Bone Marrow Nucleated Cells Research Articles

Hydroxyurea Therapy Reduces The Hypercoagulability State In a Sickle Cell Mouse Model

Several lines of evidence show that sickle cell anemia (SCA) is characterized by a hypercoagulable state. SCA patients present an elevated rate of thrombotic complications and increased biological markers of coagulation activation. In a previous study we demonstrated that therapy with hydroxyurea (HU) in SCA patients was associated with reductions in hypercoagulability markers, in addition to reductions in hemolysis, inflammation and endothelial activation markers (Colella et al Journal of Thrombosis and Haemostasis 2012). In the present study, we attempted to further investigate whether the effect of HU on hemostatic activation is dependent on fetal haemoglobin (HbF) expression, hemolysis and/or inflammation. To do so, we used the BERK murine model of SCA, a homozygous model of SCA, incapable of expressing HbF. In this BERK model, treatment with HU results in no improvement in hemolysis, due to the lack of HbF induction (Lebensburger et al Haematologica 2010). The murine SCA model was generated by transplantation of nucleated bone marrow cells from BERK mice into lethally-irradiated C57BL6 mice. Only mice expressing > 97% of human hemoglobin S (HbS) at 10 weeks after transplantation were used in the study. HU therapy at the dose of 50mg/kg (HU group) or saline alone (control mice) was administered through intraperitoneal injections 5 times per week, initiated at 11 weeks after transplantation (approximate time of sickle marrow engraftment). After a treatment period of 16 weeks, blood samples drawn from the inferior cava vein were collected and submitted to evaluation of plasma levels of thrombin-antithrombin III (TAT), a final marker of thrombin generation, in both animal groups. We also evaluated plasma levels of the inflammation marker interleukin 6 (IL6), and the endothelial marker soluble vascular cell adhesion molecule-1 (sVCAM-1). TAT, IL6 and sVCAM-1 were all measured by commercially-available ELISA kits. Statistical analyses were performed using Mann-Whitney's U test. Treatment with HU resulted in a significant reduction of TAT plasma levels (106.7 μg/L vs 138.5 μg/L; P = 0.05). Plasma levels of IL6 (7.1pg/mL vs 12.4pg/mL; P = 0.18) and sVCAM-1 (765.8 ng/mL vs 848.4 ng/mL; P= 0.43) presented non-significant reductions with HU treatment. Our results show that, in this murine SCA model, long-term HU treatment results in an improvement in the hypercoagulability state, even in the absence of HbF induction, or of a significant improvement of inflammation or endothelial activation. This indicates that HU may have a direct effect on inhibiting the activation of coagulation in SCA. Disclosures:No relevant conflicts of interest to declare.

Comparison of human mesenchymal stem cells derived from dental pulp, bone marrow, adipose tissue, and umbilical cord tissue by gene expression

Our aims were to characterize human mesenchymal stem cells isolated from various tissues by pluripotent stem cells gene expression profile. Four strains of dental pulp stem cells (DP-MSCs) were isolated from dental pulp tissue fragments adhered to plastic tissue culture dishes. Mesenchymal stem cells derived from umbilical cord tissue (UBC-MSCs) were isolated with the same technique. Bone marrow derived mesenchymal stem cells (BM-MSCs) were isolated from nucleated cells of bone marrow obtained by density gradient centrifugation. Human mesenchymal stem cells from adipose tissue (AT-MSCs) were isolated by collagenase digestion. All kinds of MSCs used in this study were cultivated in low glucose DMEM containing 5% or human platelet extract. All stem cell manipulation was performed in GMP conditions. Expression of 15 pluripotent stem cells genes on the level of proteins was measured by Proteome Profiler Human Pluripotent Stem Cell Array. Induction of MSCs to in vitro differentiation to adipocytes, osteoblasts, chondroblasts was achieved by cultivation of cells in appropriate differentiation medium. All MSCs tested were phenotypically similar and of fibroblastoid morphology. DP-MSCs and UBC-MSCs were more proliferative than bone marrow BM-MSCs and AT-MSCs. Protein expression of 15 genes typical for pluripotent stem cells distinguished them into two groups. While the gene expression profiles of BM-MSC, AT-MSCs and UBC-MSCs were similar, DP-MSCS differed in relative gene expression on the level of their products in several genes. Dental pulp mesenchymal stem cells cultivated in vitro under the same conditions as MSCs from bone marrow, adipose tissue and umbilical cord tissue can be distinguished by pluripotent stem cell gene expression profile.

Application of flow cytometry-fluorescence in situ hybridization in the measurement of relative telomere length of bone marrow CD34(+) cells in patients with myelodysplastic syndrome

This study was aimed to investigate the feasibility of flow cytometry-fluorescence in situ hybridization (Flow-FISH) in measuring the telomere length of bone marrow cell subgroups in patients with myelodysplastic syndrome (MDS). Seven newly diagnosed patients with low-risk MDS and seven nutritional anemia patients who were matched with age and sex, were enrolled in this study. Heparinized bone marrow were sampled. Taking Molt-4 cell line as internal control cells, leukocytes isolated from whole bone marrow were labeled with CD34-Alexa Fluork ® 647, then denatured by high temperature and hybridized with FITC-conjugated telomere probe. The DNA was counterstained and the relative telomere length (RTL) of nucleated cells and CD34(+) cells in bone marrow were measured by four-color flow cytometry. The results showed that CD34(+) cells could be gated for the measurement of RTL in both groups, undergoing the denaturation and hybridization. Primary analysis indicated that the RTL of bone marrow CD34(+) cells in MDS patients was significantly shorter than that of bone marrow nucleated cells (P = 0.001), and the RTL of both CD34(+) cells and nucleated cells in bone marrow of MDS patients were significantly shorter than that of control group (P = 0.020, 0.002). It is concluded that the application of Flow-FISH in the measurement of RTL of certain cell subgroup is feasible by labeling the cell with thermostable fluorescence-conjugated antibody, and this technique is worthy to be investigated further.

