CFU Assay Research Articles

Development and antimicrobial susceptibility studies of in vitro monomicrobial and polymicrobial biofilm models with Aspergillus fumigatus and Pseudomonas aeruginosa

BackgroundMixed microbial infections of the respiratory tracts with P. aeruginosa and A. fumigatus capable of producing biofilms are commonly found in cystic fibrosis patients. The primary objective of this study was to develop an in vitro model for P. aeruginosa and A. fumigatus polymicrobial biofilm to study the efficacy of various antimicrobial drugs alone and in combinations against biofilm-embedded cells. Simultaneous static cocultures of P. aeruginosa and sporelings were used for the development of in vitro P. aeruginosa-A. fumigatus polymicrobial biofilm in SD broth in 24-well cell culture plates at 35°C, and the biofilm formation was monitored microscopically and spectrophotometrically. Using P. aeruginosa-A. fumigatus sporelings cocultures we examined the effects of various antimicrobial drugs alone and in combination against polymicrobial biofilm by CFU and tetrazolium reduction assays.ResultsIn simultaneous static cocultures P. aeruginosa cells killed A. fumigatus conidia, whereas the bacterial cells showed no substantial fungicidal effect on sporelings grown for 12 h or longer at 35°C. Monospecies cultures of P. aeruginosa produced loosely adhered monomicrobial biofilm and addition of 10% bovine serum to the growth medium inhibited the formation of monomicrobial biofilm by P. aeruginosa whereas it produced tightly adhered polymicrobial biofilm in the presence of A. fumigatus mycelial growth. A. fumigatus produced firmly adherent monomicrobial and polymicrobial biofilms. A comparison of CFU and MTT assays showed that the latter is unsuitable for studying the effectiveness of antimicrobial treatment against polymicrobial biofilm. Tobramycin alone and in combination with posaconazole was highly effective against monomicrobial and polymicrobial biofilms of P. aeruginosa whereas cefepime alone and in combination with posaconazole showed excellent activity against monomicrobial biofilm of P. aeruginosa but was less effective against polymicrobial biofilm. Monomicrobial and polymicrobial biofilms of A. fumigatus showed similar susceptibility to posaconazole with and without the antibacterial drug.ConclusionsSimultaneous static coculture of A. fumigatus sporelings grown for 12 h or longer was superior to ungerminated conidia with P. aeruginosa for the development of A. fumigatus-P. aeruginosa biofilm. P. aeruginosa-A. fumigatus polymicrobial biofilm shows differential susceptibility to antimicrobial drugs whereas the susceptibility of A. fumigatus to antimicrobial drugs was unchanged.

Benzimidazole-based compounds kill Mycobacterium tuberculosis

Tuberculosis remains one of the deadliest infectious diseases, killing 1.4 million people annually and showing a rapid increase in cases resistant to multiple drugs. New antibiotics against tuberculosis are urgently needed. Here we describe the design, synthesis and structure–activity relationships of a series of benzimidazole-based compounds with activity against Mycobacterium tuberculosis (Mtb) in a replicating state, a physiologically-induced non-replicating state, or both. Compounds 49, 67, 68, 69, 70, and 72, which shared a 5-nitrofuranyl moiety, exhibited high potency and acceptable selectivity indices (SI). As illustrated by compound 70 (MIC90 < 0.049 μg/mL, SI > 512), the 5-nitrofuranyl group was compatible with minimal cytotoxicity and good intra-macrophage killing, although it lacked non-replicating activity when assessed by CFU assays. Compound 70 had low mutagenic potential by SOS Chromotest assay, making this class of compounds good candidates for further evaluation and target identification.

