Total Bone Marrow Cells Research Articles

A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques

Longitudinal bone marrow aspirates were obtained aseptically from the humerus of 36 rhesus and 6 cynomolgus macaques by using a 20G spinal needle, introduced through the bone close to the greater tuberosity. All samplings were performed without complications, and the animals showed no signs of pain or infections. The amount of total bone marrow cells obtained from each aspiration varied, in part due to animal-to-animal variation, but the yields were not affected by the sampling frequency or the length of time between each aspiration. The frequency of plasma cells in the bone marrow of each animal was also fairly stable over several longitudinal samplings while a greater, age-dependent, variation was observed between different animals.

NLRP12 suppresses host innate immune signaling to limit inflammation during emergency hematopoiesis (INM6P.404)

Abstract The hematopoietic system is capable of rapidly increasing myeloid cell production in response to tissue damage. NLRP12 is expressed in peripheral leukocytes and by osteoclasts and marrow progenitor cells. This NLR family member has been controversial but has been implicated in regulation of innate immune responses. We hypothesized that NLRP12 is a central regulator of innate immune reconstitution following an emergency hematopoietic event. To test this, wild type and Nlrp12-/- C57BL/6 female mice underwent a radiation-thermal combined injury (RCI) consisting of a 20% total body surface area, full thickness contact burn in addition to receiving a single whole body dose of 5 Gy γ-radiation. Following RCI, NLRP12-deficient mice display a lack of peripheral innate immune cell reconstitution as seen in wild type mice; specifically a ten-fold decrease in total numbers of splenic and pulmonary neutrophils and immature monocytes compared to wild type, and a three-fold increase in apoptosis of bone marrow granulocyte/monocyte progenitor cells resulting in a three-fold reduction of total bone marrow cells. Lack of NLRP12 results in a sustained increase in inflammation after injury, with significant increase in serum levels of TNFα, IL-6, IL-12, and type I interferons compared to injured, wild type mice that is inflammasome-independent. These data suggest that NLRP12 acts as an important regulator of inflammation during events that trigger immune hematopoeisis and reconstitution.

Myeloid-Derived Suppressor Cells are Generated during Retroviral Transduction of Murine Bone Marrow

Previous work by our group showed that transferring bone marrow cells transduced with an autoantigen into nonmyeloablated mice with experimental autoimmune encephalomyelitis induced immune tolerance and improved symptoms of the disease. Because this effect occurred in the absence of molecular chimerism, we hypothesized that the cells responsible did not have repopulating ability and that they were not mediating central but peripheral tolerance mechanisms. In the present study, we analyzed the immunophenotype of the cells that are generated in the transduction cultures and we evaluated the immunosuppressive activity of the main cell subpopulations produced. We show that both granulocytic (CD11b(+) Gr-1(hi)) and monocytic (CD11b(+) Gr-1(lo)) myeloid-derived suppressor cells (G- and M-MDSCs, respectively) are generated during standard 4-day γ-retroviral transduction cultures (representing about 25% and 40% of the total cell output, respectively) and that the effectively transduced cells largely consist of these two cell types. A third cell population representing about 15% of the transduced cells did not express CD45 or hematopoietic lineage markers and expressed mesenchymal stromal cell markers. Transduced total bone marrow cells and sorted M-MDSCs expressed arginase and inducible nitric oxide synthase activities, produced reactive oxygen species, and inhibited antigen-induced T-cell proliferation in vitro. Transgene-expressing MDSCs could be exploited therapeutically to induce tolerance in autoimmune diseases and in gene therapy protocols.

Determining a Clinically Relevant Strategy for Bone Tissue Engineering: An “All-in-One” Study in Nude Mice

PurposeAutologous bone grafting (BG) remains the standard reconstruction strategy for large craniofacial defects. Calcium phosphate (CaP) biomaterials, such as biphasic calcium phosphate (BCP), do not yield consistent results when used alone and must then be combined with cells through bone tissue engineering (BTE). In this context, total bone marrow (TBM) and bone marrow-derived mesenchymal stem cells (MSC) are the primary sources of cellular material used with biomaterials. However, several other BTE strategies exist, including the use of growth factors, various scaffolds, and MSC isolated from different tissues. Thus, clinicians might be unsure as to which method offers patients the most benefit. For this reason, the aim of this study was to compare eight clinically relevant BTE methods in an “all-in-one” study.MethodsWe used a transgenic rat strain expressing green fluorescent protein (GFP), from which BG, TBM, and MSC were harvested. Progenitor cells were then mixed with CaP materials and implanted subcutaneously into nude mice. After eight weeks, bone formation was evaluated by histology and scanning electron microscopy, and GFP-expressing cells were tracked with photon fluorescence microscopy.Results/ConclusionsBone formation was observed in only four groups. These included CaP materials mixed with BG or TBM, in which abundant de novo bone was formed, and BCP mixed with committed cells grown in two- and three-dimensions, which yielded limited bone formation. Fluorescence microscopy revealed that only the TBM and BG groups were positive for GFP expressing-cells, suggesting that these donor cells were still present in the host and contributed to the formation of bone. Since the TBM-based procedure does not require bone harvest or cell culture techniques, but provides abundant de novo bone formation, we recommend consideration of this strategy for clinical applications.

PU.1 promotes cell cycle exit in the murine myeloid lineage associated with downregulation of E2F1

Acute myeloid leukemia (AML) is characterized by increased proliferation and reduced differentiation of myeloid lineage cells. AML is frequently associated with mutations or chromosomal rearrangements involving transcription factors. PU.1 (encoded by Sfpi1) is an E26 transformation-specific family transcription factor that is required for myeloid differentiation. Reduced PU.1 levels, caused by either mutation or repression, are associated with human AML and are sufficient to cause AML in mice. The objective of this study was to determine whether reduced PU.1 expression induces deregulation of the cell cycle in the myeloid lineage. Our results showed that immature myeloid cells expressing reduced PU.1 levels (Sfpi1(BN/BN) myeloid cells) proliferated indefinitely in cell culture and expanded invivo. Transplantation of Sfpi1(BN/BN) cells induced AML in recipient mice. Cultured Sfpi1(BN/BN) cells expressed elevated messenger RNA transcript and protein levels of E2F1, an important regulator of cell cycle entry. Restoration of PU.1 expression in Sfpi1(BN/BN) myeloid cells blocked proliferation, induced differentiation, and reduced E2F1 expression. Taken together, these data show that PU.1 controls cell cycle exit in the myeloid lineage associated with downregulation of E2F1 expression.

