approximately 30% of AML cases currently lacking effective treatment options. As Mubritinib completed a phase I clinical trial in the context of ERBB2-positive solid tumors, our work suggests an opportunity to re-purpose Mubritinib's usage for this genetically distinct subgroup of poor outcome AML patients. DisclosuresNo relevant conflicts of interest to declare.

Protective effects of Acanthopanax senticosus - Ligustrum lucidum combination on bone marrow suppression induced by chemotherapy in mice

This study investigated the combination effects of the Acanthopanax senticosus - Ligustrum lucidum (AS-LL) herb pair on bone marrow suppression caused by chemotherapy. A bone marrow suppression model was established by intraperitoneal injection (i.p.) of cyclophosphamide (CTX, 100 mg/kg). The changes in chemical composition between the AS-LL decocted together and single were analyzed, and their effects on the bone marrow nucleated cells, peripheral blood, thymus and spleen indices, in vitro hematopoietic cell culture, ELISA and cell cycle were detected. The results showed that the contents of the main active components, such as salidroside, isofraxidin and specnuezhenide in the sample of AS-LL decocted together, increased significantly compared to singles. Moreover, AS-LL decocted together exhibited a significantly better therapeutic effect on myelosuppression induced by CTX than AS and LL alone. AS-LL decocted together significantly increased the number of bone marrow nucleated cells and displayed a good regulatory effect on peripheral blood (p < 0.01), while significantly increased the thymus index (p < 0.01) and decreased the spleen index (p < 0.01). AS-LL significantly promoted the formation of cell colonies (p < 0.05), the proliferation and differentiation of hematopoietic progenitor cells, and played a positive regulatory role in hematopoietic factors. AS-LL also reduced the proportion of G0/G1 cells, increased the ratio of S and G2/M cells, and increased the cell proliferation index (PI). All these results implied that AS-LL decocted together might be a promising food additives and therapeutic agent for myelosuppression induced by chemotherapy.

Comparisons of antithrombosis, hematopoietic effects and chemical profiles of dried and rice wine-processed Rehmanniae Radix extracts

Ethnopharmacological relevanceIn traditional Chinese medicine (TCM), Rehmanniae Radix (RR, derived from the root of Rehmannia glutinosa (Gaertn.) DC.) is commonly used as natural medicine for thousands of years, two types including the dried and rice-wine processed RR were used for different clinical purposes respectively, which were the typical case that pharmaceutical effect changed by processing in TCM. Aim of studyThe goal of this study was to investigate the differences in the antithrombosis and hematopoietic effects of extracts of dried and processed RR (DRR and PRR) in vivo, and to explore the chemical basis underlying changes of medicinal properties caused by processing. Materials and methodsThe aqueous extracts of DRR and PRR were prepared. Protective effect of varying doses of different extracts were investigated in type-I carrageenan induced mice tail thrombosis and cyclophosphamide induced myelosuppression model. The chemical composition of DRR and PRR extracts were determined by High Performance Liquid Chromatography coupled with tandem quadrupole time-of-flight Mass Spectrometry (HPLC/Q-TOF-MS). ResultsIn antithrombosis activity tests, PRR possessed less ameliorated effects than DRR in the model mouse on body temperature, tail thrombus length and blood flow. Both DRR and PRR had no significant influence on prothrombin time (PT) and activated partial thromboplastin time (APTT), only high dose DRR could decrease the content of fibrinogen (FIB) in plasma. Histological examination of lung tissue suggested that thrombosis was significantly improved in DRR-H group. For myelosuppression model, only PRR could improve peripheral hemogram, both DRR and PRR had hematopoietic effects as demonstrated by their abilities to ameliorate the bone marrow nucleated cells (BMNC) and pathology of bone marrow tissue. The hematopoietic effects of PRR were significantly more potent than that of DRR at the concentration of 9 g/kg. By comparing the chemical composition, we found that iridoid glycosides were decreased and furfural derivatives increased in DRR after processing which may be the chemical mechanism contribute to the differences in efficacy. ConclusionsAccording to the results of this research, processing with rice wine for nine cycles significantly reduced antithrombotic effects and enhanced the hematopoietic effects of DRR as demonstrated in model mice. It can scientifically explain the different effect among two types of RR in clinical through the diverse method of processing and usage. Meanwhile, the predicted activity compounds from two types of RR can be potential candidates for the treatment of thrombosis and anemia.

