Bone Marrow Nucleated Cells Research Articles

Abstract 3082: Isolation and identification of disseminated tumor cells from bone marrow

Abstract Background: Disseminated tumor cells (DTCs), detected by immunohistological staining for cytokeratins, found in the bone marrow (BM) of breast cancer patients portend a poor prognosis. Study of these cells has been hindered by their rarity in BM specimens. We have tested a microfiltration technique for the enrichment of DTCs. Methods: BM aspirates from healthy volunteers were collected and mixed with defined numbers SKBR3 breast cancer cells at a concentration of 1,000 to 10,000 tumor cells per 1x10^6 nucleated BM cells., BM was then diluted 1:10 with PBS and 7 ml was placed in CellSaveTM tubes to simulate a patient BM collection. Each specimen was processed within 24 hours of collection. Specimens were pre-filtered using a 70 µm mesh sieve (BD cat#352350) to remove large particles. The effluent was filtered through CellSieveTM microfilters, which have 7 µm diameter pores in a uniform array, of 160,000 pores in a 9 mm diameter area. Filters containing cells were post-fixed, cells permeabilized, and stained with DAPI and fluorescent antibodies specific to epithelial cytokeratins 8, 18, and 19 (FITC), EpCAM (PE), and CD45, a negative control (Cy5). Five representative 1% areas of each filter were imaged and used to estimate total cell and tumor cell counts, based on fluorescent DAPI and cytokeratin antibody staining. Results: From each BM specimen (7 ml) containing a total of 14x10^6 nucleated BM cells, post filtration samples contained only 17-21x10^3 residual, nucleated BM cells. For specimens spiked at 10 tumor cells per 1,000 BM cells, approximately 30x10^3 positively staining tumor cells were recovered (20% tumor cell recovery with approximately 170 fold enrichment). For specimens spiked at 1 tumor cell per 1,000 BM cells, approximately 7x10^3 total tumor cells were recovered (50% tumor cell recovery with approximately 400-fold enrichment). Captured cells co-stained with cytokeratins and EpCAM, but not CD45. Control BM specimens with no added tumor cells demonstrated only rare, staining cells (4 per sample), likely due to background or epithelial cell contamination. Conclusions: We have tested a microfiltration technique for enrichment of BM DTCs. Microfiltration of BM is rapid without filter clogging. Recovered DTCs can be immuno-stained, and are easily identified and distinguished from BM cells. The DTC capture efficiency varied with cancer cell concentration and ranged from 20-50% with up to 400 fold enrichment when samples contained lower numbers of tumor cells. The microfiltration enrichment assay continues to be optimized for isolation and molecular characterization of DTCs from patient BM samples, where the concentration of DTCs seldom exceeds 10 cells per 1x10^6 BM cells. Citation Format: Peixuan Zhu, Daniel Adams, Rebecca L. Aft, Sreeraj G. Pillai, Mark A. Watson, Shuhong Li, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang. Isolation and identification of disseminated tumor cells from bone marrow. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2014-3082

Isolation and purification of psoralen and isopsoralen and their efficacy and safety in the treatment of osteosarcoma in nude rats.

Modern studies have shown that psoralen has a significant inhibitory effect on tumor growth in a variety of animals and humans. To obtain coumarin compounds - psoralen and isopsoralen - from traditional Chinese medicine Psoralea corylifolia L. using chromatographic techniques and isolation and purification methods, and to observe the transplanted tumor growth inhibitory effects and adverse reactions of psoralen and isopsoralen in nude rats with osteosarcoma. Dried ripe fruits of Psoralea corylifolia L. were taken as the raw material to prepare crude extract of Psoralea corylifolia L. by ethanol reflux method. Column chromatography was used to isolate the crude extract; compounds were structurally identified based on (1)H-NMR, (13)C-NMR spectra, the two compounds were identified as psoralen andisopsoralen, and their contents were 99.7% and 99.6, respectively. Nude rat model of osteosarcoma was established; the rats were randomized into: normal saline group, psoralen low- and high-dose groups, isopsoralen low- and high-dose groups, and cisplatin group. Osteosarcoma volume and weight inhibition rates in nude rats in each group were observed; radioimmunoassay was used to determine the serum alkaline phosphatase activity; peripheral blood cell and bone marrow nucleated cell counts were determined; light microscopy was used to observe heart, liver, spleen, lung, kidney, and tumor histopathology; and electron microscopy was used to observe the fine structure of tumor cells. Tumor volume inhibition rates were 43.75% and 40.18%, respectively, in the psoralen and isopsoralen low-dose groups, and tumor weight inhibition rates were 38.83% and 37.77%. Tumor volume inhibition rates were 67.86% and 66.96%, respectively, in the psoralen and isopsoralen high-dose groups, and tumor weight inhibition rates were 49.47% and 47.87%. Psoralen and ispsoralen markedly lowered serum AKP level. Psoralen and isopsoralen induced apoptosis or necrosis of osteosarcoma. After administration of high doses of psoralen and isopsoralen, toxic reactions such as writhing, lassitude, and hypoactivity were seen. Kidney histopathology showed tubulointerstitial dilatation and congestion, and inflammatory cell aggregation in the renal intercellular space. Psoralen and isopsoralen did not cause any significant toxic side effects to the bone marrow, or other organs such as heart, lung, liver, and spleen. Psoralen and isopsoralen have growth inhibitory effects on transplanted tumor in nude rats with osteosarcoma, and can induce tumor cell apoptosis or necrosis, without significant toxic effects.