Oxidative stress leads to increased mutation frequency in a murine model of myelodysplastic syndrome

The myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it has been suggested that additional mutations lead to progression of MDS to AML, the causative agent(s) for such mutations remains unclear. Oxidative stress is a potential cause, therefore, we evaluated levels of reactive oxygen species (ROS) in NUP98-HOXD13 (NHD13) transgenic mice, a murine model for MDS. Increased levels of ROS were detected in bone marrow nucleated cells (BMNC) that express CD71, a marker for cell proliferation, as well as immature, lineage negative bone marrow nucleated cells from NHD13 mice. In addition to the increase in ROS, increased DNA double strand breaks and activation of a G2/M phase cell cycle checkpoint were noted in NHD13 BMNC. Finally, using an in vivo assay for mutation frequency, we detected an increased mutation frequency in NHD13 BMNC. These results suggest that oxidative stress may contribute to disease progression of MDS to AML through ineffective repair of DNA damage and acquisition of oncogenic mutations.

Clinical and Laboratory Profiles of 109 Patients diagnosed as Multiple Myeloma in Erbil City.

Background: Multiple myeloma is characterized by clonal expansion and accumulation of abnormal plasma cells in the bone marrow compartment. This study was conducted to determine the frequency and hematological presentation of multiple myeloma in Nanakaly Hospital in Erbil city.patients and Methods: This descriptive retrospective study was carried out at Nanakaly Hospital over a period of six years from January 2007 to August 2012. A total of 109 patients who were diagnosed with multiple myeloma all were included to analyze the clinical and laboratory profiles. All patients were selected based on preset diagnostic criteria by the WHO.Results: The study showed a male: female ratio of 1:1.1, the mean age of male patients was 57.8 years and for females were 57.9. Backache was the most frequent presentation (41.28%) of the patient then bone pain (33.94%), followed by pallor (10.1%) and bone mass (9.2%). The hematological findings showed anemia in 72% patients. Forty five percent of the bone marrow nucleated cells were plasma cells.Conclusions: Males are affected equally to females by multiple myeloma. Majority of patients present with backache, bone pain and anemia.

Anti-radiation effect of resveratrol

To explore the anti-radiation protective effect of resveratrol (RES). (60)Co-γ irradiated injury model was established. A total of 200 Kunming mice were randomly divided into 4 groups (50 in each group): Group I, II, III, and IV. Each group was sub-divided into 5 groups: a normal control (n=10), an irradiated model control group (n=10) and 3 treatment groups of RES (50, 100, and 300 mg/kg RES treatment groups, 10 in each group). RES was orally administered daily for 30 d in the RES treatment groups and 1% sodium carboxymethylcellulose was orally administered in the normal control and irradiated model group. Thereafter, except the normal control group, the mice in other groups were exposed to different dosages of (60)Co-γ once, and the gavage was continued until the end of different experimental periods. Peripheral leucocytes, nucleated bone marrow cells were counted; superoxide dismutase (SOD) activity and hemolysin in the serum were determined at different time. Under the different dosages of (60)Co-γ irradiation and the provisions of the experimental conditions, the leucocyte count was (1.69±0.82)× 10(9) and (1.61±0.51)× 10(9)/L in the 100 and 300 mg/kg RES treatment groups, which was significantly increased, when compared with the irradiated model control group [(0.73±0.69)× 10(9)/L] ( P<0.05, P<0.01 respectively). The number of nucleated bone marrow cells was (17.5±4.8) and (17.1±4.7)× 10(5)/mL in the 100 and 300 mg/kg RES treatment groups respectively, which significantly increased when compared with the irradiated model control group [(7.3±2.2)× 10(5)/mL ] ( P<0.01 ). The SOD activity was (110.41±17.04) U/ mL in the 100 mg/kg RES treatment group, which was significantly increased when compared with the irradiated model control group [(95.80±10.42) U/mL ] ( P<0.05 ). There was no significant difference in the serum hemolysin in all RES treatment groups (all P>0.05). At 100 and 300 mg/kg, RES has good anti-radiation effect.

P-250 Nucleophosmin cytoplasmic and nucleus immunohistochemical cell positivity of bone marrow biopsy sections in AML and MDS patients

