Marrow Nucleated Cells Research Articles

Endogenous production of ω-3 polyunsaturated fatty acids mitigates cisplatin-induced myelosuppression by regulating NRF2-MDM2-p53 signaling pathway.

Cisplatin is a chemotherapy medication used to treat a wide range of cancers. A common side effect of cisplatin is myelosuppression. Research suggests that oxidative damages are strongly and consistently related to myelosuppression during cisplatin treatment. ω-3 polyunsaturated fatty acids (PUFAs) can enhance the antioxidant capacity of cells. Herein, we investigated the protective benefit of endogenous ω-3 PUFAs on cisplatin-induced myelosuppression and the underlying signaling pathways using a transgenic mfat-1 mouse model. The expression of mfat-1 gene can increase endogenous levels of ω-3 PUFAs by enzymatically converting ω-6 PUFAs. Cisplatin treatment reduced peripheral blood cells and bone marrow nucleated cells, induced DNA damage, increased the production of reactive oxygen species, and activated p53-mediated apoptosis in bone marrow (BM) cells of wild-type mice. In the transgenics, the elevated tissue ω-3 PUFAs rendered a robust preventative effect on these cisplatin-induced damages. Importantly, we identified that the activation of NRF2 by ω-3 PUFAs could trigger an antioxidant response and inhibit p53-mediated apoptosis by increasing the expression of MDM2 in BM cells. Thus, endogenous ω-3 PUFAs enrichment can strongly prevent cisplatin-induced myelosuppression by inhibiting oxidative damage and regulating the NRF2-MDM2-p53 signaling pathway. Elevation of tissue ω-3 PUFAs may represent a promising treatment strategy to prevent the side effects of cisplatin.

Interferon gamma (IFN-g) continuous infusion affects engraftment in a murine transplant model

IntroductionEffects of Interferon gamma (IFN-g) on hematopoiesis have been reported as both stimulatory and suppressive. In humans, IFN-g appears to be deleterious in conditions such as chronic neutropenia, anemia of chronic disease, and aplastic anemia, suggesting some level of bone marrow suppression. Inborn errors of immunity often have an inflammatory component as part of their phenotype, and this inflammation may impact on their transplant outcomes leading to increased graft failure. We thus developed a murine model using implanted pumps to assess the impact of exogenous IFN-g administered by continuous infusion during transplantation. MethodsMurine model and IFN-g administration: Bone marrow nucleated cells (BMNCs) were collected from femurs and tibias of 8- to 10-week-old B6. SJL-Ptprca-Pep3b-/BoyJ donors (CD45.1+) and 5 million cells were injected via tail vein into 10- to 12-week-old recipient C57BL6 mice (CD45.2+) after 400cGy irradiation. Micro-osmotic pumps (Alzet®1007D/2001) were surgically implanted 2 days before transplant to provide continuous infusion of recombinant mouse IFN-g at 5ug/day for 7 days. Donor chimerism%CD45.1 + engraftment inT, B, NK, and myeloid cells was determined in peripheral blood every 2 weeks (from 4 to 16 weeks) post-transplant using cell specific monoclonal antibodies. 16 weeks after transplant, mice were euthanized, and %CD45.1+ engraftment was measured in spleen and bone marrow. ResultsAlthough the absolute peripheral blood numbers were similar (Fig.1A), the infusion of IFN-g in this non myeloablative transplant resulted in lower donor engraftment as compared to control transplants (Fig.1B). Myeloid engraftment appeared to be most affected, with a statistically significant reduction of engraftment (p < 0.005) in monocytes and granulocytes. The quantitative reduction was higher than 70% as compared to control transplants worsening over time, suggesting that the long-term repopulating cells were most affected (Fig.1C). Bone marrow was affected in a similar pattern (Fig.1D). [Display omitted] ConclusionsWe established a mixed-chimera model to assess the impact of exogenous IFN-g on engraftment. Our results provide evidence that continuously elevated IFN-g levels are deleterious for engraftment and may help explain the clinical phenomena of graft loss in this patients. Further analysis of other inflammatory pathways and IFN-g blockers are ongoing.

Effect of Recombinant Human Thrombopoietin (rhTPO) on Long-term Hematopoietic Recovery in Mice with Acute Radiation Sickness and Relative Mechanism

To investigate the effect and relative mechanism of Recombinant Human Thrombopoietin (rhTPO) on long-term hematopoietic recovery in mice with acute radiation sickness. Mice were intramuscularly injected with rhTPO (100 μg/kg) 2 hours after total body irradiation with 60Co γ-rays (6.5 Gy). Moreover, six months after irradiation, peripheral blood, hematopoietic stem cells (HSC) ratio, competitive transplantation survival rate and chimerization rate, senescence rate of c-kit+ HSC, and p16 and p38 mRNA expression of c-kit+ HSC were detected. Six months after 6.5 Gy γ-ray irradiation, there were no differences in peripheral blood white blood cells, red blood cells, platelets, neutrophils and bone marrow nucleated cells in normal group, irradiated group and rhTPO group (P>0.05). The proportion of hematopoietic stem cells and multipotent progenitor cells in mice of irradiated group was significantly decreased after irradiation (P<0.05), but there was no significant changes in rhTPO group (P>0.05). The counts of CFU-MK and BFU-E in irradiated group were significantly lower than that in normal group, and rhTPO group was higher than that of the irradiated group(P<0.05). The 70 day survival rate of recipient mice in normal group and rhTPO group was 100%, and all mice died in irradiation group. The senescence positive rates of c-kit+ HSC in normal group, irradiation group and rhTPO group were 6.11%, 9.54% and 6.01%, respectively (P<0.01). Compared with the normal group, the p16 and p38 mRNA expression of c-kit+ HSC in the irradiated mice were significantly increased (P<0.01), and it was markedly decreased after rhTPO administration (P<0.01). The hematopoietic function of mice is still decreased 6 months after 6.5 Gy γ-ray irradiation, suggesting that there may be long-term damage. High-dose administration of rhTPO in the treatment of acute radiation sickness can reduce the senescence of HSC through p38-p16 pathway and improve the long-term damage of hematopoietic function in mice with acute radiation sickness.