Familial Mastocytosis: Identification Of KIT K509I Mutation and Its In Vitro Sensitivity To Imatinib, Dasatinib and PKC412

BackgroundMast cell diseases are myeloproliferative neoplasms characterized by an abnormal proliferation and accumulation of mast cells in different tissues. The clinical presentation of mastocytosis is heterogeneous, ranging from skin-limited disease to more aggressive variants that may be associated with multiorgan dysfunction/failure and shortened survival. In a relatively high proportion of cases, the clonal nature of the disease can be established on the basis of the demonstration of gain-of-function mutations involving the tyrosine kinase (TK) domain of KIT in skin lesions and BM cells and by the factor-independent proliferation and transforming abilities of these mutations. The tyrosine kinase inhibitor Imatinib is a treatment available for mastocytosis patients; however, some KIT mutations, specially KIT D816V, confer resistance to this drug. AimsTo characterize the clinical phenotype and molecular mutations of 2 relatives with diagnosis of systemic mastocytosis (WHO 2008). We also aimed to test the in vitro sensitivity of primary bone marrow (BM) cells from both patients to tyrosine kinase inhibitors. Patients and methodsFour individuals were included in the study; two patients (case 1 [mother], and case 2 [daughter]), and the parents of case 1. DNA samples were obtained from total BM cells, CD3+ BM cells and oral mucosa of patients, and from peripheral blood of all individuals. KIT (exons 1 to 21) was submitted for Sanger sequencing analysis. Primary bone marrow cells (5X104) from the 2 patients were cultured and treated with Imatinib (5uM), Dasatinib (80nM) and PKC 412 (100nM) or with vehicle only (control cells) and submitted for proliferation (MTT) and apoptosis assays (Annexin-V/PI) at days 4, 8 and 12 of culture. ResultsCase 1 was a 33 year-old woman with a chronic history of pruritic skin rash who was referred to our outpatient service for evaluation of massive splenomegaly (25 centimeters in length) and pancytopenia. She had neither comorbidities nor any familial history of hematological malignancies. The patient had no siblings and had only one daughter (case 2). At biopsy, she showed extensive skin and bone marrow infiltration by mast cells. During follow up, the patient presented with spontaneous splenic rupture and had to undergo splenectomy, which led to the resolution of pancytopenia. She was diagnosed with Aggressive Systemic Mastocytosis. Her daughter (case 2), a 17 year-old woman, was also evaluated for an insidious history of diffuse skin rash. Skin and bone marrow biopsies showed massive infiltration by atypical mast cells and a diagnosis of Indolent Systemic Mastocytosis was made. The rare KIT K509I mutation was found in all DNA samples obtained from both patients, but not from the parents of case 1. This suggests that the KIT K509I was a germ line mutation acquired de novo by patient 1 that was subsequently transmitted to her daughter (patient 2). In vitro treatment of primary bone marrow cells harboring the KIT K509I mutation from patients 1 and 2 resulted in variable clinical response rates according to the drug used and the treatment duration. Imatinib treatment resulted in a significant reduction in proliferation (days 4, 8 and 12 of culture) and an increase in apoptosis (days 8 and 12) in cases 1 and 2 (all p≤0.03). Although Dasatinib resulted in decreased proliferation in both patients at day 12 (all p≤0.008), a significantly higher apoptosis ratio was observed only for patient 1 at day 12 of culture (p=0.03). PKC412 had a negative effect over cell growth in patient 1 (days 4 and 8) and in patient 2 (day 4) (all p≤0.03); however, no effect in apoptosis ratio was seen. ConclusionsWe herein provide a report of a KIT K509I mutation in familial mastocytosis. This mutation has been previously described in the literature in one case of familial mastocytosis. Although rare, the screening for KIT K509I mutation should be considered in all cases of familial mastocytosis. Based on in vitro studies, mastocytosis patients harboring the KIT K509I mutation could benefit from treatment with Imatinib, Dasatinib and PKC 412. However, Imatinib may be more effective in inducing neoplastic mast cells apoptosis. Both patients described were started on Imatinib in June 2013. Disclosures:No relevant conflicts of interest to declare.

Knockdown Of The Piwi - Like Protein 4 (PIWIL4) Delays Leukemic Growth and Is Associated With Gross Changes In The Global Histone Methylation Marks In Human MLL - Rearranged AML

Piwi proteins belong to a class of proteins which were shown to be critically involved in the maintenance of the self-renewal property of stem cells in lower organisms. Furthermore, it was shown that they preserve genomic integrity through epigenetic silencing of transposable elements via CpG methylation and repressive histone modifications such as H3K9me3 in close interaction with a novel class of non-coding RNA called piRNA. So far there are neither precise data on the function of Piwi proteins in human acute myeloid leukemia, nor are there reports on expression of piRNAs in this disease. In a first step we tested PIWIL gene expression levels in normal human hematopoietic cells and leukemic patient samples by qRT-PCR. Among the family of human PIWI genes, PIWIL4 showed the highest expression level and was ubiquitously expressed in normal hematopoietic stem/progenitors, mature lymphoid and myeloid cells. Importantly, PIWIL4 showed aberrantly high expression in more than 72% of the AML patients (n=68; p< 0.0001) compared to normal CD34+ bone marrow (BM) and total BM cells (n=3). Notably, in nine of the ten MLL-AF9 rearranged AML patients, PIWIL4 was 64-fold higher expressed compared to normal CD34+ BM (p<0.0001) and 8-fold higher compared to inv(16), PML-RARa or cytogenetically normal AML patients (p<0.0001). To further validate this finding we analysed gene expression data performed on CD34+ human cord blood cells transduced with MLL-AF9 (n=9) vs AML-ETO (n=6) vs MYH11 (n=3): of note, PIWIL4 showed a 6 fold increase in expression in the MLL-AF9 transduced cells compared to the other experimental arms. Stable knockdown of PIWIL4 in the MLL rearranged AML cell lines MV4-11 (MLL-AF4) and THP-1 (MLL-AF9) significantly impaired growth in vitro (n=3) reducing proliferation and clonogenic growth by 83%/93% and 91%/93%, respectively. In addition, depletion of PIWIL4 delayed onset of leukemia in NSG mice transplanted with MV4-11/ THP-1 cells transduced with shPIWIL4 compared to the scrambled control (shRNA: AML onset 48/62d after transplantation vs. 30/30 days in the scrambled control; n=4/8 per arm; p< 0.0001/p<0.001). ChIP-seq analysis revealed that depletion of PIWIL4 in the THP1 cell line results in a marked global reduction in repressive H3K9me3 marks and in an increase in activating H3K4me3 marks as compared to cells transduced with the scrambled control. RNA-seq analyses revealed over 2500 differentially expressed genes upon PIWIL4 depletion with 60% of the genes being upregulated compared to the scrambled control (p<0.05). Among them genes involved in cell cycle such as RB1, P21, TGFB1 as well as epigenetic modifiers such as SETDB1, HDAC1,2 and demethylating enzyme TDG were differentially expressed. RB1 and EED, a protein necessary for PRC2 complex function, displayed an increase in expression and loss of H3K9me3 modifications on their promoters upon knockdown of PIWIL4. To prove piRNA expression in human AML and to test any association between PIWIL4 expression and piRNA signatures, microarray analyses covering 23,677 piRNAs was performed on the MLL-AF9 rearranged THP-1 cell line, of which 14193 piRNAs showed expression levels higher than 4 (arbitrary log2 scale). PIWIL4 knockdown induced differential expression of 981 piRNAs (p≤0.01, fold change ≥2), of which 527 were downregulated and 454 upregulated. Thus, collectively, we could show for the first time that PIWIL4 expression is deregulated in human AML, affects leukemic growth, shapes epigenetic marks and impacts piRNA expression in this disease. Disclosures:No relevant conflicts of interest to declare.