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

Bartonella henselae Infection in Sickle Cell Disease Mice Is Associated with Hyperalgesia.

Sickle cell disease (SCD) is the most prevalent hematologic genetic disorder. Acute vaso-occlusive painful crisis is the hallmark of the disease and may be related to subclinical infections. Bartonellosis, a rare and neglected infection, is caused by Bartonella spp., which can be found in donated blood. These bacteria cause intraerythrocytic and endothelial infection and pain, all of which occur in SCD. It is likely that this infection is transmitted to SCD patients during transfusion from donated blood, leading to pain. We, therefore, evaluated whether Bartonella henselae infection would cause hyperalgesia in mice with SCD. SCD mice were generated by transplantation of nucleated bone marrow cells harvested from transgenic Berkeley sickle mice into 2-month-old irradiated C57BL/6 mice. We infected four SCD mice by intraperitoneal inoculation with B. henselae, and inoculated four other mice with the same volume of saline. Mechanical hyperalgesia was determined using von Frey monofilaments by two blinded observers. Thereafter, the animals were anesthetized and euthanized to collect blood, liver, and spleen samples to seek B. henselae infection by PCR. We confirmed the experimental infection in all animals by PCR. Tremors and mechanical hypersensitivity were demonstrated by SCD mice infected with B. henselae infection but not in those receiving saline. B. henselae infection may be related to pain and other symptoms in SCD.

Studies of the effects and mechanisms of ginsenoside Re and Rk3 on myelosuppression induced by cyclophosphamide

BackgroundGinsenoside Re (Re) is one of the major components of Panax ginseng Meyer. Ginsenoside Rk3 (Rk3) is a secondary metabolite of Re. The aim of this study was to investigate and compare the effects and underlying mechanisms of Re and Rk3 on cyclophosphamide-induced myelosuppression. MethodsThe mice myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide. Peripheral blood cells, bone marrow nucleated cells, and colony yield of hematopoietic progenitor cells in vitro were counted. The levels of erythropoietin, thrombopoietin, and granulocyte macrophage colony-stimulating factor in plasma were measured by enzyme-linked immunosorbent assay. Bone marrow cell cycle was performed by flow cytometry. The expression of apoptotic protein bcl-2, bax, and caspase-3 was detected by Western blotting. ResultsBoth Re and Rk3 could improve peripheral blood cells, bone marrow nucleated cell counts, thymus index, and spleen index. Furthermore, they could enhance the yield of colonies cultured in vitro and make the levels of granulocyte macrophage colony-stimulating factor, erythropoietin, and thrombopoietin normal, reduce the ratio of G0/G1 phase cells, and increase the proliferation index. Finally, Re and Rk3 could upregulate the expression of bcl-2, whereas they could downregulate the expression of bax and caspase-3. ConclusionRe and Rk3 could improve the hematopoietic function of myelosuppressed mice. The effect of Rk3 was superior to that of Re at any dose. Regulating the levels of cytokines, promoting cells enter the normal cell cycle, regulating the balance of bcl-2/bax, and inhibiting the expression of caspase-3 may be the effects of Re and Rk3 on myelosuppression.

Treatment of β654 -thalassaemia by TALENs in a mouse model.

This study explored whether TALENs-mediated non-homologous end joining (NHEJ) targeting the mutation site can correct the aberrant β-globin RNA splicing, and ameliorate the β-thalassaemia phenotype in β654 mice. TALENs vectors targeted to the human β-globin gene (HBB) IVS2-654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALENs+ /β654 mice. The gene editing and off-target effects were analysed by sequencing analysis. β-globin expression was identified by RT-PCR and Western blot analysis. Various clinical indices including haematologic parameters and tissue pathology were examined to determine the therapeutic effect in these TALENs+ /β654 mice. Sequencing analysis revealed that the HBB IVS2-654C >T point mutation was deleted in over 50% of the TALENs+ /β654 mice tested, and off-target effects were not detected. RT-PCR and Western blot analysis confirmed the expression of normal β-globin in TALENs+ /β654 mice. The haematologic parameters were significantly improved as compared with their affected littermates. The proportion of nucleated cells in bone marrow was considerably decreased, splenomegaly with extramedullary haematopoiesis was reduced, and significant decreases in iron deposition were seen in spleen and liver of the TALENs+ /β654 mice. These results suggest effective treatment of the anaemia phenotype in TALENs+ /β654 mice following deletion of the mutation site by TALENs, demonstrating a simple and straightforward strategy for gene therapy of β654 -thalassaemia in the future.