The relationship between the alkaline phosphatase network and the haematopoiesis in mice subjected to whole-body irradiation

Abstract Purpose: To investigate the relationship between the alkaline phosphatase (ALP) network of the marrow stroma and the haematopoietic regeneration after mice whole-body irradiation. Materials and methods: Three groups of mice were irradiated with a non-lethal ionising radiation dose: the fi rst one received an intraperitoneal injection of Levamisole, ALP inhibitor, 24 h before irradiation; the second one received an intraperitoneal injection of Lisinopril, haematopoiesis inhibitor, 24 h before irradiation; the third was left untreated, but irradiated. The fourth group, untreated and not irradiated, was the control. The total surface occupied by ALP positive processes, revealed by means of ALP cytochemistry in the marrow area, was evaluated semi-quantitively. Nucleated bone marrow cells were also counted. Results: ALP network began to increase 24 h after irradiation to reach a maximum after 72 h, when the bone marrow was almost become completely empty of the haematopoietic cells. This increase advances the haematopoietic recovery. This process was substantially delayed when the mice were injected with Levamisole 24 h before irradiation. On the contrary, ALP network increased strongly since the fi rst day after irradiation when the mice were injected with Lisinopril 24 h before irradiation. Conclusions: These data have indicated that the haematopoietic recovery and repopulation of the bone marrow were advanced by the ALP network recovery.

Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion.

Mesenchymal stromal/stem cell (MSC) expansion in conventional monolayer culture on plastic dishes (2D) leads to progressive loss of functionality and thus challenges fundamental studies on the physiology of skeletal progenitors, as well as translational applications for cellular therapy and molecular medicine. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow nucleated cells within 3D porous scaffolds in a perfusion-based bioreactor system. The 3D-perfusion system generated a stromal tissue that could be enzymatically treated to yield CD45- MSC. As compared to 2D-expanded MSC (control), those derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7–8 doublings) better maintained their progenitor properties, as assessed by a 4.3-fold higher clonogenicity and the superior differentiation capacity towards all typical mesenchymal lineages. Transcriptomic analysis of MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability and a significant upregulation of multipotency-related gene clusters following 3D-perfusion- as compared to 2D-expansion. Interestingly, the differences in functionality and transcriptomics between MSC expanded in 2D or under 3D-perfusion were only partially captured by cytofluorimetric analysis using conventional surface markers. The described system offers a multidisciplinary approach to study how factors of a 3D engineered niche regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems.

Concentration of Rat Bone Marrow Nucleated Cells Using Hypo-osmotic Hemolysis in Distilled Water.

Background:Bone marrow aspirates contain primarily red blood cells. To achieve efficient cell transplantation for regeneration, the red blood cells need to be removed from the aspirates. Cell isolation is typically performed using density gradient centrifugation. However, this method entails issues of clinical safety and convenience. This study describes an efficient method to concentrate bone marrow nucleated cells by hypo-osmotic hemolysis.Methods:The optimal hemolysis conditions were determined by diluting the bone marrow suspensions with distilled water in various dilution ratios. Then, the resulting cell fractions were transplanted in a rat cranial defect model to evaluate their effects on bone formation and their angiogenic effects.Results:The optimal hemolysis conditions were a 3.3-fold dilution in distilled water and a hypo-osmotic exposure time of 45 seconds. Nucleated cells obtained using this method included granulocytes and mononuclear cells. These cells contain cytoplasmic angiogenic factors, including vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor. In a rat cranial defect model, callus formation and angiogenesis were significantly increased following transplantation of concentrated marrow nucleated cells in this manner.Conclusions:These results suggest angiogenic and osteogenetic effects of transplanting marrow nucleated cells using this hypo-osmotic method.