Postnatal epithelium and mesenchyme stem/progenitor cells in bioengineered amelogenesis and dentinogenesis

Rodent incisors provide a classic model for studying epithelial–mesenchymal interactions in development. However, postnatal stem/progenitor cells in rodent incisors have not been exploited for tooth regeneration. Here, we characterized postnatal rat incisor epithelium and mesenchyme stem/progenitor cells and found that they formed enamel- and dentin-like tissues in vivo. Epithelium and mesenchyme cells were harvested separately from the apical region of postnatal 4–5 day rat incisors. Epithelial and mesenchymal phenotypes were confirmed by immunocytochemistry, CFU assay and/or multi-lineage differentiation. CK14+, Sox2+ and Lgr5+ epithelium stem cells from the cervical loop enhanced amelogenin and ameloblastin expression upon BMP4 or FGF3 stimulation, signifying their differentiation towards ameloblast-like cells, whereas mesenchyme stem/progenitor cells upon BMP4, BMP7 and Wnt3a treatment robustly expressed Dspp, a hallmark of odontoblastic differentiation. We then control-released microencapsulated BMP4, BMP7 and Wnt3a in transplants of epithelium and mesenchyme stem/progenitor cells in the renal capsule of athymic mice in vivo. Enamel and dentin-like tissues were generated in two integrated layers with specific expression of amelogenin and ameloblastin in the newly formed, de novo enamel-like tissue, and DSP in dentin-like tissue. These findings suggest that postnatal epithelium and mesenchyme stem/progenitor cells can be primed towards bioengineered tooth regeneration.

Tgif1 Regulates Quiescence and Self-Renewal of Hematopoietic Stem Cells

TG-interacting factor 1 (TGIF1) is a transcriptional repressor that can modulate retinoic acid and transforming growth factor β signaling pathways. It is required for myeloid progenitor cell differentiation and survival, and mutations in the TGIF1 gene cause holoprosencephaly. Furthermore, we have previously observed that acute myelogenous leukemia (AML) patients with low TGIF1 levels had worse prognoses. Here, we explored the role of Tgif1 in murine hematopoietic stem cell (HSC) function. CFU assays showed that Tgif1(-/-) bone marrow cells produced more total colonies and had higher serial CFU potential. These effects were also observed in vivo, where Tgif1(-/-) bone marrow cells had higher repopulation potential in short- and long-term competitive repopulation assays than wild-type cells. Serial transplantation and replating studies showed that Tgif1(-/-) HSCs exhibited greater self-renewal and were less proliferative and more quiescent than wild-type cells, suggesting that Tgif1 is required for stem cells to enter the cell cycle. Furthermore, HSCs from Tgif1(+/-) mice had a phenotype similar to that of HSCs from Tgif1(-/-) mice, while bone marrow cells with overexpressing Tgif1 showed increased proliferation and lower survival in long-term transplant studies. Taken together, our data suggest that Tgif1 suppresses stem cell self-renewal and provide clues as to how reduced expression of TGIF1 may contribute to poor long-term survival in patients with AML.

Abstract 18445: Embryonic Origins of Vascular Progenitors in the Adult Murine Aorta

Background: The vasculature is derived from many different sources during embryonic development, including neural crest, and mesoderm of splanchnic, pharyngeal and somitic origin. Vascular smooth muscle cells (VSMC) of the aorta are similarly derived from several embryonic sources, however the developmental and phenotypic heterogeneity of their progenitors is poorly understood. Methods &amp; Results: Using specialized Cre mice which irreversibly mark cells of specific embryonic origin, we isolated distinct subsets of progenitors from the aorta that are derived from neural crest (Wnt1), somite (Myf5) and another (Brachyury; T) mesoderm source. Intriguingly, a population of lineage-marked cells that express high levels of the reporter tdTomato (tdT-hi) is uniquely expressed in the aorta, and not found in blood, bone marrow or spleen. In Myf5- and T-marked lineages, this tdT-hi population gave rise to clonally-derived spheres that differentiated into VSMC, adipocytes, S100β-positive neural cell and macrophage in vitro. Consistent with the observed progenitor activity of the tdT-hi population, a subset of these cells co-express Sox2 and Sca1, known adult stem/progenitor cell markers. Further evidence of progenitor activity was demonstrated in an in vitro CFU assay where tdT-hi cells gave rise to Mac3+Ly6C+ macrophage and Ly6G+ neutrophils. As experimental cell culture is prone to artifact, and differentiated cell types may not be represented in vivo, we conducted complementary analyses in mice. Leukocytes such as macrophage, B-cells and T-cells from Myf5-Cre mice were found to be lineage-marked cells with lower levels of tdT expression (tdT-lo), suggesting derivation from somitic mesoderm and differentiation from the tdT-hi aortic progenitor. Indeed, somite-derived leukocytes were enriched over 6-fold in the arch of the aorta vs. the blood, bone marrow and spleen of these mice. Summary: These data support the existence of an aorta-resident Myf5 lineage-marked (i.e. somitic mesoderm-derived) progenitor capable of contributing to both VSMC and blood compartments. How these neural crest vs. somite- and other mesoderm-derived aortic-resident progenitors and their progeny participate in vascular pathogenesis is not yet known.