Stathmin 1 Is Involved In The Highly Proliferative Phenotype Of High-Risk Myelodysplastic Syndromes and Acute Leukemia Cells

Introduction: Stathmin 1, also known as Oncoprotein 18 (OP18) or Leukemia-associated phosphoprotein p18 (LAP18), is an important cytoplasmic microtubule-destabilizing protein that plays a critical role in the process of mitosis, proliferation and accurate chromosome segregation through regulation of microtubule dynamics. High levels of Stathmin 1 have been reported in solid tumors and have been associated with poor prognosis in various types of cancers. The identification of overactive proteins in leukemia cells, compared to normal hematopoietic cells, as well as understanding the molecular and cellular basis of the disease may provide new therapeutic opportunities. Aims: To evaluate Stathmin 1 expression in proliferating and non-proliferating hematopoietic cells, in bone marrow cells from healthy donors and from patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). In addition, we evaluated the effect of Stathmin 1 silencing on proliferation and apoptosis in the U937 acute myeloid leukemia cell line. Materials and Methods: A panel of human leukemia cell lines that included myeloid (K562, KU812, NB4, HL60, P39, HEL, U937, KG1 and THP1) and lymphoid cells (Jurkat, MOLT4, Daudi, Raji, Namalwa and Karpas 422) in exponential growth was used. Peripheral blood lymphocytes (PBL) were induced, or not, to proliferate upon PHA stimulation for 72 hours. A total of 30 healthy donors and 117 patients at diagnosis (MDS=52 [low-risk=36, high-risk=16], AML=49, and ALL=16) were included in the study. Stathmin 1 gene and protein expression was evaluated by qPCR and Western blot. Stathmin 1 was stably knocked down with specific shRNA-expressing lentiviral vector and cell growth was examined by MTT assay, clonogenicity by colony formation and apoptosis by AnnexinV/PI. Appropriate statistical analyses were performed; results are expressed as median (minimum- maximum). Results: A higher expression of Stathmin 1 was observed in all leukemia cell lines, when compared with normal non-proliferating hematopoietic cells. We also observed a marked increase in Stathmin 1 expression in PBL induced to proliferate with PHA after 72 hours. Stathmin 1 transcripts were significantly increased in total bone marrow cells from patients with AML (2.01 [0.35-8.88]; p=.0009) and ALL (2.94 [1.16-10.82]; p=.0004), compared with healthy donors (1.01 [0.38-4.08]). No difference in Stathmin 1 expression was observed between healthy donors and MDS patients. When the MDS group was stratified by the WHO classification into low and high-risk MDS, Stathmin 1 expression was significantly higher in the high-risk, when compared with low-risk MDS (1.62 [0.42–3.28] vs. 1.13 [0.36–2.61], p=.03). Similar results were found in isolated CD34+ bone marrow cells, Stathmin 1 transcripts were significantly increased in CD34+ AML cells compared with CD34+ normal cells, and in high-risk compared with low-risk MDS (all p≤.02). Interestingly, 3 out of 5 MDS patients showed a significant increase in Stathmin 1 transcripts after disease progression. Also, a significant positive correlation was observed between percentage of bone marrow blasts and Stathmin 1 expression in MDS patients (p=.03; r=.31). In U937 leukemia cells, Stathmin 1 silencing significantly reduced cell proliferation (p=.02) and clonal growth (p<.0001), but did not modulate apoptosis. Conclusions: Stathmin 1 is overexpressed in high-risk MDS and acute leukemia cells, and is upregulated during MDS progression, suggesting that Stathmin 1 plays a role in the highly proliferative phenotype. Our study adds new insights to the role of Stathmin 1 in leukemogenesis. Future studies are necessary to validate whether Stathmin 1 is a predictive marker for MDS progression, and to determinate whether Stathmin 1 is a “driver” or a “passenger” during malignant transformation. Disclosures:No relevant conflicts of interest to declare.

FMNL1 Is Downregulated In Myelodysplastic Syndromes and Is Involved In Megakaryocytic Differentiation

BackgroundMyelodysplastic syndromes (MDS) are disorders characterized by morphological dysplasia, impaired differentiation and defective cellular functions, resulting in peripheral cytopenias. FMNL1 belongs to a family of formin-related proteins, indispensable for many fundamental actin-dependent processes. Recently, FMNL1 has been described to be upregulated and play a role in the actin cytoskeleton dynamics during monocyte differentiation to macrophages. AimsThe aim of this work was to characterize FMNL1 expression in total bone marrow cells of patients with MDS comparing to normal donors. We also analyzed FMNL1 expression in erythrocytic, granulocytic and megakaryocytic differentiation, using cell line models. Finally, we evaluated the impact of inhibition of FMNL1 during megakaryocytic differentiation. MethodsA total of 49 patients with a diagnosis of MDS, receiving no treatment, and 18 samples from normal donors were included in the study, which was approved by the National Ethical Committee Board. Samples were submitted to RNA extraction after removal of erythrocytes by hemolysis. FMNL1 expression levels from cell lines or total bone marrow cells were determined by quantitative PCR (q-PCR) or Western blot. KU812 was treated 50 μM hemin and 100 μM hydroxyurea for erythrocytic differentiation. K562 was stimulated with 20nM of PMA for megakaryocytic differentiation. NB4 was treated with 10-6 M of ATRA for granulocytic differentiation. Megakaryocytic differentiation was followed by the increase in megakaryocytic marker (CD61) determined by flow cytometry and cells were also stained with May–Grunwald–Giemsa. K562 cells were transduced with lentivirus-mediated shRNA targeting LacZ or FMNL1. Apoptosis was assessed by Annexin-V/PI staining and cell cycle was evaluated by flow cytometry, both at 24 and 48 hours after induction with PHA. The statistical methods used were the age-adjusted multivariate linear regression analysis, Mann Whitney test or t test. ResultsFMNL1 expression in bone marrow samples was significantly lower in MDS when compared with normal donor cells (P=0.01), especially in the high risk group (P<0.02). Using cell line models for hematopoietic differentiation, there was a fifteen-fold increase and a five-fold increase in FMNL1 expression for megakaryocytic (P=0.002) and granulocytic differentiation (P=0.05) respectively. Western blot analysis corroborated these findings. There was no difference for erythrocytic differentiation. After PMA treatment, the level of the megakaryocytic markers CD61 was significantly lower in K562 shFMNL1 when compared with shLacZ (P=0.01). The level of CD41a (P=0.5) and CD42b (P=0.1) showed a trend toward a decrease in K562 shFMNL1 when compared with shLacZ, however not statistically significantly. Corroborating these data, K562 shFMNL1 showed abnormal megakaryocytic morphological features (larger cells with polylobulated or polysegmented nuclei and vacuolization) compared with cells shLacZ. There were no statistical differences in the apoptosis levels and cell cycle analysis between K562 shFMNL1 and shLacz cells. ConclusionsThe family of formin-related proteins has mainly been related to actin-dependent processes although little is known regarding their possible involvement in haematopoiesis. The lower FMNL1 expression in MDS BM could reflect the role of this protein in cell differentiation. We were therefore prompted to study this issue in depth using megakaryocytic differentiation as a system. In this study, we show increased expression of FMNL1 in PMA-induced megakaryocytic differentiation of K562 cells. Furthermore, knockdown of FMNL1 deregulates differentiation, suggesting that FMNL1 is required in order to maintain the effective megakaryocytopoiesis in MDS. Although FMNL1 silencing effectively down-regulated CD61 expression, CD41a and CD42b were reduced to a lesser extent. The effect of FMNL1 cannot be explained by modifications of the cell cycle or apoptosis during differentiation; and is probably due to the effect in changes in the dynamic remodeling of the cytoskeleton. Disclosures:No relevant conflicts of interest to declare.