Background Nucleophosmin (NPM) is phosphoprotein in nucleolus and shuttles continuously between nucleus and cytoplasm. Major role of NPM is to mediate nuclear export of ribosome components to the cytoplasm and to control centrosome duplication and it also interacts with oncosupressors p53 and p19Arf thus controlling cell proliferation and apoptosis. NPM mutations are involved in pathogenesis of some AML cases. Introduction The abnormal mutated NPM1 protein shows an aberrant cytoplasmic localization in leukemic cells (NPMc), whereas the wild-type protein is mainly located in the nucleolus and on the nuclear membrane. Approximately one-third of cases of denovo acute myeloblastic leukemia (AML) carry mutations in the c-terminal region of NPM (NPM1) gene. NPM1 mutations are rare in myelodisplasia (MDS) but in some reports NPM mutations and NPMc cell bone marrow cell positivity were also found in MDS. Purpose Aim of the study were to analyze cell NPM immunohistochemical expression in nuclei (NPMn) and cytoplasm (NPMc) in bone marrow (BM) sections of 7 patients with de novo AML and 9 MDS patients. Materials and Methods Immunohistochemical procedures were performed on paraffin sections cut from B5-fixed/EDTA-decalcified BM biopsies. Sections were subjected to micro waving/antigen retrieval, pH9 and immunostained with anti-NPM monoclonal antibody (clone 376) using immuno alkaline phosphatase technique. NPMc and NPMn cell positivity between AML and MDS patients were statistically compared with Mann-Whitney and Chi-square tests. From patients informed consent was obtained. Results NPMn cell positivity was seen in 6/7 AML and in 8/9 MDS patients. NPMc was detected in 5/7 AML and in 8 out of 9 MDS patients. Percentages of NPMn of BM cells were higher in MDS than in AML patients with borderline statistical significance. NPMn immunoexpression above 30% of BM positive cells was more frequently (with statistical significance) observed in MDS than in AML patients (p< 0.05). Conclusions Higher NPMn cell positivity (above 30% of nucleated BM cells) in MDS is probably linked to higher number of normal BM cells in MDS. Lower NPMn cell positivity in AML is maybe associated to higher number of leukemic cells and also to NPM functional loss in progression and transformation of MDS to AML. NPMc was also observed in most of our AML and MDS patients. This finding is in a concordance with some recent reports also implicating that that NPM1 mutations may be also seen in MDS, but further studies are needed to analyze correlation of NPMc cell positivity and NPM1 mutations in MDS patients

Combined flow cytometric assessment of CD45, HLA‐DR, CD34, and CD117 expression is a useful approach for reliable quantification of blast cells in myelodysplastic syndromes

Quantification of bone marrow (BM) blasts by cytomorphology is essential for the diagnosis of myelodysplastic syndromes (MDS). Owing to its subjectivity and the potential impact of dysplastic features on accurate identification of blast cells, more objective approaches are required, multiparameter flow cytometry (MFC) being a particularly promising approach in this regard. However, no consensus exists about the optimal combination of markers and strategy to be used. BM blast counts from 74 MDS patients were evaluated by morphology versus four different MFC phenotypic criteria: "CD34⁺", "CD34⁺ and/or CD117⁺", "CD34⁺, and/or CD117⁺ HLA-DR⁺", and "CD34⁺ and CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-/lo) " cells. For each criterium, the percentage of blasts was calculated using either all BM nucleated cells or non-erythroid CD45⁺ cells as denominator. RESULTS.: The number of "CD34⁺ and/or CD117⁺ HLA-DR⁺"cells showed the highest correlation and agreement with morphological counts, only a minor proportion of cases being misclassified by MFC vs. morphology for the >5% and >10% classification thresholds. In turn, a CD34⁺ phenotype was insufficient to correctly identify and quantify blasts. Conversely, usage of non-erythroid BM cells as denominator, or inclusion of "CD34⁺ and/or CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-lo") cells were both associated with overestimated blast counts. Quantification of "CD34⁺ and/or CD117⁺ HLA-DR⁺" cells (from all nucleated BM cells) by MFC is an efficient method for the enumeration of blasts in MDS. However, caution should be taken with replacing morphology by MFC blast counts; its combined use may rather provide complementary information increasing the accuracy and reproducibility of BM blast cell counts in these patients.

A Retrospective Analysis of Cytogenetic and Clinical Characteristics in Patients With Multiple Myeloma

BackgroundCytogenetic alterations in patients with multiple myeloma (MM) represent important risk factors in terms of prognosis. In this study, the impact of the cytogenetic aberrations of MM on patient clinical features and outcome was investigated. MethodsConventional cytogenetic analysis with R-banding technique and molecular cytogenetic characterization by interphase fluorescence in situ hybridization (FISH) were used to detect aberrant chromosomal arrangements, including 17p13 and 13q14 deletions, 14q32 rearrangement and 1q21 amplification, in bone marrow nucleated cells from 65 patients. ResultsAbout 16.9% of patients showed aberrations by conventional cytogenetic analysis, whereas 49.2% of patients showed aberrations by interphase FISH analysis. Abnormalities of 13q14, 1q21, 14q32 and 17p13 were detected in 27.7%, 13.8%, 16.9% and 29.2%, respectively. Patients with a 13q14 deletion or combined with 17p13 deletion frequently had a late stage of the disease, and tended to have elevated serum levels of β2 microglobulin and lower levels of albumin. The progression-free survival and overall survival of FISH-positive patients were lower than for those without detectable abnormalities, especially in the conventional chemotherapy arm. ConclusionsThese findings demonstrate that myeloma cells are prone to exhibiting a complex aberration and that FISH is superior to conventional cytogenetic analysis with a higher detection rate of chromosomal abnormalities. Patients with a 17p13 or 13q14 deletion, 14q32 rearrangement and 1q21 amplification were more likely to have a poor prognosis for MM.