Imaging flow cytometry-based multiplex FISH for three IGH translocations in multiple myeloma

Three types of chromosomal translocations, t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32), are associated with prognosis and the decision making of therapeutic strategy for multiple myeloma (MM). In this study, we developed a new diagnostic modality of the multiplex FISH in immunophenotyped cells in suspension (Immunophenotyped-Suspension-Multiplex (ISM)-FISH). For the ISM-FISH, we first subject cells in suspension to the immunostaining by anti-CD138 antibody and, then, to the hybridization with four different FISH probes for genes of IGH, FGFR3, MAF, and CCND1 tagged by different fluorescence in suspension. Then, cells are analyzed by the imaging flow cytometry MI-1000 combined with the FISH spot counting tool. By this system of the ISM-FISH, we can simultaneously examine the three chromosomal translocations, i.e, t(4;14), t(14;16), and t(11;14), in CD138-positive tumor cells in more than 2.5 × 104 nucleated cells with the sensitivity at least up to 1%, possibly up to 0.1%. The experiments on bone marrow nucleated cells (BMNCs) from 70 patients with MM or monoclonal gammopathy of undetermined significance demonstrated the promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive compared with standard double-color (DC) FISH examining 200 interphase cells with its best sensitivity up to 1.0%. Moreover, the ISM-FISH showed a positive concordance of 96.6% and negative concordance of 98.8% with standard DC-FISH examining 1000 interphase cells. In conclusion, the ISM-FISH is a rapid and reliable diagnostic tool for the simultaneous examination of three critically important IGH translocations, which may promote risk-adapted individualized therapy in MM.

Expression Changes of Hypoxia-Inducible Factor-1α in G-CSF Induced Hematopoietic Stem Cell Mobilization

To investigate the expression and its relative mechanism of hypoxia-inducible factor-1α(HIF-1α) in bone marrow(BM) of mice during G-CSF mobilization of hematopoietic stem cells (HSC) . Flow cytometry was used to detect the proportion of Lin-Sca-1+ c-kit+ (LSK) cells in peripheral blood of C57BL/6J mice before and after G-CSF mobilization. And the expression of HIF-1α and osteocalcin (OCN) mRNA and protein were detected by RQ-PCR and immunohistochemistry. The number of osteoblasts in bone marrow specimens of mice was counted under the microscope. The proportion of LSK cells in peripheral blood began to increase at day 4 of G-CSF mobilization, and reached the peak at day 5, which was significantly higher than that of control group (P<0.05). There was no distinct difference in the expression of HIF-1α mRNA between bone marrow nucleated cells and osteoblasts of steady-state mice (P=0.073), while OCN mRNA was mainly expressed in osteoblasts, which was higher than that in bone marrow nucleated cells (P=0.034). After mobilization, the expression level of HIF-1α increased, but OCN decreased, and the number of endosteum osteoblasts decreased. The change of HIF-1α expression was later than that of OCN and was consistent with the proportion of LSK cells in peripheral blood. The expression of HIF-1α in bone marrow was increased during the mobilization of HSC mediated by G-CSF, and one of the mechanisms may be related to the peripheral migration of HSC induced by osteoblasts inhibition.

An Automated Pipeline for Differential Cell Counts on Whole-Slide Bone Marrow Aspirate Smears

The pathologic diagnosis of bone marrow disorders relies in part on the microscopic analysis of bone marrow aspirate (BMA) smears and the manual counting of marrow nucleated cells to obtain a differential cell count (DCC). This manual process has significant limitations, including the analysis of only a small subset of optimal slide areas and nucleated cells, as well as interobserver variability due to differences in cell selection and classification. To address these shortcomings, we developed an automated machine learning-based pipeline for obtaining 11-component DCCs on whole-slide BMAs. This pipeline uses a sequential process of identifying optimal BMA regions with high proportions of marrow nucleated cells, detecting individual cells within these optimal areas, and classifying these cells into 1 of 11 DCC components. Convolutional neural network models were trained on 396,048 BMA region, 28,914 cell boundary, and 1,510,976 cell class images from manual annotations. The resulting automated pipeline produced 11-component DCCs that demonstrated a high statistical and diagnostic concordance with manual DCCs among a heterogeneous group of testing BMA slides with varying pathologies and cellularities. Additionally, we demonstrated that an automated analysis can reduce the intraslide variance in DCCs by analyzing the whole slide and marrow nucleated cells within all optimal regions. Finally, the pipeline outputs of region classification, cell detection, and cell classification can be visualized using whole-slide image analysis software. This study demonstrates the feasibility of a fully automated pipeline for generating DCCs on scanned whole-slide BMA images, with the potential for improving the current standard of practice for utilizing BMA smears in the laboratory analysis of hematologic disorders.