Pharmacological Inhibition Of Histone Deacetylase (HDAC) 1, 2 Or 3 Have Distinct Effects On Cellular Viability, Erythroid Differentiation, and Fetal Globin (HbG) Induction

Induction of HbG is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and fetal hemoglobin (HbF) protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). While a variety of selective HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by variable efficacy and concerns over off-target side-effects observed in clinical trials, potentially due to inhibition of HDAC3. Additionally, it remains to be determined if HDAC1 or HDAC2 is the preferred therapeutic target. In this work we present data that investigates the effects of selective inhibitors of HDAC1, 2, or 3 on cytotoxicity, erythroid differentiation, and HbG induction in cultured human CD34+ bone marrow cells.Acetylon Pharmaceuticals has generated a library of structurally distinct compounds with a range of selectivity for each of HDAC1, 2, or 3 (Class I HDAC) as determined in a biochemical assay platform. From our initial chemical series, we identified ACY-822 as a Class I HDAC inhibitor with IC50 values of 5, 5, and 8 nM against HDAC1, 2, and 3, respectively. In contrast, ACY-1112 is 30-fold selective for HDAC1 and 2, with IC50 values of 38, 34, and 1010 nM against HDAC1, 2, and 3, respectively. Treatment of cells for 4 days with ACY-822 (1 μM) resulted in a 20-fold decrease in cell viability, while ACY-1112 (1 μM) treatment resulted in a minimal reduction in viability (1.2-fold) and a 2-fold increase in the percentage of HbG relative to other beta-like globin transcripts. This result suggests that pharmacological inhibition of HDAC3 is cytotoxic and is consistent with the therapeutic rationale for the design selective inhibitors of HDAC1 and 2.To investigate if HDAC1 or HDAC2 is the preferred therapeutic target, we utilized a second series of structurally distinct compounds. We identified ACY-957 as an HDAC1/2 selective compound biased towards HDAC1 with IC50 values of 4, 15, and 114 nM for HDAC1, 2, and 3, respectively. In contrast ACY-1071 showed balanced HDAC1 and 2 selectivity with IC50 values of 27, 24, and 247 nM for HDAC1, 2, and 3, respectively. Treatment of cells for 6 days with 1 μM of ACY-957 or ACY-1071 resulted in a 3-fold increase in the percentage of HbG relative to other beta-like globin transcripts. However, we found that ACY-957 treatment resulted in an approximately 3-fold decrease in cell viability after 6 days of treatment, while ACY-1071 treatment resulted in a minimal reduction (1.2-fold) in cell viability. Decreased cell viability observed with ACY-957 was associated with a reduction of cells positive for the erythroid differentiation markers CD71 and glycophorinA. This result is consistent with the Mx-Cre mouse model where HDAC1KO; HDAC2het had reduced numbers of erythrocytes, thrombocytes, and total bone marrow cells, while the HDAC1het; HDAC2KO was unaffected (Wilting RH, EMBO Journal, 2010).Our results suggest that compounds with a pharmacological profile of increased selectivity towards HDAC2 inhibition versus HDAC1 may be less cytotoxic and minimize effects on differentiation, while still inducing HbG in human CD34+ bone marrow cells. Disclosures:Shearstone:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

Prognostic Impact Of MLL5 transcript Levels On Outcome Of Patients With Acute Promyelocytic Leukemia Treated With All-Trans Retinoic Acid and Anthracycline-Based Chemotherapy: An International Consortium On Acute Promyelocytic Leukemia Study