Expressing trend of NME3 protein in acute myeloid leukemia HL-60 cells and patients' bone marrow

To verify the differential expression of non-metastasis cell 3 (NME3) protein in HL-60 cells when they were induced to differentiate into monocyte and granulocyte like cells, and study its value in diagnosis of acute myeloid leukemia, all-trans retinoic acid (ATRA) and a new steroidal drug NSC67657 were employed to induce acute myeloid leukemia HL-60 cells into monocyte and granulocyte like cells. Then the cell differentiating direction was observed by chemical staining, the degree of differentiation was determined by surface antigen CD11b/CD14 detection, and the apoptosis was excluded by phosphatidylserine valgus analysis, by which cellular differentiating model was constructed. Furthermore, RT-PCR and Western blot were employed to verify the differentially expression of NME3 before and after differentiation of HL-60 cells. At last, samples from bone marrow nucleated cells of 26 patients with myeloid leukemia, which were diagnosed definitely by clinical doctors, and 5 normal people were chosen. Then the expressing trend of NME3 protein in these testing groups was analyzed by means of comparison. The results showed that ATRA (2 µmol/L for 5 d) and NSC67657 (10 µmol/L for 5 d) could induce HL-60 cells to differentiate into monocyte and granulocyte like cells above 90% without cell apoptosis. The expression of NME3 gene and protein were down-regulated by the inducers, which was accorded with the screening results that was got using proteomics technology in the former research. The expression of NME3 protein in bone marrow from acute myeloid leukemia patients was elevated significantly as compared to normal persons. It is concluded that the expression level of NME3 protein is down-regulated after cellular differentiation, according with the changing trend in leukemia patients, which imply that NME3 protein may be a potential biomarker for diagnosis of acute myeloid leukemia.

Enhanced hematopoietic protection from radiation by the combination of genistein and captopril

The hematopoietic system is sensitive to radiation injury, and mortality can occur due to blood cell deficiency and stem cell loss. Genistein and the angiotensin converting enzyme (ACE) inhibitor captopril are two agents shown to protect the hematopoietic system from radiation injury. In this study we examined the combination of genistein with captopril for reduction of radiation-induced mortality from hematopoietic damage and the mechanisms of radiation protection. C57BL/6J mice were exposed to 8.25Gy 60Co total body irradiation (TBI) to evaluate the effects of genistein and captopril alone and in combination on survival, blood cell recovery, hematopoietic progenitor cell recovery, DNA damage, and erythropoietin production. 8.25Gy TBI resulted in 0% survival after 30days in untreated mice. A single subcutaneous injection of genistein administered 24h before TBI resulted in 72% survival. Administration of captopril in the drinking water, from 1h through 30days postirradiation, increased survival to 55%. Genistein plus captopril increased survival to 95%. Enhanced survival was reflected in a reduction of radiation-induced anemia, improved recovery of nucleated bone marrow cells, splenocytes and circulating red blood cells. The drug combination enhanced early recovery of marrow progenitors: erythroid (CFU-E and BFU-E), and myeloid (CFU-GEMM, CFU-GM and CFU-M). Genistein alone and genistein plus captopril protected hematopoietic progenitor cells from radiation-induced micronuclei, while captopril had no effect. Captopril alone and genistein plus captopril, but not genistein alone, suppressed radiation-induced erythropoietin production. These data suggest that genistein and captopril protect the hematopoietic system from radiation injury via independent mechanisms.

Accumulation of prelamin A compromises NF-κB-regulated B-lymphopoiesis in a progeria mouse model.

BackgroundAlteration in the immune system is one of the most profound aspects of aging. Progressive changes in the number of B lymphocyte progenitors during aging have been reported but the underlying mechanisms are still elusive. A heterozygous G608G mutation in the LMNA gene leads to a deletion of 50 amino acids in lamin A protein, termed progerin, and is the predominant cause of Hutchinson-Gilford progeria syndrome (HGPS). Lack of Zmpste24, a metalloproteinase responsible for prelamin A processing, leads to progeroid features resembling HGPS. Therefore Zmpste24-deficient mice provide an ideal mouse model to study the impact of lamin A and (premature) aging on the aging-related decline of B lymphopoiesis.ResultsAnalysis of bone marrow (BM) nucleated cells revealed a decline of early B cell progenitors in Zmpste24−/− mice. BM transplantation in a congenic strain completely rescued the defects in B lymphopoiesis, indicating that the decline in B cell progenitors in Zmpste24−/− mice is attributable to defective BM microenvironments rather than to cell-intrinsic defects. Further investigation revealed downregulation of a set of important early B lymphopoiesis factors in Zmpste24−/− bone marrow stromal cells (BMSCs), such as Vcam-1, SDF-1α, Flt3L and TSLP, and most of them are under transcriptional control of NF-κB signaling. Though TNFα stimulates IκBα degradation and NF-κB nuclear translocation in Zmpste24−/− BMSCs, NF-κB fails to stimulate IκBα re-expression, which mediates a negative feedback loop of NF-κB signaling in wild-type BMSCs.ConclusionsOur data demonstrate a cell-extrinsic defect of B cell development in a progeroid mouse model and a critical role for lamin A in the regulation of NF-κB signaling and cytokines that are essential for lymphopoiesis.