Risk factor analysis for bone marrow histiocytic hyperplasia with hemophagocytosis: an autopsy study.

The excessive release of inflammatory cytokines occasionally induces life-threatening hemophagocytosis referred to as hemophagocytic syndrome (HPS). A similar condition, histiocytic hyperplasia with hemophagocytosis (HHH), is often seen in bone marrow collected during autopsy. Unlike HPS, the pathogenesis of HHH remains unclear. Therefore, we performed a clinicopathological analysis of HHH from 70 autopsy cases at the University of Fukui Hospital. HHH was detected in 29 of 70 autopsies (41.4 %) and was significantly complicated with hematological diseases (p < 0.05) and sepsis (p < 0.05). The percentage of macrophages in bone marrow (BM) nucleated cells was significantly increased in HHH (p < 0.001). Data from medical records indicated no significant changes, except for the minimum values of white blood cell counts (p < 0.05) and platelet counts (p < 0.05) in HHH patients as compared with non-HHH patients. Concentrations of inflammatory mediators including IL-1β, IL-6, and IL-8 were significantly increased in HHH patients. Multivariate risk factor analysis identified hematological diseases (odds ratio (OR), 11.71), ≥15 % BM macrophages (OR, 9.42), sepsis (OR, 7.77), and high serum IL-6 levels (OR, 1.00) as independent risk factors for HHH. HHH with hypocellular BM, the most aggressive form of HHH, was recognized in 8 of 29 HHH patients and was associated with ≥25 % BM macrophages (p < 0.001), leukocytopenia (p < 0.05), and high IL-8 levels (p < 0.05). None of the HHH patients fulfilled the diagnostic criteria of HPS. These findings suggest that HHH is a different entity from HPS and that it preferentially develops under conditions of excessive inflammation and its associated risks, such as hematological diseases and sepsis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00428-014-1592-8) contains supplementary material, which is available to authorized users.

Polysaccharide isolated from Parmelia tinctorum ameliorates ionizing irradiation-induced damage in mice.

In this study, WPT-A, a type of water-soluble homogeneous lichen polysaccharide, was isolated and purified from Parmelia tinctorum. We investigated whether WPT-A has radioprotective effects when administered before total-body irradiation (TBI). Mice were treated with WPT-A via intraperitoneal injection (i.p.) once per day for three consecutive days prior to 7, 7.5, 8.5, 10 or 10.5-Gy TBI. Our results indicated that the survival rate was enhanced at a range of levels of TBI. The calculated dose reduction factor (DRF) was 1.2. White blood cell (WBC) counts, spleen colony forming units (CFU-S) and bone marrow nucleated cell (BMNC) counts were used to investigate the radioprotective effects of WPT-A on the hematopoietic system. The treatment groups received WPT-A at 20, 50 and 80 mg/kg b.w. doses before 6.5-Gy TBI and showed significantly higher BMNC and WBC counts compared with the radiation-only group. The groups administered 50 and 80 mg/kg b.w. WPT-A showed a significant increase in CFU-S compared with the radiation-only group. We also carried out a single cell gel electrophoresis assay to explore the radioprotective effects of WPT-A on DNA damage. The results from single-cell gel electrophoresis of peripheral blood leukocytes showed that WPT-A attenuated radiation-induced DNA damage. These results indicate a potential use for WPT-A as a radioprotector.

Effect of GW003 on the granulocyte macrophage colony formation ability of bone marrow cells in vitro

The aim of this study was to investigate the effect of GW003 on the ability of granulocyte colony forming in vitro of bone marrow cells. The bone marrow samples was collected from normal rhesus, the patients with leukemia in stages of remission and chemotherapy respectively, and the nucleated cells were separated and cultured for 12 days after addition of different concentrations of GW003 or rhG-CSF, or G-CSF mutant. Then the amount of colony-forming unit-granulocyte-macrophage was counted. The results indicated that GW003 could enhance the ability of bone marrow nucleated cells of rhesus to forming CFU-GM in vitro, and its effect was much better than that of rhG-CSF or G-CSF mutant at the same concentration(®). The GW003 showed dose-response relationship to CFU-GM level (r = R(2) = 0.965, P = 0.003, in a certain concentration), the GW003 also could enhance CFU-GM formation of marrow nucleated cells in leukemic patients, especially for patients receiving chemotherapy. The GW003 could relieve the marrow suppression caused by chemotherapy significantly. It is concluded that the GW003 can significantly improve the ability of bone marrow cells to form granulocyte colony in vitro as well as effectively alleviate bone marrow suppression.