BackgroundThe MLL5 gene encodes a histone methyltransferase implicated in positive control of several genes related to hematopoiesis. Its close relationship with retinoic acid–induced granulopoiesis suggests that the deregulated expression of MLL5 might lead acute promyelocytic leukemia (APL) blasts to become less susceptible to differentiation-inducing ATRA effects. Here, we retrospectively determined the MLL5 transcript levels in samples from APL patients enrolled in the International Consortium on Acute Promyelocytic Leukemia (IC-APL) trial and analyzed its relationship with clinical and laboratory features, hematologic recovery, relapse, and survival. The results of the IC-APL have been previously reported (Blood 2013, 121(11) pp: 1935). In brief, complete hematological remission (CR) was achieved by 153/180 patients enrolled in Brazil, Mexico, Chile and Uruguay and after a median follow up of 28 months, the 2-year cumulative incidence of relapse (CIR), overall survival (OS) and disease-free survival (DFS) were 4.5%, 80% and 91%, respectively.Design and MethodsOne hundred and twenty-one APL patients (age, 15-73y) from seven different Brazilian institutions and treated according to the IC-APL protocol were included. The treatment schedule was identical to that adopted in the PETHEMA/HOVON LPA2005 trial, except for the replacement of idarubicin by daunorubicin. ATRA treatment was initiated immediately in all cases in which the diagnosis of APL was suspected based on morphology. As normal controls, total bone marrow (BM, n=8) and peripheral blood (PB, n=101) cells from healthy donors (age, 18-60y) were collected and mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. Additionally, 28 CBF-leukemia samples were included. Gene expression profile was analyzed by real-time quantitative PCR using the ABL FusionQuant Standard Kit as an endogenous control. Based on the continuous distribution of MLL5/ABL expression on APL samples (Figure 1), we adopted the median value of MLL5/ABL expression as cut-off to dichotomize APL patients in “low” and “high” MLL5 transcript levels.ResultsMLL5 expression was not different between APL, CBF-leukemia and healthy donors samples (Figure 1; P=0.19). There was no relevant difference between APL patients with low (n=62) and high (n=59) MLL5 transcript levels with respect to clinical and laboratory features, although high MLL5 transcript levels were more likely to be present in female gender (P=0.029). Overall, 102 (84%) achieved CR. Patients with low MLL5 transcript levels had significantly lower CR rate than patients with high MLL5 transcript levels (74% vs 95%; P=0.002). Twelve patients (10%) experienced early mortality (i.e., death during induction therapy) due to hemorrhage (n=6; 50%), disease progression (n=1; 8.3%), thrombosis (n=1; 8.3%), therapy-related infection (n=3; 25%) and differentiation syndrome (n=1; 8.3%); MLL5 transcript levels had no impact on early mortality (P=0.155). With a follow-up of 33 months among survivors (range, 1-72 months), patients with low MLL5 transcript levels had significantly lower 2-year OS (P=0.005) and 2-year DFS rate (P=0.037) than patients with high MLL5 transcript levels. Up to January 2013, a total of six relapses (5%) had been recorded. The 2-year CIR among patients with low and high MLL5 transcript levels was 14% (95%CI: 5% to 27%) and 2% (95%CI: 0.1% to 11%), respectively (P=0.04). We have further evaluated the prognostic impact of MLL5 transcript levels in those patients who remain alive after induction therapy (107 patients). Low MLL5 transcript levels were predictive of lower CR rate (P=0.042) and 2-year OS rate (P=0.009), but had no impact on DFS (P=0.106).ConclusionOur results show that MLL5 transcript levels may predict lower remission rate, short survival and higher risk of relapse in APL patients treated with ATRA and anthracycline-based chemotherapy. This is the first report describing the MLL5 expression as a prognostic factor in APL; nevertheless, our results should be confirmed in an independent cohort. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Downregulation Of SPINT2 Expression In Stromal Cells From Myelodysplastic Syndrome Patients Increases HGF and SDF-1 Secretion and Promotes Cell-Cell Adhesion By Alteration In Integrin Expression

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, an important role is also played by the hematopoietic microenvironment niche that has as key component the mesenchymal stromal cells (MSC). The MSC of MDS patients have morphological and functional abnormalities. Recently, our group identified new possible target genes involved in MDS pathophysiology through microarray analysis of MSC from MDS patients. An interesting underexpressed gene found was SPINT2, a gene that encodes a transmembrane protein which inhibits the hepatocyte growth factor activator (HGFA), the enzyme responsible for the conversion of hepatocyte growth factor (HGF) into its active form. SPINT2 is downregulated in some types of solid cancer and correlated with their prognostic and progression; however, the functional role of SPINT2 in MDS remains unknown. We herein investigated the role of SPINT2 in MSC, studying HGF and SDF1 secretion and cell adhesion with normal CD34+, P39 and U937 cells by silencing SPINT2 gene in HS5 and HS-27a stromal cell lines. We also investigated, using qPCR, SPINT2 and HGF mRNA expression in MSC and total bone marrow (BM) cells from 56 untreated MDS patients (WHO 35 low-risk, 21 high-risk) and 28 healthy donors. To inhibit SPINT2, specific shRNA expressing lentiviral vectors targeting SPINT2 gene or no specific sequence were used. The HGF and SDF1 secretion in cell supernatant from the cells silenced or not for SPINT2 was measured by BioPlex after 6, 12, 24 and 48h. The cell-cell adhesion of CD34+, P39 or U937 cells onto transduced stromal cells and the adhesion molecule profile were analyzed by flow cytometry. We observed a significant decrease in SPINT2 mRNA expression of MDS MSC (P=0.006) and MDS BM cells (P=0.03) compared to normal cells. Further, HGF mRNA expression of MDS MSC was significantly increased (P=0.01) compared to normal cells. Spearman analysis showed a negative significant correlation between SPINT2 and HGF expressions (P=0.01;r2=0.60). In both stromal cell lines, SPINT2 inhibition resulted in a significant increase in HGF secretion after 24 and 48h and a significant increased secretion of SDF1 after 48h. Moreover, SPINT2 silencing induced a significant increased adherence of CD34+, P39 and U937 cells onto stromal cells probably due to the alteration in integrin expression, since an increase in CD49b and CD49d and decrease in CD49e expressions were also observed in cells silenced for SPINT2. Considering that SPINT2 limits signaling via HGF pathway by inhibiting HGF activation through HGFA, the SPINT2 underexpression in MDS would allow the conversion of the inactive HGF monomer to an active heterodimer. Interestingly, a significant decrease of SPINT2 and increase of HGF expressions were observed in MSC of MDS patients, compared to normal cells. Moreover, recent studies have demonstrated that HGF serum levels are significantly increased in MDS patients and dependent on MDS severity. In addition, SPINT2 inhibition in stromal cells significantly increased HGF secretion by these cells. The increased HGF secretion can result in an autocrine regulation which induces the production and secretion of SDF1 by stromal cells themselves. Cytokines secretions provided by MSC are required for adhesion, survival and proliferation of HSC cells. In this way, in MDS MSC, SPINT2 underexpression and, consequently, increased HGF and SDF1 secretion, may lead to an increased adhesion between MSC and normal or malignant HSC cells. Corroborating our hypotheses, we found overexpression of integrins CD49b and CD49d, which mediate cell-MSC interaction, and a decrease in CD49e expression, an integrin that promotes interaction with extracellular matrix. The interaction onto MSC contributes to the maintenance of the stem and malignant cell properties, such as self-renewal, survival and proliferation. Cytokine secretion and cell adhesion onto MSC is important for MDS physiology. Hence, we demonstrate for the first time that, in MDS, SPINT2 plays a role in the HGF and SDF1 secretion by MSC, resulting in an alteration in cell-cell adhesion and molecule adhesion profile. In view of these data, the SPINT2 expression alteration in MDS MSC may constitute a particular mechanism of MDS pathophysiology and maintenance of self-renewal, homing and proliferation of HSC and malignant clones in MDS.Support: FAPESP, CNPq Disclosures:No relevant conflicts of interest to declare.