CDX2 regulates Human Leukemogenesis Via Modulation Of The Wnt-Signaling Pathway

IntroductionThe caudal-type homeobox (Cdx) gene family has been mainly studied during early development for its role in axial elongation and antero-posterior patterning. More recently, Cdx genes were shown to regulate embryonic hematopoiesis by interactions with the canonical Wnt pathway and Hox genes. The role of Cdx genes in adult hematopoiesis remains poorly understood. Adult hematopoietic stem and progenitor cells derived from healthy murine bone marrow (BM) express low levels of Cdx1 and Cdx4 but not Cdx2. However, the majority (>80%) of human acute myeloid (AML) and lymphoid leukemias (ALL) were shown to express the human homologue CDX2, and ectopic induction of Cdx2 expression was sufficient to robustly induce myeloid leukemia in murine bone marrow cells. On the molecular level, the leukemogenic activity of Cdx2 was associated with modulation of Hox and Klf4 gene expression (Faber et al, 2013). The current study further explores the role of CDX2 in leukemogenesis by analyzing the effects of CDX2 expression induction or repression on human healthy and malignant hematopoietic cells and its molecular effects on the Wnt signaling pathway known to regulate Cdx genes during embryonic development. MethodsHuman bone marrow or mobilized peripheral blood derived CD34+ cells as well as the human leukemic cell lines SKM-1, NOMO-1, EOL-1 and NALM16 were exposed to lentiviruses containing CDX2 overexpression, shRNAs against CDX2 or control constructs. Efficient modulation of CDX2 expression was verified on gene expression level by qRT-PCR and on protein level by immunoblot analysis. CDX2 modified and control cells were subjected to growth, colony forming (CFU), cell cycle, flow cytometry and qRT-PCR gene expression analysis assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rγnull (NSG) mice. To explore the effect of Dickkopf-1 (DKK1), recombinant human DKK1 protein or carrier was supplemented to the methylcellulose in CFU assays. ResultsshRNA-mediated knockdown of CDX2 in leukemic cell lines lead to reduced growth (SKM-1, NALM16) and CFU formation (SKM-1 cells). Consistently, CDX2 knockdown SKM-1 cells showed lower ability to repopulate NSG mice and, upon subcutaneous injection in the flank, gave rise to much smaller tumors when compared to control cells, supporting the notion that CDX2 plays roles in human leukemogenesis. In contrast to the data published in mice, healthy human CD34+ cells transduced to overexpress CDX2 were unable to induce leukemia upon transplantation in NSG mice within an observation period of 5 months. On the molecular level, CDX2 modified cells showed differential expression of Klf4 and Hox but also Wnt pathway associated genes. Notably, robust induction of the canonical Wnt-inhibitory molecule DKK1 was observed in both healthy CD34+ stem/progenitor and leukemic cells upon CDX2 induction, while CDX2 suppression showed opposite effects. Analysis of the DKK1 promotor region revealed an interspecies conserved putative binding site for CDX2 as well as multiple HOX gene binding sites, suggesting that CDX2 can modulate DKK1 expression directly but also via its downstream HOX genes. Importantly, CFU assays performed on CDX2-knockdown cells showed a rescue of colony formation upon stimulation with DKK1 protein as compared to treatment with carrier only, demonstrating that the observed molecular interaction is functionally relevant in human leukemic cells. In contrast, control leukemic cells treated with DKK1 showed reduced CFU formation, indicating that CDX2 might act through DKK1 activation to fine-tune Wnt signal activation to the dosage that best promotes leukemogenesis and leukemic cell growth and survival. ConclusionTaken together, our data indicate that CDX2 employs DKK1 activation to modulate the Wnt signaling pathway and thereby growth, clonogenic capacity as well as in vivo tumorigenicity of human leukemia cells. In contrast to murine cells, CDX2 activation requires cooperative molecular events in order to induce leukemia in human healthy stem and progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Mir-199b, a Novel Tumor Suppressor miRNA In Acute Myeloid Leukemia With Prognostic Implications In FAB-M5 Subtype