Single-Cell Multiomics Reveal Hypomethylating Agent Partially Restores Hematopoiesis in a LR-MDS Patient at the Cost of Leukemic Transformation

Background Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by ineffective hematopoiesis. Hypomethylating agents (HMA) such as decitabine and azacytidine (AZA) induce clinical responses in both lower-risk (LR) and higher-risk groups; however, the treatment eventually fails and can be followed by leukemic transformation. The mechanism underlying transient clinical benefit from low-dose HMA (restoration of hematopoiesis) is not fully understood. Here we used cutting-edge single-cell multi-omic approaches to describe the abnormal clonal architecture and transcriptome in LR-MDS and how they are altered following HMA therapy. Results First, we defined genomic differences between aged and young healthy hematopoietic stem and progenitor cells (HSCPs) by building a single-cell atlas of human HSCP with molecularly-defined concentrations of relevant CITE seq antibodies. 350k high-quality bone marrow nucleated cells were obtained from four young donors; varying race and gender. A 6-fold molecular titration of 278 CITE-seq antibodies determined those which are functional and which contribute to clustering. Next, we repeated the captures on samples from aged healthy people (n=3), MDS patients (n=4) and conducted differential analysis on cell states and transcriptomes. Compared to young marrow, healthy marrow from elderly donors revealed global upregulation of inflammatory signals and innate immunity. In erythroid progenitors (ERP) we identified abnormally high expression of the transcription factors KLF1 and GATA1, but lower than normal expression of their hemoglobin target genes. At the same time altered erythropoiesis in the elderly appeared to be compensated by increased frequency of multilineage progenitors and more mature erythroblasts mitigating clinical anemia. Comparing MDS to aged marrow, while most cell populations from MDS showed significant gene expression changes, the amplitude was dwarfed by abnormal mRNA processing gene expression in ERP and erythroblasts. Moreover, in de novo LR-MDS samples and an anemia sample (without known mutations) further downregulation of hemoglobin genes in the erythroid lineage was accompanied by decreased erythroblast numbers leading to anemia. To study the impact of AZA on LR-MDS, we longitudinally profiled a single MDS patient (pre-HMA, post-HMA, sAML transformation, cycle 2 VEN+AZA) (Panel 1). We found that 6 cycles of AZA treatment depleted HSC; whereas lymphoid-primed multipotential progenitors (LMPP), megakaryocyte-erythroid progenitors (MEP), and less mature erythroblasts were enriched after AZA therapy (Panel 2 left). Using genotyping of the transcriptome (GoT) (Panel 2 right), we defined cells with mutations to find that not only SRSF2-relevant pathways (eg. mRNA processing, and mRNA splicing) are molecularly corrected (downregulated) but also there are fewer mutant cells among primitive stem cells and lineage-committed progenitors. However, mutant ERP and erythroblasts accumulated and pathways were more active suggesting the blockade of differentiation at terminal erythropoiesis is not rescued, consistent with the clinical observation that the patient was still RBC transfusion-dependent despite platelet recovery. Notably, these mutant erythroid cells, while defective, never transformed to leukemia. The expansion of mutant multilineage progenitors and LMPP preceded AML transformation and were eliminated (as evidenced by flow and CITE-seq) following two cycles of VEN+AZA. Conclusions: Age-matched references are critical in differential analysis of hematological diseases like AML or MDS. With matched elderly donor data sets, we were able to molecularly isolate functional disruption of erythropoiesis and megakaryopoiesis underlying anemia and thrombocytopenia, respectively, observed in an LR-MDS patient. AZA rescued the dysregulated signals in SRSF2-mutated LR-MDS patient HSPCs, but not in ERP and erythroblasts, and therefore only partially restored hematopoiesis. However, as AZA drove differentiation of both wildtype and mutant HSCP, accumulation of mutant multilineage progenitors and LMPPs contributed to progression to AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Heme Oxygenase-1 Inhibition the Cytotoxicity of NK Cells to AML By Downregulating HLA-C