Deficiency in Fpr2 results in reduced numbers of Lin−cKit+Sca1+ myeloid progenitor cells

The Lin-c-Kit+ Sca-1+ cell population in the bone marrow (BM) serves as the direct precursor for differentiation of myeloid cells. In this study, we report that deficiency in Fpr2, a G protein-coupled chemoattractant receptor in mice, is associated with reduced BM nucleated cells, including CD31+Ly6C+ (granulocytes and monocytes), CD31-/Ly6Cint (granuloid cells), and CD31-/Ly6Chigh (predominantly monocytes) cells. In particular, the number of Lin-c-Kit+Sca-1+ (LKS) cells was reduced in Fpr2-/- mouse BM. This was supported by observations of the reduced incorporation of intraperitoneally injected bromodeoxyuridine by cells in the c-Kit+ population from Fpr2-/- mouse BM. Purified c-Kit+ cells from Fpr2-/- mice showed reduced expansion when cultured in vitro with stem cell factor (SCF). SCF/c-Kit-mediated phosphorylation of P38, STAT1, Akt (Thr-308), and Akt (Ser-473) was also significantly reduced in c-Kit+ cells from Fpr2-/- mice. Furthermore, Fpr2 agonists enhanced SCF-induced proliferation of c-Kit+ cells. Colony-forming unit assays revealed that CFU-granulocyte-macrophage formation of BM cells from Fpr2-/- mice was significantly reduced. After heat-inactivated bacterial stimulation in the airway, the expansion of c-kit+ Sca-1+ cells in BM and recruitment of Ly6G+ cells to the lungs and CD11b+Ly6C+TNFα+ cells to the spleen of Fpr2-/- mice was significantly reduced. These results demonstrate an important role for Fpr2 in the development of myeloid lineage precursors in mouse BM.

Examination of the cellular mechanisms of leukocyte elevation by 10.6\xa0\u03bcm and 650\xa0nm laser acupuncture-moxibustion

To investigate the cell cycle and cellular mechanisms of leukocyte elevation by laser acupuncture in rats with cyclophosphamide (CTX)-induced leukopenia. Sixty-six rats were randomized into six groups: normal, model control group, sham treatment group, 10.6 μm laser treatment group, 650 nm laser treatment group, and 10.6 μm–650 nm compound laser treatment group. Eleven rats were used in the normal group and 55 were models that were injected with cyclophosphamide to induce leukopenia. For the three laser treatment groups, 10.6-μm and 650-nm lasers, and 10.6-μm–650-nm compound lasers were used to irradiate the DU14 (Dazhui) and bilateral ST36 (Zusanli) for 5 min each. The sham laser group received the same operation as the laser group but without irradiation. The normal group and model group were not treated. Differences in the number of nucleated cells in the femoral bone marrow, and cell cycle and cellular apoptosis of peripheral leukocytes in rats in various groups were compared. Compared with the model group and the sham laser group, the number of nucleated cells in the femoral bone marrow in the 10.6-μm laser, 650-nm laser, and 10.6-μm–650-nm compound laser group was significantly increased after treatment (P = 0.001, 0.002, 0.034, respectively) and did not show any significant difference with the normal group (P = 0.964, P = 0.838, P = 0.287, respectively). The number of cells in G2 phase in the 10.6 μm laser group was similar to that of the normal group (P = 0.973). The number of cells in G2 phase in the model, sham, 650-nm laser group, and 10.6-μm–650-nm compound laser group were significantly lower than in the normal group and 10.6-μm laser group (P = 0.016, P = 0.023, P = 0.044, P = 0.039, respectively). In the model group and the sham treatment group, the apoptosis rates of peripheral leukocytes were increased compared with the normal group (P = 0.001), while the proportion of cells in the G2 phase was significantly lower than in the normal group (P = 0.016), and the proportion of cells in S phase was higher than in the normal group (P = 0.014). The incidence of apoptosis in peripheral blood cells in the three laser treatment groups did not show any statistically significant difference when compared with the normal group (P > 0.05). Treatment with the 10.6-μm, 650-nm, and 10.6-μm–650-nm compound lasers increased the number of nucleated cells in the bone marrow, decreased the unfavorable effects of cyclophosphamide on the cell cycle, induced the cell cycle towards proliferation, decreased apoptosis, improved the intramedullary hematopoietic system, and increased peripheral leukocyte count.