Acute myeloid leukemia (AML) manifests a marked heterogeneity in both response to therapy and patient survival, observations that likely reflect its varied pathogenesis. Dysregulation of miRNAs that can act as tumor suppressors or oncogenes can result in tumorigenesis. One miRNA of interest is human miR-199b-5p, which is an intragenic miRNA encoded in the Dynamin 1 gene. Previously we demonstrated that miR-199b was significantly downregulated in AML and targets Podocalyxin and Discoidin Domain Receptor 1. Herein we investigated the functional role of miR-199b in AML and its prognostic implications. We first examined the relative miR-199b expression in steady state hematopoiesis and showed CD33+ myeloid progenitors had the highest miR-199b expression (p<0.03). In order to test whether suppression of miR-199b results in myeloproliferation, human bone marrow derived CD34+ cells were transduced with anti-miR-199b or control lentivirus particles. To examine the effect of miR-199b silencing on colony forming abilities, a CFU assay was performed. At day 16, silencing of miR-199b in CD34+ cells resulted in significant increases in CFU-GM colonies. We then investigated the effect of decreased miR-199b on hematopoietic stem cells (HSC) proliferation via aldehyde dehydrogenase assay. Anti-miR-199b CD34+ cells exhibited a significant increase in HSC numbers compared to control CD34+ cells demonstrating a proliferative advantage for HSC with decreased miR-199b. Via The Cancer Genome Atlas (TCGA), we analyzed the molecular and clinical characteristics of 166 AML cases to investigate a prognostic role for miR-199b. The Kaplan-Meier curves for high and low expression values of miR-199b and the observed distribution of miRNA expression revealed the highly expressed group had significantly better survival outcomes (p<0.016, log rank test). In addition, miR-199b expression varied by cytogenetic risk category in the least squared mean plot, with significantly higher miR-199b expression for the favorable category compared to the intermediate and poor categories combined (p<0.0001). Therefore, low expression of miR-199b predicts worse survival outcome and higher cytogenetic risk in AML. Additionally, there was significant difference between miR-199b expression across the AML subtypes with particularly low expression found in the M5 subtype. Furthermore, M5 subtype showed a poor prognosis with a one-year survival rate of only 25%, compared with 51% survival in the overall sample (p<0.024). With AML patients grouped into M5 versus other subtypes to look at the dichotomized miR-199b variable, we found that all of the M5 patients have low miR-199b expression (p<0.0001). Due to miR-199b significantly correlating with M5 subtype, the M5 cell line THP-1 was utilized to determine the epigenetic regulation of miR-199b in AML. Recently, epigenetic alterations such as histone modifications and DNA methylation have been shown to deregulate miRNA expression. Therefore, treatment of THP-1 cells for 24 hours with HDAC inhibitors AR-42 (2uM), Panobinostat (0.7uM), or Decitabine (demethylating agent, 5uM) showed miR-199b expression was significantly elevated upon AR-42 and Panobinostat treatment (p<0.0002 and p<0.0001 respectively). Furthermore, AR-42 and Panobinostat treated cells showed drastic apoptosis in both treatments. Thus, epigenetic regulation of miR-199b appears to be via histone modification. To further understand the hematopathological consequences of decreased miR-199b in the onset and development of myeloid leukemia, we employed a transduce / transplant mouse model. The Lin-Sca-1+Kit+ (LSK) population was isolated from Ly5.2 C57Bl6 mice, transduced with anti-miR-199b or control lentivirus particles, and transplanted into Ly5.1 C57Bl6 recipient mice. Short term (3.5 weeks) peripheral blood analysis revealed that loss of miR-199b results in significant increase of % neutrophils (p<0.001), % monocytes (p<0.04), and % eosinophils (p<0.02). Further analysis to determine the role of miR-199b in leukemogenesis is ongoing via this mouse model. Taken together, for the first time our results demonstrate that in vivo loss of miR-199b can lead to myeloproliferation and in vitro HDAC inhibitors can restore miR-199b-5p expression and promote apoptosis. Lastly, low miR-199b-5p in AML patients leads to worst overall survival and has prognostic significance for FAB-M5 subtype. Disclosures:No relevant conflicts of interest to declare.