Backgroud and objective: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells with abnormal differentiation of myeloid primordial naive cells, which has the highest incidence in adult Acute leukemia with a low cure rate . Clinically, less than one-third of adult AML patients can achieve sustained remission after cytarabine combined with anthracycline chemotherapy, and over 50% relapse after remission. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is still the only way to cure leukemia clinically. However, due to age limitation, infection, graft-versus-host disease, and other complications, it is not suitable for all AML patients In recent years, numerous novel drugs have provided more options for the treatment of relapsed and refractory AML while the overall response rate is unsatisfactory and there is no significant advantage in improving overall survival Exploring the mechanism affecting AML relapse and refractory treatment, formulating effective coping strategies are the key to overcome the treatment bottleneck of AML Methods: Bone marrow nucleated cells were obtained from 15 newly diagnosed AML patients, 15 recurrent AML patients and 15 normal controls. The expressions of HO-1 and HLA-C were detected by quantitative reverse transcription PCR (RT-PCR) and Western blot. In vitro cell experiments, we constructed SiHO-1 lentiviral vector to transfect AML cell lines, cocultured NK cells with it, measured the change of CD107a expression on NK cell surface, and silenced HLA-C expression with small interfering RNA. The silenced AML cell lines were co-cultured with NK cells, and CD107a secretion on NK cells was measured by flow cytometry. Results: HO-1 expression in AML patients was higher than that in healthy donors (P<0.05), while HLA-C expression was lower than that in healthy donors (P<0.01). Additionally, the plasma HO-1 levels gradually increased, while the HLA-C levels gradually decreased from healthy donors to patients with relapsed AML. In addition, Pearman correlation analysis indicated that HLA-C expression was significantly correlated with HO-1 expression (r=0.635, P<0.001). In AML cell lines, HO-1 silencing enhanced the cytotoxicity of NK cells to AML cells (P＜0.01), while HO-1 overexpression weakened the cytotoxicity of NK cells to AML cells (P＜0.05). The expression of CD107a in NK cells after HLA-C silencing was lower than the control group in U937 and THP-1 cell line. (P＜0.05). Conclusion: decreased HLA-C expression is associated with increased HO-1 expression during the progression of AML. Besides, the inhibition of HO-1 promotes HLA-C expression, which can therefore promote cytotoxicity of NK cells to AML cells.

An Automated Pipeline for Cell Differentials on Whole-Slide Bone Marrow Aspirate Smears

Abstract Current pathologic diagnosis of benign and neoplastic bone marrow disorders relies in part on the microscopic analysis of bone marrow aspirate (BMA) smears and manual counting of nucleated cell populations to obtain a cell differential. This manual process has significant limitations, including the limited sample of cells analyzed by a conventional 500-cell differential compared to the thousands of nucleated cells present, as well as the inter-observer variability seen between differentials on single samples due to differences in cell selection and classification. To address these shortcomings, we developed an automated computational platform for obtaining cell differentials from scanned whole-slide BMAs at 40x magnification. This pipeline utilizes a sequential process of identifying BMA regions with high proportions of marrow nucleated cells that are ideal for cell counting, detecting individual cells within these optimal regions, and classifying cells into one of 11 types within the differential. Training of convolutional neural network models for region and cell classification, as well as a region-based convolutional neural network for cell detection, involved the generation of an annotated training data set containing 10,948 BMA regions, 28,914 cell boundaries, and 23,609 cell classifications from 73 BMA slides. Among 44 testing BMA slides, an average of 19,209 viable cells per slide were identified and used in automated cell differentials, with a range of 237 to 126,483 cells. In comparing these automated cell differential percentages with corresponding manual differentials, cell type-specific correlation coefficients ranged from 0.913 for blast cells to 0.365 for myelocytes, with an average coefficient of 0.654 among all 11 cell types. A statistically significant concordance was observed among slides with blast percentages less or greater than 20% (p=1.0x10-5) and with plasma cell percentages less or greater than 10% (p=5.9x10-6) between automated and manual differentials, suggesting potential diagnostic utility of this automated pipeline for malignancies such as acute myeloid leukemia and multiple myeloma. Additionally, by simulating the manual counting of 500 cells within localized areas of a BMA slide and iterating over all optimal slide locations, we quantified the inter-observer variability associated with limited sample size in traditional BMA cell counting. Localized differentials exemplify an average variance ranging from 24.1% for erythroid precursors to 1.8% for basophils. Variance in localized differentials of up to 44.8% for blast cells and 36.9% for plasma cells was observed, demonstrating that sample classification based on diagnostic thresholds of cell populations is variable even between different areas within a single slide. Finally, pipeline outputs of region classification, cell detection, cell classification, and localized cell differentials can be visualized using whole-slide image analysis software. By improving cell sampling and reducing inter-observer variability, this automated pipeline has potential to improve the current standard of practice for utilizing BMA smears in the diagnosis of hematologic disorders.

Effect of Ligustri Lucidi Fructus on myelosuppression in mice induced by cytoxan.

In this study, we aimed to demonstrate the therapeutic effect of Ligustri Lucidi Fructus on chemotherapy-induced myelosuppression and elucidate its mechanism. A pharmacological study was conducted to investigate the mechanism of the inhibiting effects of Ligustri Lucidi Fructus on cyclophosphamide-induced bone marrow suppression in mice. HPLC was used to measure the chemical components. We demonstrated that medium and high doses of Ligustri Lucidi Fructus increased the amount of white blood cells and bone marrow nucleated cells (p < 0.05) in the cyclophosphamian-induced mouse model, and at the same time reduced granulocyte-macrophage-colony stimulating factor and thrombopoietin in the serum of myelosuppression mice (p < 0.01). Medium and high doses of Ligustri Lucidi Fructus can also adjust the thymus index and spleen index(p < 0.05). Ligustri Lucidi Fructus regulates the balance of bcl-2/bax, inhibits the expression of Caspase-3 and meanwhile stimulates the expression of mitogen-activated protein (MEK) and phospho extracellular regulated protein kinases (p-ERK) on the MAPK pathway. Five chemical constituents of Ligustri Lucidi Fructus, which may be related to myelosuppression, were analyzed. The content of specnuezhenide was 0.281%, that of ligustroflavone was 0.004%, that of salidroside was 0.094%, that of hydroxytyrosol was 0.060% and that of tyrosol was 0.069%. The effect of Ligustri Lucidi Fructus on myelosuppression after chemotherapy may be related to its multicomponent and multitarget nature. Ligustri Lucidi Fructus may be a promising potential drug for treatment after chemotherapy.