Matrix-Associated Chondroplasty: A Novel Platelet-Rich Plasma and Concentrated Nucleated Bone Marrow Cell–Enhanced Cartilage Restoration Technique

Bone marrow stimulation techniques such as microfracture for the treatment of articular cartilage defects so far solely reproduce mechanically inferior fibrous cartilage tissue, which might result in unsatisfactory clinical results at midterm. The combination of microfracture and biomaterials—for example, autologous matrix-induced chondrogenesis technology—has not yet proved that the disadvantages of the marrow stimulation techniques can be overcome. At present, only laboratory-cultivated autologous chondrocytes are able to restore a biomechanically superior cartilage layer and might lead to superior functional results. However, the costs are high and the patient must undergo a 2-stage procedure. By selecting the appropriate cell fraction in conjunction with a controlled release of differentiating growth factors, sufficient cartilage regeneration might be achievable on the basis of bone marrow aspirate as well. We thus describe an advanced surgical technique for the treatment of articular cartilage defects based on platelet-rich plasma and bone marrow aspirate concentrate to overcome these drawbacks.

Safety and tolerability of intrathecal delivery of autologous bone marrow nucleated cells in children with cerebral palsy: an open-label phase I trial

Background aimsCerebral palsy (CP) is related to severe perinatal hypoxia with permanent brain damage in nearly 50% of surviving preterm infants. Cell therapy is a potential therapeutic option for CP by several mechanisms, including immunomodulation through cytokine and growth factor secretion. MethodsIn this phase I open-label clinical trial, 18 pediatric patients with CP were included to assess the safety of autologous bone marrow–derived total nucleated cell (TNC) intrathecal and intravenous injection after stimulation with granulocyte colony-stimulating factor. Motor, cognitive, communication, personal-social and adaptive areas were evaluated at baseline and 1 and 6 months after the procedure through the use of the Battelle Developmental Inventory. Magnetic resonance imaging was performed at baseline and 6 months after therapy. This study was registered in ClinicaTrials.gov (NCT01019733). ResultsA median of 13.12 × 108 TNCs (range, 4.83–53.87) including 10.02 × 106 CD34+ cells (range, 1.02–29.9) in a volume of 7 mL (range, 4–10.5) was infused intrathecally. The remaining cells from the bone marrow aspirate were administered intravenously; 6.01 × 108 TNCs (range, 1.36–17.85), with 3.39 × 106 cells being CD34+. Early adverse effects included headache, vomiting, fever and stiff neck occurred in three patients. No serious complications were documented. An overall 4.7-month increase in developmental age according to the Battelle Developmental Inventory, including all areas of evaluation, was observed (±SD 2.63). No MRI changes at 6 months of follow-up were found. ConclusionsSubarachnoid placement of autologous bone marrow–derived TNC in children with CP is a safe procedure. The results suggest a possible increase in neurological function.

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

Activation of Monocyte-Derived Cells in the Bone Marrow of Myelodysplastic Syndrome

Object: To study the relationship between monocyte/histiocyte activation and myelodysplastic syndrome (MDS).Methods: Analyzing ultrastructure and myeloperoxidase reaction of nucleated cells in bone marrow from 59 cases of MDS by transmission electron microscopy. Four groups of MDS were subdivided on the basis of their content of activated inflammatory cells – morbid hematopoiesis with minimal inflammatory cell activation (MH-MICA); MDS with monocytic system activation (MSA); MDS with lymphocyte activation (LCA); and MDS with granulocyte activation (GCA).Results: About 20, 22, 7, and 10 cases were classified as MH-MICA (34%), MSA (37%), LCA (12%), and GCA sub-types (17%), respectively. About 3, 5, 0, and 3 cases from MH-MICA, MSA, LCA, and GCA, respectively, underwent leukemic transformation within 2 years.Conclusion: The findings suggest that activation of inflammatory cells in bone marrow is an important feature of MDS, and that monocytes/histocytes are perhaps the most prominent cellular participants in the pathogenesis of MDS.