Cyclophosphane effect on peripheral blood leukocytes and organs of immune system in experimental animals

Event Abstract Back to Event Cyclophosphane effect on peripheral blood leukocytes and organs of immune system in experimental animals Anatoliy P. Godovalov1*, Olga V. Lebedinskaya2, Elena A. Lebedinskaya2, Dmitriy V. Suvorov2, Alexandra N. Razdobreeva2 and Andrey V. Khorinko2 1 Acad. E.A. Wagner Perm State Medical Academy, immunology, Russia 2 Acad. E.A. Wagner Perm State Medical Academy, histology, embriology and cytology, Russia The aim of investigation was the analysis of cyclophosphane (CP) effect on the immune system organs, on peripheral blood leukocytes in experimental animals. The study was conducted in 20 male rats (284±20 g). CP was administered intraperitoneally at a concentration of 100 mg/kg of body weight, 4 fold with 24h interval. Blood samples were taken from the tail vein to determine the total leukocyte count and leukocyte formula. Pathomorphological changes in bone marrow, thymus, spleen and lymph nodes were analyzed through 48 h after final CP injection. It was shown that leukocyte number reduced by 50% on the 2nd day after the first CP injection, and by 99% - in subsequent two days. After the final CP injection the recovery of initial leukocyte number was not detected. Following the first CP introduction there was noted statistically significant increase in segmented neutrophils that were sharply declined after the 2nd cytostatic injection and were not further recovered. The amount of monocytes and lymphocytes was lowered 24h after the first injection of cyclophosphane and subsequently continued to reduce. The alteration in the functional activity was determined in phagocytic cells. The statistically significant reduction in thymus, spleen and lymph node weight, was detected, as well as degenerative changes therein and decrease nucleated cells in bone marrow. Thus as a result of investigation the development of induced immunosuppression was revealed that involved the lowering in blood leukocyte count, alteration of their composition and functional activity, progression of degenerative changes in the organs of immune system. Keywords: cyclophosphane, Immunosuppression, degenerative changes, phagocytic cells, leukocyte formula Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Godovalov AP, Lebedinskaya OV, Lebedinskaya EA, Suvorov DV, Razdobreeva AN and Khorinko AV (2013). Cyclophosphane effect on peripheral blood leukocytes and organs of immune system in experimental animals. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00593 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Mr. Anatoliy P Godovalov, Acad. E.A. Wagner Perm State Medical Academy, immunology, Perm, Russia, AGodovalov@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Anatoliy P Godovalov Olga V Lebedinskaya Elena A Lebedinskaya Dmitriy V Suvorov Alexandra N Razdobreeva Andrey V Khorinko Google Anatoliy P Godovalov Olga V Lebedinskaya Elena A Lebedinskaya Dmitriy V Suvorov Alexandra N Razdobreeva Andrey V Khorinko Google Scholar Anatoliy P Godovalov Olga V Lebedinskaya Elena A Lebedinskaya Dmitriy V Suvorov Alexandra N Razdobreeva Andrey V Khorinko PubMed Anatoliy P Godovalov Olga V Lebedinskaya Elena A Lebedinskaya Dmitriy V Suvorov Alexandra N Razdobreeva Andrey V Khorinko Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Spleen-Specific Development of Germinal Centers in Rats Treated with Antithyroid Drugs

The antithyroid drugs (ATDs) methimazole (MMI) and propylthiouracil (PTU) have been used for treatment of hyperthyroidism for more than several decades, despite the fact that they are associated with adverse drug reactions that are thought to be autoimmune mediated. We therefore examined histopathologic responses in the immune system in male and female rats given MMI (2, 20 and 200 mg/kg/day, p.o., in experiment 1; 200 mg/kg/day, p.o., in experiment 3) or PTU (25 and 250 mg/kg/day, p.o., in experiment 2; 200 mg/kg/day, p.o., in experiment 3) for two weeks. In experiments 1 and 2, highest doses of MMI and PTU induced histopathologic changes in the spleen consistent with those in experiment 3 without any changes in the other peripheral lymphoid organs and tissues. In experiment 3, histopathological evaluation of the spleen along with hematological and bone marrow examinations were performed. In both male and female rats, MMI or PTU induced histopathological changes in the spleen characterized by development of germinal centers and an increase in the number of IgG-positive plasma cells in the red pulp; these changes were most prevalent in the MMI-treated female rats. Total red and white blood cell counts were decreased in the MMI-treated male and female rats; lymphocytes and monocytes were lower in male and female rats, respectively. Bone marrow nucleated cells were significantly lower in the MMI-treated males. This is the first study to demonstrate that ATDs induce spleen specific B-cell reactions in rats

Development of a Highly Efficient Method for Isolating Very Small Embryonic-Like Stem Cells Identified in Adult Mouse Bone and Their Stem Cell Characteristics.