Generation Of Mature Erythroid Cells From Human Embryonic Stem Cell Derived Sacs and Optimization Of Erythroid Cell Generation

Human embryonic stem cells (hESCs) represent a potential alternative source for red blood cells (RBCs) for transfusion therapy. However, in vitro production of erythroid cells from hESCs has two critical limitations: low-efficiency of erythroid cell generation and absent production of adult type β globin. Previously, hESC-derived erythroid cells were generated by co-culture with feeder cells through embryoid body formation, and these erythroid cells produced embryonic type ε globin or fetal type γ globin. Recently, embryonic stem cell-derived sacs (ES sacs) were generated with hemangioblast markers and generated functional platelets (Takayama, Blood. 2008). We hypothesized that hESC-derived β globin producing erythroid cells could be efficiently produced through the hemangioblast-like ES sac and developed new strategies to generate mature erythroid cells in vitro from hESCs using the ES sac.ES sacs were generated from H1 hESCs using ES sac media containing 15% fetal bovine serum supplemented with 20ng/ml VEGF for 14-16 days. Spherical cells in suspension in these ES sacs were harvested, and erythroid cells were generated using a modified human erythroid massive amplification culture for 2 weeks (Migliaccio, Blood cells Mol Dis. 2002). We evaluated total cell counts, hematopoietic stem/progenitor cell marker (CD34) expression, and erythroid cell marker (CD235a) expression. In addition, we analyzed globin types in erythroid cells by hemoglobin electrophoresis.After erythroid differentiation from suspension cells within ES sacs (containing 12.39% CD34+ cells), we generated CD235a+ erythroid cells (55.47%). Additionally, we confirmed morphology of erythroid cells by cytospin examination and erythroid colony formation by CFU assays using harvested suspension cells within the ES sac. To improve upon erythroid cell differentiation during ES sac production, we added SCF, IL-3, Flt-3L, TPO, and EPO to the ES sac media from day 9-15. The number of CD34+ cells increased 2.7 fold (19x10e3 cells vs. 7x10e3 cells) by supplementation with SCF, IL-3 and EPO while the number of CD235a+ cells increased 6.6 fold (239x10e3 cells vs. 36x10e3 cells) after erythroid differentiation. These data suggest that cytokine stimulation can induce erythroid differentiation during ES sac production.We then hypothesized that efficient erythroid cell generation might be further promoted by shifting the balance between hematopoietic cell differentiation and endothelial cell differentiation in the hemangioblast-like ES sac. Thus, we added BMP4 to ES sac media from day 6-15 to improve hematopoietic cell differentiation, and we compared no VEGF with normal concentration VEGF (20ng/ml) from day 0-15 to reduce endothelial cell differentiation. After supplementation with BMP4, CD34+ cell numbers increased 3.1 fold (125x10e3 cells vs. 40x10e3 cells) among suspension cells in the ES sac, while the number of CD235a+ cells increase 2.6 fold (646x10e3 cells vs. 250x10e3 cells) after erythroid differentiation. In the no VEGF condition, the number of CD34+ cells increased 3.0 fold (118x10e3 cells vs. 40x10e3 cells) among suspension cells in ES sac, while the number of CD235a+ cells increased 2.8 fold (710x10e3 cells vs. 250x10e3 cells). These data suggest that erythroid differentiation during ES sac production was induced by not only stimulating hematopoietic cell differentiation but also suppressing endothelial cell differentiation.Finally, we evaluated hemoglobin types among hESC-derived erythroid cells differentiated from ES sacs without cytokine supplementation. We detected embryonic type hemoglobin (27.9%), fetal type hemoglobin (61.3%), and adult type hemoglobin (10.8%) by hemoglobin electrophoresis. These data suggests that ES sac-derived erythroid cells produce more adult type hemoglobin as compared to previous strategies.In conclusion, we demonstrated for the first time that erythroid cells can be efficiently generated from hESCs derived ES sacs and further improved by cytokine supplementation. Our data suggest that ES sacs have fractions of definitive hematopoiesis which can induce generation of mature red blood cells producing adult type hemoglobin. Disclosures:No relevant conflicts of interest to declare.