Role Of Tumor Necrosis Factor α In Bone Marrow Niche Dysfunction Of Patients With Systemic Lupus Erythematosus (SLE)

Objectives and backgroundSLE is a chronic systemic autoimmune disorder associated with autoantibodies and cytopenias. There are few studies of the pathogenesis of anemia of chronic inflammation in lupus. This study addresses the pathogenesis of the hematological manifestations of lupus. We have shown that SLE bone marrow (BM) exhibits striking death of niche and hematopoietic cells associated with tumor necrosis factor-α (TNFα) over-production. Here, we further examined the pathogenesis of hematological BM niche dysfunction. MethodsPathology records over the past 10 years from the University of Florida were reviewed and BM aspirates/core biopsies from 6 SLE patients were identified for further study. Wright-Giemsa stained BM aspirate smears and cytospin preparations, hematoxylin and eosin (H&E)-stained and reticulin stained BM core biopsies were reviewed. Immunohistochemistry (IHC) for TNFα, cleaved caspase-3, and CD71 was performed on core biopsies and expression levels were quantified morphometrically. BM specimens from individuals undergoing staging for lymphoma were selected as controls. ResultsFive of 6 SLE patients had nephritis and 3 were direct Coombs+ (one with hemolytic anemia). Mean hemoglobin was 9.1g/dL in SLE patients and 11.9g/dL in controls. Mean WBC was 3800/mm3 in patients and 8200/mm3 in controls, and mean platelet counts were 133,000/mm3 and 258,000/mm3, respectively. BM aspirates exhibited numerous apoptotic cells, erythroid dyspoiesis, plasmacytosis, hemophagocytosis, and phagocytosis of nuclear material by mature neutrophils (LE cells). Numerous LE cells were seen in 5/6 SLE BM aspirates. Compared to normal BM biopsies, SLE BM biopsies exhibited hypocellularity, erythroid dyspoiesis, polyclonal plasmacytosis, mild reticulin fibrosis, BM stromal damage/disorganization and 3 out of 6 with interstitial lymphoid aggregates. IHC with anti-cleaved caspase-3 antibodies, a specific marker of cell death, revealed numerous caspase-3+ cells in a range of 20-40% of total BM cells in contrast to control BMs of <5%. Interestingly, a large majority of osteoblasts or lining cells in BM osteal niches were caspase-3+ in SLE BM, whereas caspase-3+ cells were rare in control BM biopsies. Using double IHC (TUNEL assay plus anti-CD71 or myeloperoxidase antibodies), prominent apoptotic erythroid precursors with less extensive cell death of the myeloid and megakaryocytic lineages was found in SLE BM core biopsies. In SLE, caspase-3+ cells occupied 9-12% of the total BM area vs. 1-2% in control BM (p<0.001). As TNFα promotes Fas-mediated apoptosis and may damage hematopoietic precursors and/or stromal cells, we examined its production in SLE BM. IHC with anti-TNFα antibodies revealed intense intra- and extracellular staining surrounding neutrophils and monocytes, and the extent of TNFα staining was dramatically higher in SLE patients' BM biopsies than in controls. Staining was particularly intense adjacent to presumptive osteoblasts lining the surface of bone trabeculae, though there also was staining in interstitial regions. There was little TNFα staining of control BM. Morphometric analysis revealed 10-18% of the area in SLE BM intensely stained with anti-TNFα vs. 1-3.5% in controls (P < 0.001, Student t-test), suggesting that TNFα overproduction may cause BM niche dysfunction. ConclusionBM TNFα-mediated niche cell apoptosis is likely to be involved in the pathogenesis of SLE-associated hematological abnormalities. Disclosures:No relevant conflicts of interest to declare.

Ineffective Erythropoiesis With Increased Neutrophils In Old Mice Overexpressing Hmga2 cDNA

HMGA2 is frequently overexpressed in hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and primary myelofibrosis (PMF), and occasionally in myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). We recently reported that transgenic mice overexpressing truncated Hmga2 cDNA (ΔHmga2 mice) showed increased numbers in all lineages of peripheral blood (PB) cells, hypercellular bone marrow (BM), and splenomegaly in young adults of 3 months old (Ikeda et al, Blood, 2011). ΔHmga2 mice also showed growth advantage of hematopoietic stem cells (HSCs) in serial BM transplants and of committed progenitors in colony-replating assays. Thus, overexpression of HMGA2 may contribute to clonal expansion in PNH or myeloproliferation in PMF and MDS/MPN. Although these disorders show disease progression and development of leukemia in the long term, consequences of long-term overexpression of HMGA2 in hematopoiesis is unknown. Therefore, in this study, we investigated hematopoiesis in old ΔHmga2 mice at 15 months old. We found an increase in PB neutrophils in 16 old ΔHmga2 mice compared with 8 old wild-type (WT) mice (mean ± SD; 4.3 ± 1.7 vs. 2.4 ± 0.6 x109/L, p<0.01) as well as young mice. Strikingly, in contrast to young mice, we found a decrease in PB red blood cells of older ΔHmga2 mice compared with WT mice (8.5 ± 0.5 vs. 9.3 ± 0.4 x1012/L, p<0.01), which was associated with an increase in the mean corpuscular volume (MCV, 48.5 ± 3.7 vs. 42.1 ± 1.8 fL, p<0.01). PB total leukocyte and platelet counts were similar between old ΔHmga2 mice and old WT mice. Total BM cell counts were higher in 11 old ΔHmga2 mice compared with 12 WT mice (2.1 ± 0.4 vs. 1.4 ± 0.5 x107/femur, p<0.01). Spleens were 2-fold heavier in old ΔHmga2 mice (n=11) compared with old WT mice (n=12, p<0.01). Histology showed hypercellular BM with erythroid dysplasia, but neither fibrosis nor leukemia, in 5 old ΔHmga2 mice. Absolute numbers of BM lineage-Sca-1+c-kit+ HSCs were higher in 5 old ΔHmga2 mice compared with 3 old WT mice (73 ± 31 vs. 23 ± 10 x103/femur, p=0.036). In addition, proportions of CD71+Ter119+ erythroblasts were higher in 3 old ΔHmga2 mice compared with 3 WT mice (11.8 ± 1.2 vs. 5.1 ± 1.1%, p<0.01), although absolute number of megakaryocyte-erythrocyte progenitors did not show a statistical difference (137 ± 36 vs. 96 ± 18 x103/femur, p=0.1). Progenitor assay also showed increased numbers of BFU-E colonies in 3 old ΔHmga2 mice compared with 3 WT mice (25 ± 2 vs. 13 ± 2, p<0.01), but new BFU-E colonies did not grow from replating of these colonies of old ΔHmga2 mice, in contrast to young ΔHmga2 mice. Annexin V staining showed increased spontaneous apoptosis after 24-hour incubation in Ter119+CD71+ cells of 3 old ΔHmga2 mice compared with 3 old WT mice (26.6 ± 2.7 vs. 14.3 ± 7.0%, p=0.033). In MACS-sorted Ter119+ erythroid cells, expression of anti-apoptotic Bcl-xl mRNA in old ΔHmga2 mice was lower compared with old WT mice (3.3 ± 1.2 vs. 5.3 ± 0.3, p=0.016), while that in young ΔHmga2 mice were higher than young WT mice (6.9 ± 0.4 vs. 3.5 ± 0.3, p<0.01), suggesting the possibility that some changes, including expression of Bcl-xl affected sensitivity of erythroid cells to apoptosis during aging, resulted in ineffective erythropoiesis. In conclusion, long-term expression of HMGA2 may lead to neutrophilia with ineffective erythropoiesis, which may mimic MDS/MPN rather than myelofibrosis, possibly through impairment of Bcl-xl in erythroid cells during a long course. Ineffective erythropoiesis may also in part explain BM failure in PNH. Disclosures:No relevant conflicts of interest to declare.