Abstract 2345 Background:Ratajczak and his colleagues identified a unique population of very small embryonic-like (VSEL) stem cells in adult mouse bone marrow (BM) (Leukemia 2006:20;857). These VSELs are; 1) very small (∼4 μm); 2) express pluripotent stem cell markers, such as Oct4, Nanog, SSEA-1, and Rex-1; 3); responsive to a SDF-1 gradient; 4) possess large nuclei that contain euchromatin. It is very interesting to note that VSELs possess the potential to differentiate into 3 germ layers in vitro and in vivo, thereby contributing to tissue/organ regeneration. These VSELs were isolated as lineage-negative (Lin−), Sca-1-positive (Sca-1+), CD45-negative (CD45−) cells by FACS. However, the incidence of VSELs in BM-derived mononuclear cells is ∼0.01%. Therefore, it is difficult to isolate VSELs very effectively. This study describes our recently developed highly efficient method for isolating VSELs using enzymatic treatment of murine bone. Materials and Methods:Murine BM nucleated cells (BMNC) were isolated from BM flushed from the pairs of femurs and tibiae of 8 week-old C57BL/6 mice. Erythrocytes were removed using a hypotonic solution. Then the remaining bone tissues were thoroughly washed using PBS- with 2% FCS. These bone tissue specimens were crushed in a mortar and then incubated in cell dissociation buffer containing a-medium with 5% FCS supplemented with 1.5 mg/ml type I collagenase and 2 mg/ml dispase at 37°C for 1 hour. Next, the BMNCs and bone-derived nucleated cells (BDNCs) were stained with various monoclonal antibodies, including anti-lineages, anti-CD45, anti-Sca-1, anti-CXCR4, anti-CD133, and anti-PDGFRα, and then were used for subsequent FACS analyses. Results:The R1 gate was set on the FSC channel using 4 and 10 μm synthetic beads, based on the predicted very small size of VSELs. The VSELs were isolated from BMNCs and BDNCs by multicolor FACS, as a population of Lin−Sca-1+CD45− cells (Fig. 1A). The incidences of VSELs in the BMNCs and BDNCs were 0.001% and 0.1%, respectively. Therefore, the enzymatic treatment of bone tissues yielded about 100 times the efficiency for the isolation of VSELs (Fig. 1B). The bone-derived (BD) VSELs were small (< 5 μm) and possessed a relatively large nucleus surrounded by a narrow rim of cytoplasm. They expressed CD133, but not PDGFRα. However, they weakly expressed CXCR4. The gene expression profiles were analyzed using real time quantitative PCR (RQ-PCR) to evaluate the expression of ES cell markers (Oct4, Nanog, Rex1, Dppa3), HSC (KSL) markers (c-kit, Tal1, GATA2), and MSC markers (Nestin, Ang1, CXCL12, VE-Cadherin). Unexpectedly, BD VSELs expressed high levels of Nestin and Cadherin. However, they expressed weak levels of Oct4 and Nanog. The gene expression profile of the BD VSELs was clearly distinct from the well-defined populations of ES cells, KSL cells, and MSCs. Interestingly, the number of these BD VSELs significantly increased after the induction of liver injury by carbon tetrachloride administration. They were then most likely mobilized into the peripheral blood (PB). G-CSF did mobilize KSL cells into PB, as previously reported. However, G-CSF did not mobilize the BD VSELs. The effects of sRANKL on the mobilization of BD VSELs were examined in vivo. Interestingly, the number of BD VSELs significantly increased 2–3 days after the administration of sRANKL. However, the number of VSELs in PB did not increase. These results suggest that BD VSELs actively proliferated after liver injury and bone resorption. Conclusion:The present data suggest that the majority of the Lin−Sca-1+CD45− cells reside in the bone tissue. BD VSELs resemble BM-derived VSELs. However, a RQ-PCR analysis revealed that the gene expression profile of BD VSELs was different from those of the previously reported BM-derived VSELs. Further studies will therefore be required to elucidate their stem cell characteristics and the potential relationship between BD VSELs and BM-derived VSELs. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Residual Disease Detection by Flow Cytometry Predicts Risk of Relapse and Overall Survival in Patients with Acute Myeloid Leukemia Following Reduced Intensity- and Myeloablative- Allogeneic Hematopoietic Cell Transplantation

AbstractAbstract ▪4098▪This icon denotes a clinically relevant abstractRelapse after allogeneic hematopoietic cell transplantation (HCT) is usually incurable for acute myeloid leukemic (AML) patients. Residual disease (MRD) monitoring pre- and post-HCT may improve relapse prediction, allowing the targeted implementation of post-HCT interventions to at risk patients when disease burden is sufficiently low for these to be effective. Previous studies have shown that MRD detection by multiparameter flow cytometry (MRD MFC) at either pre- (Leung et al 2012, Walter et al 2011) or post- transplant (Yan et al 2012) timepoints is prognostic for myeloablative HCT outcome in AML. Quantification of hematopoietic populations enriched for leukemic stem cells such as CD34+CD38low or lymphoid-primed multi-potential progenitor-like (LMPP-like) (Lin-CD34+CD38lowCD90-CD45RA+) (Goardon et al 2011) may improve the specificity of MFC MRD assays.In this study we retrospectively evaluated the predictive value of MRD MFC at both pre- and post- HCT timepoints in a cohort of unselected AML/high risk myelodysplasia (MDS) patients (n=44) who underwent reduced intensity conditioning (RI n= 32, median age 59, range 34–70) or myeloablative (MA n=12, median age 28, range 19–47) HCT between June 2010 and November 2011 (Table 1). MFC MRD was assessed both by detection of standard leukemic- aberrant-immunophenotypes (LAIPs) (identified at presentation and/or relapse) and quantification of CD34+CD38low and LMPP-like progenitors (LSC-enriched progenitors, LSC-EP).Pre HCT, 37 patients (MA = 11, RI = 26) were assessable for MFC MRD. 15 (41%) were MRD positive (LAIP MRD+) and 47% (7/15) (MA = 37.5%, 3/8; RI = 57%, 4/7) of these relapsed post HCT compared to 8% (MA = 0%, RI = 9%) of MRD negative patients (LAIP-MRD-), (Fig 1 p 0.03).Post HCT, 34 patients (MA = 9, RI =25) were assessable for MFC MRD. 10 (37%) had detectable LAIP MRD positivity between 2 and 9 months post HCT. 80% of these relapsed (MA = 60%; RI = 100%) with a median disease free survival (DFS) post HCT of 5 months (MA = 4 months; RI = 5 months); there were no relapses in the 17 patients who remained LAIP MRD- at a median follow-up of 20 months (range 9–26). (p=0.0006, Figure 2a). Presence of MRD post-transplant was associated with significantly poorer overall survival (p=0.005).Although 1 patient with high MRD (in CR, LAIP >1%, LSC-EP +) pre HCT relapsed <3 months post HCT, in 8 of 9 other relapses MRD positivity detected at ≥ 2 months post HCT preceded clinical relapse by >1 month (median time to relapse from MRD detection of 1.5 months, range 1–6; OS, median 4 months, range 1 - not reached).CD34+CD38low progenitors (34+38low) were < 0.03% of bone marrow nucleated cells in the majority of patients. Detectable 34+38low were mainly CD45RA+ so in most cases correlated with LMPP-like quantitation. Pre HCT, 34+38low were detectable in 40% of patients who went on to relapse and in only 9% of those who have not yet relapsed. Post HCT, 34+38lowpositivity preceded frank relapse by ≥1 month in 60% of patients who relapsed. Only 6% of patients who have not yet relapsed had detectable 34+38low. LSC-LEP positivity appears prognostic for DFS and OS (Figure 2b) but for a lower frequency of relapses compared to LAIP MRD positivity (60% v 80%). Conclusions:These data suggest that post HCT MFC detection of LAIP MRD is predictive of relapse in RI as well as MA HCT. LSC-LEP quantitation may be prognostic in a subset of patients. Pre HCT MRD might be more predictive of relapse in RI than MA HCT. However, post HCT MRD positivity precedes most clinical relapses by a time window which may be sufficient for interventions such as azacytidine or donor lymphocyte infusion (DLI) when disease burden is still low. These results provide a basis for the use of MFC residual disease detection pre and post HCT to inform treatment decisions in reduced intensity as well as myeloablative HCT.Table 1Patient DemographicsTotalLAIP MRD pre HCTLAIP MRD post HCT+ve-ve+ve-veNo. of patients4415141017Median Age (range)55 (19–70)41(19–66-)60 (26–66)44(19–66)61(20–66)SexFemale185537Male26109710Type of HCTReduced intensity32812514Myeloablative127253Remission status pre HCTCR1309121013CR2/CR3135203Refractory11001CytogeneticsFavourable11001Intermediate35713713Adverse87133 [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Effector CD4+CD45RA−CD25brightFoxp3bright Regulatory T Cell (eTreg) Distribution Is Significantly Impaired in Chronic Myelomonocytic Leukemia (CMML) and Correlates with TET 2 Mutational Status.