Carbohydrates and ginsenosides in shenmai injection jointly improve hematopoietic function during chemotherapy-induced myelosuppression in mice

BackgroundShenmai injection (SMI), a traditional Chinese medicine (TCM) injection prepared from Red ginseng and Ophiopogon japonicus, is widely used in clinics to treat chemotherapy-induced myelosuppression. Similar to other TCM injections, SMI contains a high amount of carbohydrates (fructose, sucrose, and maltose) in addition to the bioactive substances, specifically ginsenosides (Rg1, Re, and Rb1). To date, the role of these carbohydrates in the hematopoietic function of SMI remains unclear.PurposeWe aimed to investigate the hematopoietic effects and potential mechanisms of SMI and its components, focusing on the carbohydrates present in SMI.Experimental design/methodsFirst, we evaluated the hematopoietic effect of SMI on 5-fluorouracil (5-FU)-induced myelotoxicity in a tumor-bearing mouse model. Then we prepared mixtures of ginsenosides and carbohydrates according to their proportions in SMI and evaluated their hematopoietic function in mice with 5-FU-induced myelosuppression. Finally, hematopoiesis-related molecular networks were built based on RNA sequencing (RNA-seq) of the bone marrow stromal cells (BMSCs), and the potential mechanisms of carbohydrates and ginsenosides were evaluated.ResultsSMI attenuated 5-FU-induced myelotoxicity in tumor-bearing mice. Both ginsenosides and carbohydrates increased the bone marrow nucleated cell (BMNC) count and improved the bone marrow morphology in myelosuppressive mice; they promoted the proliferation of BMSCs derived from those myelosuppressive mice. Bioinformatics analyses revealed ECM-receptor interaction, Hippo signaling, and Wnt signaling are common pathways regulated by both ginsenosides and carbohydrates; Gstt1, Gstp2, Gsta4 and Oplah in Glutathione metabolism pathway and Cd19, Cd79a, and Cd79b in B cell receptor pathway are uniquely regulated genes related to carbohydrates but not ginsenosides.ConclusionsCarbohydrates may collaborate with ginsenosides and contribute to the hematopoietic function of SMI. Carbohydrates could be considered as a bioactive component in this TCM injection.Graphical

Animal Model Establishment and Its Mechanism of Cytarabine-Iduced Myelosuppression

To establish an optimized model of bone marrow suppression induced by cytarabine (Ara-C) in C57BL/6 mice and preliminarily explore the mechanism of myelosuppression based on the cycle and apoptosis of BMNC. C57BL/6 mice were intraperitoneally injected with Ara-C 50, 100 and 200 mg/kg for 7 days, respectively. The survival rate and body weight of C57BL/6 mice were monitored. The number of peripheral blood cells and bone marrow nucleated cells (BMNC) was detected, and the morphology of bone marrow, thymus and spleen were measured on the 7th, 14th and 21st day of the experiment. The cycle and apoptosis of BMNC were also detected by flow cytometry. Ara-C 200 mg/kg caused 46.7% mortality in mice, and other doses had no significant effect on mortality. All doses of Ara-C induced bone marrow suppression in mice, as shown by a decrease in the number of peripheral blood cells (WBC, Neu, RBC, PLT) and BMNC (P<0.05), decrease in bone marrow hyperplasia, accompanied by immunosuppression and compensatory hematopoiesis of the spleen, and the above manifestations and duration were dose-dependent. Among them, the myelosuppression caused by Ara-C 50 mg/kg recovered quickly, and caused by Ara-C 200 mg/kg was too severe. The result of flow cytometry showed that Ara-C could cause S and G2/m arrest and increased apoptosis in BMNC. Ara-C can induce myelosuppression in mice with a dose-dependent severity and duration, and the model of myelosuppression with Ara-C 100 mg/kg is more optimized. The mechanism is related to the inhibition of BMNC proliferation and the promotion of apoptosis.

Therapeutic effect of autologous bone marrow cells injected into the liver under the guidance of B‑ultrasound in the treatment of HBV‑related decompensated liver cirrhosis.

The 5-year mortality rates associated with decompensated liver cirrhosis (DLC) can reach 50%, which suggests that this condition poses a serious health risk. In previous studies conducted by our group, autologous bone marrow nucleated cells (ABMNCs) were used to treat HIV-positive patients with DLC through the right omental vein; however, trauma and poor compliance were encountered. In the present study, the percutaneous liver approach to inject ABMNCs under the guidance of B-ultrasound was employed for the treatment of DLC. A total of 108 patients with DLC were retrospectively divided into the routine drug treatment group (control group; 30 cases), the right omental vein infusion of ABMNCs group (observation group 1; 38 cases) and the B-ultrasound-guided liver injection of ABMNCs group (observation group 2; 40 cases). After treatment, the liver synthesis (prothrombin time, albumin and ascites) and secretion functions (total bilirubin) in observation groups 1 and 2 were significantly improved compared with those of the control group (P<0.01) and the bone marrow function was also significantly improved compared with that of the control group (P<0.01). While, the bone marrow function (white blood cell, platelet, and hemoglobin) in observation group 1 was significantly improved compared with that of observation group 2 at the end of treatment (P<0.01). After a 1-year follow-up, the case fatality rate was 2.5% (1/40) in observation group 2, which was significantly lower than the 20% fatality rate (6/30) recorded in the control group (P<0.05). The injection of ABMNCs into the liver under the guidance of B-ultrasound was significantly better than conventional drug therapy in treating DLC. This approach has obvious advantages such as no hospitalization, minimal trauma, rapid recovery and good compliance, all of which make it worthy of application in primary hospitals.