Aberrant Upregulation Of Alternatively Spliced Isoforms Of Son In Myeloid Leukemia: Implications In Hematopoiesis and Leukemogenesis

▪SON is a large, poorly characterized nuclear speckle protein. The key structural domains comprising the SON protein are the serine/arginine-rich (RS) domain, single stranded RNA-, and double stranded RNA-binding domains, in addition to several long repeating amino acid sequences likely to facilitate its scaffolding function. Previously, we identified SON as a key factor for efficient RNA splicing of diverse genes related to cell cycle, DNA repair, and survival signaling pathways (Molecular Cell, 2011, 42:185). We also demonstrated that SON inhibits transcriptional activation of the promoter associated to the miR-23a-27a-24 cluster, which harbors a regulator for the expression of GATA-2, a known hematopoietic stem cell regulator (J. Biol. Chem. 2013, 288:5381). Moreover, SON is highly expressed in hematopoietic tissues/organs, and particularly upregulated in hematopoietic stem cells and leukemic blasts. These results suggest potential roles of SON in both normal hematopoiesis and hematological malignancies. Based on the nucleotide sequence of the SON gene, several databases predict that the primary transcript of SON contains alternative exons, and inclusion of these alternative exons during RNA splicing may generate C-terminally truncated short isoforms (splice variants) which lack RNA-binding motifs. However, expression of these short SON isoforms has not been confirmed in any types of cells and functions of the isoforms remain unexplored.Using 3' rapid amplification of cDNA end (3' RACE), we confirmed that short SON isoforms are indeed expressed in hematopoietic cells, and the included alternative exon provides the 3' UTR and polyadenylation signal different from those of full-length SON transcript. To further examine SON isoform expression, we designed primers targeting specific alternative exons of SON and screened the level of full-length SON and the short isoforms. Our quantitative PCR data showed that lineage marker negative (Lin-) bone marrow cells highly express full-length SON, but not short isoforms, when compared to total bone marrow cells. Interestingly, we found that the levels of short SON isoforms are noticeably high in acute myeloid leukemia (AML) patient bone marrow cells, while full-length SON is downregulated when compared to normal bone marrow cells. These expression patterns were consistent with data analyses in a cancer microarray database (Oncomine). Furthermore, we found that short isoforms, but not full-length SON, were downregulated during PMA-induced differentiation of HL-60 and K562 cells. These results suggest that while full-length SON is the major form in normal hematopoietic stem cells/progenitors, the production of short SON isoforms through alternative exon inclusion is aberrantly activated in leukemic cells, likely due to a factor associated with impaired differentiation.To address whether the short SON isoforms affect the function of full-length SON in RNA splicing, we examined RNA splicing efficiency using a minigene containing SON-dependent exon-intron junction. As we expected, the short isoform alone, which lacks RNA-binding motifs, was not able to process minigene RNA splicing. However, to our surprise, co-expression of a short isoform together with full-length SON caused an increase in spliced RNA product from a minigene. These results suggest that short isoform expression potentiates the full-length SON function during exon recognition. Taken together, our results reveal that inclusion/skipping of the alternative exons within the primary SON transcript is abnormally regulated in myeloid leukemia, resulting in upregulation of short splice variants of SON. Furthermore, fine-tuning of RNA splicing efficiency by short SON isoforms implicates potential roles of these isoforms in RNA processing and global gene expression during hematopoiesis and leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Fast Detection Of MYD88-L265P Mutation By PCR-RFLP In Chronic Lymphoproliferative Disorders

IntroductionWaldenstrom's macroglobulinemia (WΜ) is an incurable disorder characterized by bone marrow infiltration from neoplastic lymphoplasmacytic B-lymphocytes and monoclonal IgM production. Recent data suggest a possible causal relationship of MYD88-L265P mutation with the pathogenesis of the disease (Treon Sp et al. N Eng J Med 2012;367:826-33). Additional studies have conformed that MYD88-L265P is characteristic of WΜ, but it is also rarely present in other chronic lymphoproliferative disorders (LPDs) (Jiménez C et al. Leukemia 2013,doi: 10.1038/leu.2013.62; Varettoni M et al. Blood 2013;121:2522-8). The purpose of this study was to analyse the prevalence of the aforementioned mutation in patients with WM and other LPDs, using a fast and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol. MethodsGenomic DNA was extracted from the bone marrow of 11 patients with WΜ, 12 patients with B-cell chronic lymphocytic leukemia (CLL), 8 with splenic lymphoma with villous lymphocytes (SLVL). Furthermore, consecutive samples of peripheral blood and bone marrow isolated CD19+ cells, derived from a patient with monoclonal IgM gammopathy of undetermined significance (MGUS-IgM), were also retrospectively analysed for the presence of MYD88-L265P defect. The detection of the mutation was performed by PCR amplification of the MYD88 exon 5 region, followed by RFLP analysis, since the presence of the mutation results in the generation of BsiEI restriction enzyme site. PCR-RFLP results were also confirmed by direct sequencing of purified CD19+cells. ResultsThe MYD88-L265P mutation was detected in 10 patients with WΜ (90.9%), in 1 patient with SLVL with markers of lymphoplasmatocytoid differentiation (12.5%) and was absent in all CLL patients. Interestingly, our PCR-RFLP protocol exhibited a greater sensitivity than direct sequencing, when applied to total bone marrow cells (12.5% vs 25%). Moreover, the patient with MGUS-IgM displayed the MYD88-L265P mutation in isolated CD19+cells of both bone marrow and peripheral blood in all consecutive samples and remains in a stable condition for the last 7 years. ConclusionPCR-RFLP is a rapid, sensitive and reliable technique for the detection of MYD88-L265P mutation in patients with WM and lymphomas with lymphoplasmatocytoid differentiation. Additionally, the presence of MYD88-L265P mutation in MGUS-IgM in the absence of disease progression for many years, suggests that this genetic defect alone is not sufficient to lead to overt neoplastic disease. Disclosures:No relevant conflicts of interest to declare.