AbstractAbstract 2808 Background:Naturally occurring CD4+CD45RA+CD25highFoxp3+ regulatory T cells (rTregs) and in particular the highly suppressive effector CD4+CD45RA−CD25brightFoxp3bright Tregs (eTregs) are critical to limit impaired immune responses. On the other hand, in advanced stages of cancers, increased numbers of Tregs are found in the tumor microenvironment, where they probably suppress antitumor immune responses. Likewise, expansion of Tregs in the bone marrow (BM) and peripheral blood (PB) has been shown in high risk MDS and AML (Kordasti, Blood, 2007). CMML, the most frequent MDS/MPN, displays both MDS and MPN features, has heterogeneous but generally poor prognosis and is characterized by a large number of somatic gene mutations, the most common being those of ASXL1 and TET2 genes. While ASXL1 mutations confer poor prognosis (Gelsi-Boyer, Br J Haematol, 2010) the prognostic impact of TET2 mutations is more controversial. Here we report that distribution of eTreg is impaired among patients with CMML and that high eTREG counts are associated with TET2 mutations. Methods:Between February 2011 and July 2012, 33 CMML patients and 25 healthy controls were studied. PBMC were purified from blood samples on Ficoll-Hypaque gradients to determine populations of resting CD4+CD45RA+FoxP3+CD25+ Tregs (rTregs) and effector CD4+CD45RA−CD25brightFoxp3bright Tregs (eTregs) by flow cytometry. For CMML patients DNA was extracted from total BM nucleated cells or PB CD14+monocytes obtained by immunomagnetic selection and subjected to PCR analysis for target genes. Results:Median age of the 33 patients analyzed was 75 years (range 47–88), M/F: 20/13. Twenty nine pts (87%) had CMML 1 and 4 (13%) had CMML 2. Median WBC count was 8.4 G/l (range 3.1–39.4), median blood monocytes 1.9 G/l (range 1–9.3), and median BM blasts 5% (range 2–22). Twenty four (73%) pts had WBC<13G/l and 9 (27%) WBC≥13G/l. Eight (23%) pts had splenomegaly (SMG) and 2 (6%) had extra medullary disease (EMD) (skin involvement exclusively). Abnormal karyotype was found in 11 (33%) pts, including 3 isolated +8, 3 isolated -Y, but no −7 or complex abnormality. In 29 pts screened for gene mutations, 16 (55%) TET2, 10 (35%) SRSF2, 7 (24%) ASXL1, 3 (10%) JAK2, 3 (10%) SF3B1, 3 (10%) ZRSR2, 3 (10%) CBL, 2 (6%) IDH2, 2 (6%) RAS, 2 (6%) RUNX1, 1 (3%) FLT3, and 1 (3%) DNMT3a mutations were found. No pt received cytoreductive treatment before Treg analysis.Overall median numbers of eTregs (1.97% (range 0–11.8%) vs 1.81% (0.4–3.76%), p= 0.96) and rTregs (0.79% (0.1–9.32%) vs 0.94% (0.15–2.33%), p=0.75) did not significantly differ in CMML pts and controls. Nevertheless, distribution of eTregs was significantly impaired among CMML pts compared to controls with 9 pts vs 1 control having <1% eTreg and 9 pts vs 2 controls having >3% eTreg (Table 1). Interestingly, eTegs were significantly increased among the 16 pts with TET2 mutations (2.95% (0.82–11.8%) vs 1% (0–4.8%), p=0.0057), potentially increased in SRSF2 mutated pts (3.05% (0–11.8%) vs 1.86% (0.3–5.71%) p=0.12) and similar in pts carrying ASXL1 mutations (2.23% (0–5.33%) vs 2.03% (0.3–11.8%), p=0.82). Impairment of eTreg distribution was not correlated with WBC count, PB monocytosis, Hb, plt count, BM blast %, karyotype, SMG, EMD and survival. By contrast, rTregs were distributed in CMML patients as in controls. Conclusion:Our analysis shows for the first time significantly impaired distribution of activated Tregs in CMML pts, and particularly in CMML with TET2 mutation, without association to specific prognostic factors or survival. As clonal TET2 mutations also occur in lymphocytes of MDS and CMML, our results may suggest the existence of a control loop connecting myeloid and regulatory T cell lineages, which needs further investigation. Treg differentiation and function are influenced by Foxp3 promoter methylation. Therefore generalized hypomethylation secondary to loss of TET2 function in stem cells could result in Treg expansion, which in turn could impact myeloid differentiation and/or function. In any case, Treg expansion may participate in the state of immunosuppression associated to a specific group of CMML.Table 1:Distribution of Treg populations in CMML pts and controls.RangeCTRL/NoCMML/NoPCD4+CD45RACD25brightFoxp3brightTregs (eTregs)0–1%190.0041–3%2215>3%29CD4+CD45RA+CD25highFoxp3+Tregs (rTregs)0–1%14210.3951–4%1110>4%02 Disclosures:Fenaux:Novartis: Honoraria, Research Funding.