Grain-Sized Moxibustion Heightens the AntiTumor Effect of Cyclophosphamide in Hepa1-6 Bearing Mice.

Objective The side effects of chemotherapy as a treatment of liver cancer cannot be ignored. Grain-sized moxibustion, a characteristic external therapy, has been shown to reduce the toxic and side effects of chemotherapy and regulate the immune function. The purpose of this study was to explore the synergistic antitumor activity of grain-sized moxibustion combined with cyclophosphamide (CTX). Methods A hepatoma 1–6 (Hepa1-6)-bearing mouse model was established by injecting mice with Hepa1-6 cancer cells. CTX and grain-sized moxibustion on Dazhui (DU14), Zusanli (ST36), and Sanyinjiao (SP6) were used for treatment, and mouse survival status, body weight, and tumor growth, weight, and volume were measured. White blood cells (WBCs) and bone marrow nucleated cells (BMNCs) were quantified. The spleens and livers of Hepa1-6-bearing mice were pathologically examined and scored. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured with enzyme-linked immunosorbent assay (ELISA) kits, and protein and mRNA expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) in tumor tissues were measured with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) techniques. Results Both grain-sized moxibustion and CTX could restrain the growth of Hepa1-6 tumors, reducing both tumor volume and weight; the combined treatment had a greater effect. Grain-sized moxibustion down-regulated the expression of proliferation genes Ki67 and PCNA, weakened the proliferation ability of Hepa1-6 tumor cells, inhibited tumor growth, and enhanced the antitumor effect of CTX. In addition, grain-sized moxibustion significantly improved the signs of CTX-induced toxicity (including weight loss, leukopenia, bone marrow suppression, and hepatotoxicity), down-regulated serum AST and ALT levels, reduced spleen and liver inflammation, and improved liver and spleen indices. Conclusion Grain-sized moxibustion can synergize with CTX to enhance the antitumor effect of CTX and alleviate its toxic and side effects. It may be a promising adjuvant therapy to chemotherapy.

Effect of Interleukin-6 Gene Deletion on Radiation-Induced Mouse Hematopoietic Injury and Relative Mechanism

To study the effect of interleukin-6 (IL-6) gene deletion on radiation-induced hematopoietic injury in mice and relative mechanism. Before and after whole body 60Co γ-ray irradiation, it was analyzed and compared that the difference of peripheral hemogram, bone marrow hematopoietic stem and progenitor cells conts in IL-6 gene knockout (IL-6-/-) and wild-type (IL-6+/+) mice and serum IL-6 and G-CSF expression levels in above- mentioned mouse were detected. Moreover, 30 days survival rate of IL-6-/- and IL-6+/+ mice after 8.0 Gy γ-ray irradiation were analyzed. IL-6 levels in serum of IL-6+/+ and IL-6-/- mice were respectively (98.95±3.85) pg/ml and (18.36±5.61) pg/ml, which showed a significant statistical differences (P<0.001). There were no significant differences of peripheral blood cell counts and G-CSF level in serum between IL-6+/+ and IL-6-/- mice before irradiation (P>0.05). However, the number of leukocytes, neutrophils, lymphocytes, monocytes, platelets in peripheral blood and G-CSF level in serum of IL-6-/- mice were significantly decreased at 6 h after 8.0 Gy γ-ray irradiation compared with that of IL-6+/+ mice. On days 30 after 8.0 Gy γ-ray irradiation, the survival rate of IL-6+/+ and IL-6-/- mice was 62.5% and 12.5%, and the mean survival time of dead mice was 16.0±1.0 and 10.6±5.3 days, respectively. On days 14 after 6.5 Gy γ-ray irradiation, bone marrow nucleated cells in IL-6+/+ and IL-6-/- mice were respectively (10.0±1.2)×106 and (8.3±2.2)×106 per femur. Compared with IL-6+/+ mice, the proportion of Lin-Sca-1-c-kit+ (LK) in bone marrow of IL-6-/- mice had no significant change (P>0.05), but the proportion of Lin-Sca-1+c-kit+ (LSK) was significantly decreased (P<0.05). IL-6 plays an obvious role in regulating hematopoietic radiation injury, and IL-6 deficiency can inhibit the radiation-induced increase of endogenous G-CSF level in serum, aggravates the damage of mouse hematopoietic stem cells（HSC） and the reduction of mature blood cells in peripheral blood caused by ionizing irradiation, resulting in the shortening of the survival time and significant decrease of the survival rate of mice exposed to lethal dose radiation.