Hematopoietic effects and mechanisms of Fufang E׳jiao Jiang on radiotherapy and chemotherapy-induced myelosuppressed mice

Ethnopharmacological relevanceFufang E׳jiao Jiang (FEJ), which has been widely used in clinic to replenish qi (vital energy) and nourish blood, is a famous traditional Chinese medicine formula made up of Colla corii Asini (Donkey-hide gelatin prepared by stewing and concentrating from the hide of Equus asinus Linnaeus.), Radix Codonopsis Pilosulae (the root of Codonopsis pilosula (Franch.) Nannf.), Radix Ginseng Rubra (the steamed and dried root of Panax ginseng C.A. Mey.), Fructus Crataegi (the fruit of Crataegus pinnatifida Bunge) and Radix Rehmanniae Preparata (the steamed and sun dried tuber of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey.). The present study aimed to investigate the hematopoietic effects of FEJ on myelosuppressed mice induced by radiotherapy and chemotherapy systematically and to explore the underlying hematopoietic regulation mechanisms. MethodsThe myelosuppressed mouse model was induced by 60Co radiation, cyclophosphamide and chloramphenicol. FEJ was then administered by i.g. at the dosages of 5, 10, or 20mL/kg·d for 10d. The numbers of blood cells from peripheral blood and bone marrow nucleated cells (BMNC) were counted. Body weight and the thymus and spleen indices were also measured. The numbers of hemopoietic progenitor cells and colony-forming unit-fibroblast (CFU-F) were measured in vitro. The ratio of hematopoietic stem cells (HSC) in BMNC, cell cycle and apoptosis of BMNC were determined by flow cytometry. The histology of femoral bone was examined by H&E staining. The levels of transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-6 (IL-6) in serum were measured by ELISA. IL-1β, IL-3, IL-6 mRNA levels in spleen were detected by real-time quantitative PCR (RT-qPCR). In addition, bone marrow stromal cells (BMSC) were cultured in vitro followed by treatment with different doses of FEJ (2.5, 5, 10μL/mL) for 48h. Then the levels of cytokines (IL-6, SCF, GM-CSF) in the conditioned media and their mRNA levels in BMSC were determined by ELISA and RT-qPCR, respectively. ResultsFEJ could significantly increase the numbers of peripheral blood cells and BMNC, and reverse the loss of body weight and the atrophy of thymus and spleen in a dose-dependent manner. The quantities of hemopoietic progenitor cells and CFU-F in bone marrow were also significantly increased in a dose-dependent manner after FEJ administration. A high-dose FEJ of 20mL/kg·d could significantly increase the ratio of HSC in BMNC, promote bone marrow cells entering the proliferative cycle phase (S+G2/M) and prevent cells from proceeding to the apoptotic phase. FEJ could also improve the femoral bone marrow morphology. Furthermore, FEJ could increase the levels of GM-CSF and IL-3 and reduce the level of TGF-β in serum, and enhance the expressions of IL-1β and IL-3 mRNA in spleen. Lastly, the levels of cytokines (IL-6, SCF, GM-CSF) in the conditioned media and their mRNA levels in BMSC were elevated after treatment with FEJ. ConclusionsFEJ was clearly confirmed to promote the recovery of bone marrow hemopoietic function in a myelosuppressed mouse model, which may be attributed to (i) improving bone marrow hematopoietic microenvironment; (ii) facilitating the cell proliferation and preventing BMNC from apoptosis; (iii) stimulating the expressions of IL-1β, IL-3, IL-6, SCF and GM-CSF and inhibiting the expression of TGF-β.

Preliminary Study of Autologous Bone Marrow Nucleated Cells Transplantation in Children With Spinal Cord Injury

The objective of this study was to assess the safety and efficacy of transplanting bone marrow nucleated cells (BMNCs) to treat children with complete interruption of spinal cord (SC) continuity. The present study was conducted from 2005 to 2011. The inclusion criteria were a magnetic resonance imaging-confirmed complete interruption of SC continuity and no improvement in neurological status within 6 months after standard therapy. Bone marrow was isolated from the iliac ala and submitted to BMNC isolation. Subsequently, the cell suspension was administered into the SC cavity and intravenously. In total, 18 of 19 intraspinal and intravenous BMNC transplantation procedures performed caused no adverse events. One case was connected with transient bradycardia. The experimental therapy showed no late complications in the 1- to 6-year follow-up evaluation period. Neurological improvement was observed in two patients who received multiple implantations. One patient demonstrated improved superficial sensation from Th3 to Th12/L1 and a restored bladder-filling sensation. In the other case, superficial sensation was improved from C2 to C5, and the respiratory drive, the swallowing reflex, and tongue movements were restored. Spasticity and quality of life were improved in three of five patients. In addition, skin pressure ulcers healed and did not recur. Our preliminary results demonstrate the safety and feasibility of BMNC transplantation in children with complete SC injury. The results indicate that a certain degree of neurological and quality-of-life improvement can be attained by children with chronic complete SC injury who receive multiple BMNC implantations.