Detection of disseminated tumor cells in the bone marrow of breast cancer patients using multiplex gene expression measurements identifies new therapeutic targets in patients at high risk for the development of metastatic disease

Disseminated tumor cells (DTCs) detected in the bone marrow (BM) of breast cancer patients identify women at high risk of recurrence. DTCs are traditionally detected by immunocytochemical staining for cytokeratins or single gene expression measurements, which limit both specificity and sensitivity. We evaluated the Nanostring nCounter™ platform for multi-marker, gene expression-based detection and classification of DTCs in the BM of breast cancer patients. Candidate genes exhibiting tumor cell-specific expression were identified from microarray datasets and validated by qRT-PCR analysis in non-malignant human BM and identical samples spiked with predefined numbers of molecularly diverse breast tumor cell lines. Thirty-eight validated transcripts were designed for the nCounter™ platform and a subset of these transcripts was technically validated against qRT-PCR measurements using identical spiked BM controls. Bilateral iliac crest BM aspirates were collected and analyzed from twenty breast cancer patients, prior to neoadjuvant therapy, using the full 38-gene nCounter™ code set. Tumor cell-specific gene expression by nCounter™ was detected with a sensitivity of one cancer cell per 1 × 10(6) nucleated BM cells after optimization. Measurements were quantitative, log linear over a 20-fold range, and correlated with qRT-PCR measurements. Using the nCounter™ 38-gene panel, 6 of 8 patients (75 %) who developed metastatic disease had detectable expression of at least one transcript. Notably, three of these patients had detectable expression of ERBB2 in their BM, despite the fact that their corresponding primary tumors were HER2/ERBB2 negative and therefore did not receive trastuzumab therapy. Four of these patients also expressed the PTCH1 receptor, a newly recognized therapeutic target based on hedgehog signaling pathway inhibition. The presumptive detection and classification of DTCs in the BM of breast cancer patients, based on sensitive and quantitative multi-marker detection of gene expression using the nCounter™ platform, provide an opportunity to both predict early distant recurrence and, more importantly, identify opportunities for preventing the spread of disease based on the expression of unique, therapeutically actionable gene targets. This study demonstrates the application of a new technology for multiplexed gene expression-based detection of DTCs in the BM of breast cancer patients and identifies at least two therapeutically targetable genes that are frequently expressed in the BM of patients who develop metastatic disease.

MYELOID CELL 11β-HSD1 REGULATES THE INFLAMMATORY RESPONSE DURING MYOCARDIAL INFARCT HEALING AND PROTECTS THE HEART FROM DETRIMENTAL REMODELLING AFTER IRRADIATION AND BONE MARROW TRANSFER

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is responsible for intracellular regeneration of glucocorticoids. 11β-HSD1-deficient mice exhibit enhanced angiogenesis, inflammatory cell recruitment and heart function after myocardial infarction (MI). This study aimed...

Is CD34 truly a negative marker for mesenchymal stromal cells?

The prevailing school of thought is that mesenchymal stromal cells (MSC) do not express CD34, and this sets MSC apart from hematopoietic stem cells (HSC), which do express CD34. However, the evidence for MSC being CD34(-) is largely based on cultured MSC, not tissue-resident MSC, and the existence of CD34(-) HSC is in fact well documented. Furthermore, the Stro-1 antibody, which has been used extensively for the identification/isolation of MSC, was generated by using CD34(+) bone marrow cells as immunogen. Thus, neither MSC being CD34(-) nor HSC being CD34(+) is entirely correct. In particular, two studies that analyzed CD34 expression in uncultured human bone marrow nucleated cells found that MSC (BMSC) existed in the CD34(+) fraction. Several studies have also found that freshly isolated adipose-derived MSC (ADSC) express CD34. In addition, all of these ADSC studies and several other MSC studies have observed a disappearance of CD34 expression when the cells are propagated in culture. Thus the available evidence points to CD34 being expressed in tissue-resident MSC, and its negative finding being a consequence of cell culturing.