Haploidentical bone marrow transplantation in patients with relapsed or refractory severe aplastic anaemia in the USA (BMT CTN 1502): a multicentre, single-arm, phase 2 trial

Relapsed severe aplastic anaemia is a marrow failure disorder with high morbidity and mortality. It is often treated with bone marrow transplantation at relapse post-immunosuppressive therapy, but under-represented minorities often cannot find a suitably matched donor. This study aimed to understand the 1-year overall survival in patients with relapsed or refractory severe aplastic anaemia after haploidentical bone marrow transplantation. We report the outcomes of BMT CTN 1502, a single-arm, phase 2 clinical trial done at academic bone marrow transplantation centres in the USA. Included patients were children and adults (75 years or younger) with severe aplastic anaemia that was refractory (fulfilment of severe aplastic anaemia disease criteria at least 3 months after initial immunosuppressive therapy) or relapsed (initial improvement of cytopenias after first-line immunosuppressive therapy but then a later return to fulfilment of severe aplastic anaemia disease criteria), adequate performance status (Eastern Cooperative Oncology Group score 0 or 1, Karnofsky or Lansky score ≥60%), and the presence of an eligible related haploidentical donor. The regimen used reduced-intensity conditioning (rabbit anti-thymocyte globulin 4·5 mg/kg in total, cyclophosphamide 14·5 mg/kg daily for 2 days, fludarabine 30 mg/m2 daily for 5 days, total body irradiation 200 cGy in a single fraction), related HLA-haploidentical donors, and post-transplantation cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis. Additionally, for GVHD prophylaxis, mycophenolate mofetil was given orally at a dose of 15 mg/kg three times a day up to 1 g three times a day (maximum dose 3000 mg per day) from day 5 to day 35, and tacrolimus was given orally or intravenously from day 5 to day 180 as per institutional standards to maintain a serum concentration of 10-15 ng/mL. The primary endpoint was overall survival 1 year after bone marrow transplantation. All patients treated per protocol were analysed. This study is complete and is registered with ClinicalTrials.gov, NCT02918292. Between May 1, 2017, and Aug 30, 2020, 32 patients with relapsed or refractory severe aplastic anaemia were enrolled from 14 centres, and 31 underwent bone marrow transplantation. The median age was 24·9 years (IQR 10·4-51·3), and median follow-up was 24·3 months (IQR 12·1-29·2). Of the 31 patients who received a transplant, 19 (61%) were male and 12 (39%) female. 13 (42%) patients were site-reported as non-White, and 19 (61%) were from under-represented racial and ethnic groups; there were four (13%) patients who were Asian, seven (23%) Black, one (3%) Hawaiian/Pacific Islander, and one (3%) more than one race, with seven (23%) patients reporting Hispanic ethnicity. 24 (77%) of 31 patients were alive with engraftment at 1 year, and one (3%) patient alive with autologous recovery. The 1-year overall survival was 81% (95% CI 62-91). The most common grade 3-5 adverse events (seen in seven or more patients) included seven (23%) patients with abnormal liver tests, 15 (48%) patients with cardiovascular changes (including sinus tachycardia, heart failure, pericarditis), ten (32%) patients with gastrointestinal issues, seven (23%) patients with nutritional disorders, and eight (26%) patients with respiratory disorders. Six (19%) deaths, due to disease and unsuccessful bone marrow transplantation, were reported after transplantation. Haploidentical bone marrow transplantation using this approach results in excellent overall survival with minimal GVHD in patients who have not responded to immunosuppressive therapy, and can expand access to bone marrow transplantation across all populations. In clinical practice, this could now be considered a standard approach for salvage treatment of severe aplastic anaemia. Attention to obtaining high cell doses (>2·5 × 108 nucleated marrow cells per kg of recipient ideal bodyweight) from bone marrow harvests is crucial to the success of this approach. US National Heart, Lung, and Blood Institute and US National Cancer Institute.

The Protective Effect of Sweet Potato Root Tuber on Chemotherapy-Induced Thrombocytopenia.

Sweet potato (Ipomoea batatas L.) is one of the leading crops worldwide, containing high nutritional components such as fiber and polyphenols. Root tuber of Simon 1 (SIMON), a cultivar of sweet potato, is a folk food in China with a hemostasis function but lacking experimental data support. Now the protective effect of SIMON on chemotherapy-induced thrombocytopenia (CIT), a serious complication of cancer treatment, is investigated for the first time by a CIT mouse model induced by intraperitoneal injection of carboplatin. As a result, SIMON raises the number of peripheral platelets, white blood cells, and bone marrow nucleated cells in CIT mice significantly. Besides, carboplatin-induced atrophy of the thymus, spleen, and disordered metabolism of the inflammatory immune system and glycerophospholipids are also reversed by SIMON. Phytochemical analysis of SIMON indicates 16 compounds including eight phenolic derivatives, which might be associated with its anti-CIT bioactivity. Sweet potato (SIMON) may be an efficient function food in the prevention of bleeding disorders.