Successful allogeneic hematopoietic stem cell transplantation with reduced-intensity conditioning for B-cell prolymphocytic leukemia in partial remission

B-cell prolymphocytic leukemia (B-PLL) is a rare, chemotherapy-resistant lymphoid neoplasm. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a potentially curative treatment for B-PLL, the number of B-PLL patients who have been treated with allo-HSCT is small and its efficacy has not been established. We report the case of a 59-year-old woman with B-PLL in partial remission, who was successfully treated with allo-HSCT following reduced-intensity conditioning (RIC). She was initially treated with four courses of combination chemotherapy consisting of rituximab, fludarabine, cyclophosphamide, and mitoxantrone, which resulted in partial remission with residual tumor cells comprising 7 % of bone marrow nucleated cells. Although the residual B-PLL cells appeared to be indolent, as the disease progressed slowly during partial remission, these cells lost CD20 expression, and the prognosis of the patient was considered to be poor with conventional chemotherapy. She was therefore given RIC, followed by allo-HSCT from an HLA-matched sibling donor. Her clinical course following allo-HSCT was uneventful, and she remained in complete remission at 32 months post-transplantation. Although the therapeutic strategy for B-PLL should be determined based on the severity of the disease, RIC with allo-HSCT may be a therapeutic option for indolent B-PLL when the long-term prognosis of patients is markedly poor.

Methodological study on the animal model of aplastic anemia

Aplastic anemia (AA) animal models can be established by the methods such as physics,chemistry,physical chemistry,immunology and others.However,there are some disadvantages in these animal AA models.On the basis of clinical manifestations,peripheral blood cells,reticulocyte count,bone marrow nucleated cell count,pathology,lymphocyte subsets and immune function and other aspects from animal model,a comprehensive analysis of these four methods was taken,to find a new simple,effective and stable animal model of AA. Key words: Aplastic anemia; Animal model

Effect of Danggui Buxue Tang on immune-mediated aplastic anemia bone marrow proliferation mice

To investigate the pharmacological effects of Danggui Buxue Tang (DBT) on immune-mediated aplasia anemia mice. The model of immune-mediated aplasia anemia mice was induced by means of 60Co γ-ray irradiation and mixed cells of thymus and lymphnode of DBA/2 mice infusion through tail vein, the parameters tested indices were as following: blood picture, bone marrow nucleated cell count (BMNC), murine colony-forming unit-megakaryocytes (CFU-GM) of bone marrow cells, murine colony-forming unit-erthroid (CFU-E) and burst forming unit-erythroid (BFU-E). The results showed that DBT could not only withstand significantly decreation of blood cells by immune-mediated, but also stimulate on the growth of bone marrow colony cell and increase the weight of hemopoietic progenitor of bone marrow. Therefore, DBT had an obvious treat effect on immune-mediated aplasia anemia models mice.

Recombinant Human Thrombopoietin Promotes The Recovery Of Megakaryocyte Lineage In Patients With Severe Aplastic Anemia Receiving Immunosuppressive Therapy