The Ex-Vivo Tolerance Induction To TLR7 In Donor Lymphocytes Prevents aGvHD In a Mismatched Transplantation Mouse Model

Acute Graft vs Host Disease (aGvHD) is the major cause of non-relapse mortality after allogeneic stem cell transplantation limiting the effectiveness of this therapy. The efficacy of the conventional anti-GvHD prophylaxis and treatment is unpredictable while it leads to generalized immunosuppression and subsequently, to increased susceptibility to opportunistic infections. Toll-like Receptors (TLRs) are able to detect exogenous pathogen-associated molecular patterns (PAMPs) as well as a variety of endogenous self-antigens released as danger signals during “sterile tissue injury”. Polymorphisms of the genes encoding TLR4 and TLR9 have been associated with a higher incidence of GvHD in hematopoetic stem cell transplant recipients. TLRs are widely known for their ability to mainly activate cells of the immune system, however, regulatory mechanisms of TLR tolerance induction have recently also been recognized; repeated exposure to low doses of TLR ligands «desensitize» the host following challenge with the same or other TLR agonists. We investigated the ex-vivo tolerance induction to TLR 2, 4, 7 after extended exposure of mouse splenocytes (mSPLCs) to low-dose specific TLR agonists (Pam3CKS4, LPS, R848 respectively) in culture followed by a high-dose challenge with the specific TLR agonists. Successful tolerance induction to all TLRs tested, was assessed in vitro by ELISA, measuring the TNFa levels in the culture supernatant of mouse splenocytes (p≤0.05). Additionally, the optimal dose and the duration required for each agonist to induce tolerance in SPLC culture was determined. Furthermore, TLR7 stimulation with the optimal dose of TLR7 ligand, R848, in purified, immunomagnetically separated, subsets of human PBMCs, demonstrated that TLR7 tolerance induction is mostly mediated by T-lymphocytes (p&lt;0.001). Subsequently, we examined whether the ex-vivo tolerance induction in donor’s lymphocytes can prevent aGvHD in a mismatched transplantation mouse model (C57BL/6 donors to irradiated Balb/c recipients). In this model, aGvHD usually appears within 45 days post-transplantation. We tested 4 groups (I-IV) that received: 5Χ106 T-cell depleted total bone marrow cells (TCD-BM, control group Ι), TCD-BM cells plus 30x106 SPLCs (group II), TCD-BM cells plus mSPLCs after pre-treatment with R848 (group III), TCD-BM cells plus mSPLCs after pre-treatment with LPS (group IV). The clinical assesement of aGvHD was made by a max 10-point scoring system based on alterations in 5 parameters (weight loss, fur texture, posture, physical activity and skin integrity). No animal from groups II and IV survived beyond day 25 post transplantation (15/15), whereas groups I and III showed a significantly higher survival rate with 16 of the 21 mice still being alive at the end of the experiment (day 67, p&lt;0.001). The mean body weight loss on day 18 post-transplant was 32% in groups II and IV and 12% in groups I and III (p=0.001). The max mean aGvHD score was 7.5-8 in groups II and IV and 0.5-1.8 in groups I and III (days 18-25, p&lt;0.001). Detailed histopathology in skin, liver and gastrointestinal tract sections demonstrated severe aGvHD lesions accompanied by disruption of the normal tissue architecture in groups II, IV and normal findings in groups I, III. In conclusion, ex-vivo donor lymphocyte tolerance induction to TLR7, but not TLR4, could serve as a potential and clinically applicable tool of aGvHD prophylaxis. The ex vivo approach abrogates the need for direct administration of TLR7-specific agonists to humans. Disclosures: No relevant conflicts of interest to declare.

Early Growth Response-1 (EGR-1) and Nuclear Factor of Activated T Cells (NFAT) Cooperate to Mediate CD40L Expression in Megakaryocytes and Platelets

Increasing evidence implicates circulating platelets as mediators of chronic inflammatory and autoimmune diseases via the expression and release of CD40L, an important modulator of inflammation and adaptive immune responses traditionally associated with activated T cells. Emerging evidence suggests that platelet CD40L is dynamically regulated in several chronic inflammatory and autoimmune diseases and may mediate progression and secondary pathology associated with those disease states. The present study identifies NFATc2 as a key transcriptional modulator of CD40L expression in megakaryocytes and inflammatory activity of platelets. Furthermore, the current data show that EGR-1, a member of the early growth response family of zinc finger transcription factors, modulates NFATc2-dependent regulation of CD40L expression in megakaryocytes. Our novel demonstration that in vivo biochemical or genetic inhibition of NFATc2 activity in megakaryocyte diminishes platelet CD40L implicates the NFATc2/EGR-1 axis as a key regulatory pathway of inflammatory and immunomodulatory activity in platelets and represents a target for the development of therapeutics for the potential treatment of chronic inflammatory and autoimmune diseases.

Stem Cell Technologies Based on Hemangioblast Technology Focusing on Human Blood Cells

The in vitro generation of hematopoietic stem cells (HSCs) and mature hematopoietic cells from hemangioblast derived from embryonic stem (ES) or induced pluripotent stem (iPS) cells promises to provide an alternative source of cells that could replace total bone marrow cells or HSC-enriched fractions. This mini-review deals with innovation related to hemangioblast-based methods for blood cells production as disclosed in recent patent literature and current barriers to clinical translation are discussed.

Effect on lifespan of high yield non-myeloablating transplantation of bone marrow from young to old mice

Tissue renewal is a well-known phenomenon by which old and dying-off cells of various tissues of the body are replaced by progeny of local or circulating stem cells (SCs). An interesting question is whether donor SCs are capable to prolong the lifespan of an aging organism by tissue renewal. In this work, we investigated the possible use of bone marrow (BM) SC for lifespan extension. To this purpose, chimeric C57BL/6 mice were created by transplanting BM from young 1.5-month-old donors to 21.5-month-old recipients. Transplantation was carried out by means of a recently developed method which allowed to transplant without myeloablation up to 1.5 × 108 cells, that is, about 25% of the total BM cells of the mouse. As a result, the mean survival time, counting from the age of 21.5 months, the start of the experiment, was +3.6 and +5.0 (±0.1) months for the control and experimental groups, respectively, corresponding to a 39 ± 4% increase in the experimental group over the control. In earlier studies on BM transplantation, a considerably smaller quantity of donor cells (5 × 106) was used, about 1% of the total own BM cells. The recipients before transplantation were exposed to a lethal (for control animals) X-ray dose which eliminated the possibility of studying the lifespan extension by this method.