P964: IMMUNOPHENOTYPED-SUSPENSION-MULTIPLEX FISH BY IMAGING FLOW CYTOMETRY FOR THE SIMULTANEOUS DIAGNOSIS OF THREE PIVOTAL IGH TRANSLOCATIONS IN MULTIPLE MYELOMA

Background: The cytogenetic abnormalities including t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32) are associated with the molecular profiles, clinical features, and treatment outcome in multiple myeloma (MM). Although the double-color interphase fluorescence in situ hybridization (DC-FISH) is well established to detect those translocations, the conventional method can analyze only one abnormality on each slide. Aims: To analyze multiple chromosomal abnormalities simultaneously with high sensitivity and specificity, we aimed to develop a new diagnostic modality of four-color FISH for multiple translocations in immunophenotyped cells in suspension using imaging flow cytometry (immunophenotyped-suspension-multiplex (ISM)-FISH). Methods: For the ISM-FISH, we first subjected Carnoy-fixed cells in suspension to immunostaining using a brilliant violet 421-conjugated anti-CD138 antibody. Then, we hybridized with four different FISH probes, i.e., Texas Red (TxRed)-conjugated probe for IGH, fluorescein isothiocyanate (FITC)-conjugated probe for FGFR3, Gold-conjugated probe for cyclin D1 (CCND1), and Cy5-conjugated probe for c-MAF, in suspension. Then, more than 2.5 × 104 nucleated cells for each sample were analyzed with imaging flow cytometer MI-1000 (Sysmex) by six channels: one for immunophenotyping, four for FISH and one for bright field image. In the process of imaging flowcytometric analysis, we sorted CD138-positive cell fraction, computed the distance between FISH probes using originally developed spot counting tool, and determined to be positive for translocation when the distance is under the threshold. Results: By this ISM-FISH system, we simultaneously examined three chromosomal translocations, including t(4;14), t(14;16), and t(11;14), in three MM cell lines, KMS-11, KMS-21-BM, KMS-26, and successfully detect the abnormalities. Using different proportions of transformation-positive MM cells spiked with transformation-negative leukemic cells, HL-60, ISM-FISH showed a sensitivity of less than 0.1%. Next, ISM-FISH and conventional DC-FISH were performed for bone marrow nucleated cells from 70 patients with MM or monoclonal gammopathy of undetermined significance (MGUS) and the experiments showed a promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive than standard DC-FISH, examining 200 interphase cells with its optimal sensitivity of up to 1.0%. Furthermore, the ISM-FISH showed a positive concordance of 83.3% and negative concordance of 97.4% using standard DC-FISH, examining 200 interphase cells. Furthermore, in three patients (4%), t(4;14) or t(11;14) were positive by ISM-FISH but negative by DC-FISH examining 200 cells. Extensive DC-FISH study examining additional 1,000 cells showed positive in these ISM-FISH-positive/DC-FISH (200cells)-negative samples, suggesting higher sensitivity of ISM-FISH than that of the standard DC-FISH. Image:Summary/Conclusion: This study developed the new diagnostic system of ISM-FISH, which enables the simultaneous diagnosis of three clinically pivotal chromosomal translocations, t(4;14), t(14;16), and t(11;14), in MGUS and MM. This system may facilitate rapid and reliable cytogenetic diagnosis and promote patient-oriented therapy according to the type of chromosomal translocation in the clinical practice setting.

High Counts in Hematologic Malignancies Predict Low Metaphase Yield for Cytogenetic Analysis.

This study examined the impact of various preanalytical variables on metaphase yield in hematologic malignancies. Marrow samples from patients with hematologic malignancies that were subjected to cytogenetic analysis were categorized into two groups: one with samples that yielded an adequate number of metaphases, defined as at least 20, and a second with a low number of metaphases (LNM), having fewer than 20 metaphases. Age, sex, bone marrow nucleated cell (MNC) count, and peripheral blood counts (hemoglobin, total WBC count, and platelet count) were analyzed for an association with LNM. Of 455 samples, 17% (79/455) belonged to the LNM group, including 6% (27/455) that yielded no metaphases. MNCs and WBCs were higher in the LNM group (P < .001 for both). MNCs were higher in LNM groups in both acute myeloid leukemia (P = .008) and acute lymphoblastic leukemia (P = .001). Receiver operating characteristic curves showed moderate prediction of MNC and WBC counts for LNM with areas under the curves of 0.7. Other analyzed parameters showed no significant associations with LNM. Low metaphase yields occur frequently in hematologic malignancies with high counts. This could reflect biological characteristics of these malignancies that merit further investigation.

Glucocorticoid-induced expansion of classical monocytes contributes to bone loss

Classical monocytes are commonly involved in the innate inflammatory response and are the progenitors of osteoclasts. Excess endogenous glucocorticoids (GCs) can increase the levels of classical monocytes in blood and bone marrow. The role of this cell population in high-dose exogenous GC-induced osteoporosis (GIOP) remains to be elucidated. In this study, GIOP was established in rats and mice by daily methylprednisolone injection, and monocyte subsets were analyzed by flow cytometry. We demonstrated that classical monocytes accumulate in bone marrow during GIOP. Similarly, the monocyte proportion among bone marrow nucleated cells was also increased in patients with steroid treatment history. We sorted classical monocytes and analyzed their transcriptional profile in response to GCs by RNA sequencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that classical monocytes isolated from GC-treated rats exhibited osteoclast differentiation potential. Deletion of classical monocytes by clodronate liposome treatment prevented GIOP via inhibition of osteoclastogenesis and restoration of CD31HiendomucinHi vessels. Regarding the molecular mechanism, classical monocytes express high levels of glucocorticoid receptors. In vitro treatment with GCs increased both the percentage and absolute number of monocytes and promoted their proliferation. In summary, classical monocytes mediated GC-induced bone loss and are a potential target for therapeutic intervention in GIOP treatment.