▪ ObjectiveTo assess the effectiveness of recombinant human thrombopoietin (rhTPO) in severe aplastic anemia (SAA) patients receiving immunosuppressive therapy (IST). MethodsEighty SAA patients receiving IST during a period from January 2007 to December 2011 were included in this retrospective analysis. Thirty-two subjects also received rhTPO treatment (15,000 U, three times a week, subcutaneously). The remaining 48 patients did not receive rhTPO treatment. The choice of using (or not using) rhTPO was based on physician discretion, and more importantly, patient will (partly based on financial capability to afford the medication). rhTPO was discontinued when platelet count returned to normal range. Hematologic response, bone marrow recovery, transfusion interval (platelet or red-cells), and the time to transfusion-free status were compared. ResultAt 6th months after the treatment, hematologic response along at least one lineage was achieved in 65.6% of the subjects receiving rhTPO vs. 41.7% in those who did not receive rhTPO (p=0.04). Response rate of megakaryocyte and erythroid lineage at 3rd months was also higher in subjects receiving rhTPO than in those who did not (43.8% vs. 10.4%, p=0.001; 50.0% vs. 20.8%, p=0.006). The mean number of megakaryocyte per bone marrow slide was higher in subjects receiving rhTPO than those who did not (9.7±3.1 vs. 2.6±4.2, p=0.002) after three months. The percentage of nucleated erythroid cells in bone marrow was also higher in subjects receiving rhTPO after three months (22.2±13.2% vs. 13.6±13.9% in those who did not receive rhTPO; p=0.007). The percentage of reticulocytes in peripheral blood was higher in subjects receiving rhTPO (1.9±1.4% vs. 0.7±0.4% in those who did not receive rhTPO; p=0.001) after three months. Myeloid percentage in bone marrow did not differ at any time points (3, 6, or 9 months). The need for platelet transfusion was lower in subjects receiving rhTPO (transfusion interval: 13.8±14.3 vs. 6.9±5.2 and 26.3±28.9 vs. 15.7±13.1 days in those who did not receive rhTPO during the first three and six months, respectively; P=0.004, P=0.03). The need for red cell transfusion was also lower in subjects receiving rhTPO (interval: 32.5±22.0 vs. 11.9±7.2 days and 50.4±27.9 vs. 23.9±20.1 days during the first three and six months, respectively; p=0.001 P=0.009). Time to independence from platelet transfusion was significantly shorter in subjects receiving rhTPO (99.9±49.9 vs. 156.3±14.5 days in those who did not receive rhTPO; p=0.01). ConclusionrhTPO improves hematologic response and promotes bone marrow recovery in SAA patients receiving IST. Disclosures:No relevant conflicts of interest to declare.

Early Bone Marrow Examination, On The Fifth Day Of Induction For AML, Is Highly Predictive Of Response

▪Rapid eradication of blasts from peripheral blood during induction therapy in AML is associated with remission and overall survival. We prospectively evaluated the value of a bone marrow (BM) examination on day 5 of induction therapy in unselected newly diagnosed AML patients assigned for intensive induction. Seventy nine patients were enrolled during a 29-months period in six medical centers in Israel. BM blasts were evaluated by two independent observers and in most cases with the support of FACS analysis. Induction protocols administrated were 3+7 with daunorubicin 60 or 90 mg/m2 or clofarabine 40mg/m2 for five days [58 (73%), 10(13%) and 11(14%) patients, respectively]. Patients' median age was 59 (range 22-78) years. Among surviving patients the actual follow-up time was longer than 9 months in 25(48%) patients and longer than 12 months in 16 (30%) patients. Cytogenetic results are available for 70 patients [2(3%), 47(67) and 21(30%) with standard, intermediate and poor risk, respectively]. Flt3ITD mutation was present in 11(13.9%) patients and NPM1 mutation, in 12 who presented with normal karyotype and were negative for FLT3-ITD.Treatment protocol was not altered on the basis of day 5 BM examination which was repeated on day 12-14. Residual disease (>10% blasts) was detected and a re-induction protocol (repeat 3+7 at doses of 60 or 45mg/m2 at day 14 or clofarabine 30mg/m2 on day 21) was administered in 20(25%) patients. Overall, 50(72%) patients achieved remission (CR1). Post-remission therapies varied according to patient age and risk factors. Overall, 29(37%) patients underwent allogeneic stem cell transplantation. During the study period, 27(34.2%) patients succumbed, 5(6.3%) during induction, 8(10%) while in remission and 12(15%) following relapse.Day 5 blast counts higher than 10% of all BM nucleated cells predicted for the identification of residual leukemia on day 14 regardless of BM cellularity. Moreover, day 5 counts lower than 10% of all BM nucleated cells predicted for the achievement of CR1 with a single induction course. The 10% cutoff for BM day 5 blast count accurately identified these patients with a sensitivity of 80% and specificity of 74%. Kaplan Meir analysis demonstrated superior overall survival (OS) and lower relapse rate (figure 1a&b) of rapid responders than those with more than 10% blasts in day 5 BM. The 2-year anticipated OS is 80% and 35% (p=0.05), respectively. For the 50 patients who achieved CR1, the predicted 2-year relapse rate was as low as 8% for patients with a low day 5 BM blast count and 82% in the group of higher day 5 BM blasts (p=0.004). [Display omitted] Bone marrow day 5 aspiration was not always informative and in seven cases where biopsy was not available the blast content couldn't be assessed. Of the 72 patients with a valid day 5 blasts count, 24 (33%) were rapid responders in whom blasts were less than 10% of BM nucleated cells. On day 14, a lower than 10%, blast count was recognized in 54% of patients. However, 11/16 (69%) of patients who despite high blast counts at day 5 had a low count at day 14, either failed to achieve remission or relapsed. Only 5 patients presented with a low blast count at day 5 had a day 14 blasts count higher than 10%. Rapid response on day 5 was not associated with age or molecular status; yet, there was a trend (p=0.08) towards lower frequency of poor cytogenetics among these patients. Surprisingly, mean presenting WBC was higher among rapid responders. ConclusionBone marrow blast count on the fifth day of induction during chemotherapy is a powerful predictor of AML prognosis, irrespective of other pre-treatment risk factors. Larger studies, with longer follow up are required to determine its role in clinical management. Disclosures:No relevant conflicts of interest to declare.

Imatinib Inhibits SCF-Induced Development Of Human Mast Cells In Vitro and Induces Profound and Selective Mast Cell Deficiency In Patients With Ph+ CML

Although mast cells (MC) play an important role as effector cells of allergic reactions and may contribute to vascular repair processes after a thromboembolic event, their physiological role in healthy tissues is currently unknown. In mice, several ´MC-deficiency models´ have been developed. In these models, MC-deficiency is usually triggered by a lack of KIT or lack of a functional KIT-ligand, stem cell factor (SCF). Currently, no comparable human model is available. We examined the in vitro and in vivo effects of the KIT-targeting drug imatinib on growth and differentiation of MC. Imatinib was found to inhibit SCF-induced development of human MC from cord blood-derived progenitors in an in vitro long-term culture assay (100 ng/ml SCF, day 28). The effects of imatinib on MC differentiation in this assay were dose-dependent (IC50: 0.01 µM). Correspondingly, efficacious long-term treatment of CML patients with imatinib (400 mg daily) resulted in a profound reduction of MC numbers. Whereas no substantial decrease in the numbers of MC was seen within the first year of treatment, the numbers of tryptase+ and KIT+ MC in the bone marrow (BM) decreased to less than 5% of pre-treatment values in 23 patients who were in continuous complete molecular response (CMR) for at least 2 years during imatinib therapy (numbers of tryptase+ BM cells: pre-treatment values: 21.1±13.7% of nucleated BM cells vs CMR-values in the same patients: 0.5±0.6% vs normal BM: 3.8±1.6%; p<0.001). Tryptase mRNA levels and KIT mRNA levels in the BM also decreased significantly during treatment with imatinib in these patients (p<0.01). Moreover, during treatment with imatinib, serum tryptase concentrations decreased to low or even undetectable levels (before therapy: 32.0±11.1 ng/ml vs CMR: 3.4±1.8 ng/ml, p<0.01). Other myeloid lineages in the BM, known to develop independently of KIT, were not affected by imatinib therapy. Remarkably, imatinib-induced MC deficiency was not accompanied by any specific symptoms or side effects (no increased rate of bacterial infections, thromboembolic events or secondary neoplastic lesions). In a next step, we examined MC numbers in C57BL/6J mice and cellular histamine levels in BALB/c mice treated with imatinib. In these experiments, we found that treatment with imatinib (60 mg/kg/day vs control saline by intraperitoneal=i.p. injection over 42 days; 4 mice per group) leads to a significant decrease in the numbers of MC (skin MC numbers per high power field in control mice: 36.7±10.1 versus imatinib-treated mice: 15.7±4.6, p<0.001). MC numbers and cellular histamine levels in peritoneal samples of BALB/c mice treated with imatinib (50 mg/kg/day i.p. vs control saline; 3 mice each) also decreased substantially during treatment with imatinib (histamine levels on day 24: imatinib: 9.2±1.4 ng/106 cells vs control: 75.7±2.1 ng/106cells, p<0.001). In conclusion, our data show that imatinib produces a profound MC deficiency in mice and men, without causing a particular clinical condition or syndrome. Based on these observations, we hypothesize that MC may be less relevant in physiologic processes and tissue homeostasis in healthy organs than has so far been assumed. Part of the work, including data on mast cells in BM sections, has been presented at EHA 2013. However, in our current ASH abstract, we have included additional data on mast cell numbers and histamine levels in 2 different mouse models. These mouse data have not been presented at EHA or any other congress before. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.