STS1 and STS2 Are Important Regulators Of Hematopoietic Stem Cells

The receptor tyrosine kinases FLT3 and KIT are highly expressed on the surface of leukemic blasts in most patients with acute myeloid leukemia. Although about one third of patients display activating mutations in FLT3 (and more rarely in KIT), the majority of patients have no mutations in FLT3 or KIT. Previously, we demonstrated that Cbl functions as the E3 ligase for both FLT3 and KIT, and that ligase-inactivating mutations of Cbl stabilize FLT3 and KIT on the cell surface by preventing endocytosis and degradation (Sargin et al, Blood 2007). Furthermore, we demonstrated that expression of E3-ligase deficient Cbl mutants led to the development of a myeloproliferative disease in a murine bone marrow transplantation model (Bandi et al, Blood 2009). However, Cbl mutations are rarely found in AML.Here, we investigated the role of the Cbl regulators suppressors of T-cell signaling 1 and 2(STS1 and STS2) in stabilizing wild-type FLT3 and KIT on the cell surface of hematopoietic stem and progenitor cells (HSPCs). STS1 is ubiquitously expressed, while STS2 expression is restricted to the hematopoietic tissue. STS1 and STS2 constitutively bind to Cbl, while their binding to FLT3 and KIT is dependent on ligand-activation by FL and SCF, respectively. Interestingly, STS1 (but not STS2) functions as a tyrosine phosphatase for both ligand-activated FLT3 and KIT. This required the PGM domain of STS1, as PGM point mutant of STS1 did not dephosphorylate FLT3 or KIT. In line with this, knockdown of STS1 using stably expressing shRNA constructs showed a significant hyperphosphorylation of FLT3 and KIT.By using STS1/STS2 single and double knockout mice, we analyzed the effects of STS1 and STS2 on hematopoietic stem and progenitor cells in vivo. We found that deficiency of STS1 causes an increase of both absolute number and frequency of LSK (lineage marker-, KIT+, Sca1+) cells, which contain HSPCs. This phenotype was even more pronounced in STS1 and STS2 double knockout (dKO) mice, and is mainly attributable to the short term hematopoietic stem cell (ST-HSC) and multipotent progenitor (MPP) cell population, as defined by both standard and SLAM markers. Colony assays using primary bone marrow cells revealed a significantly higher colony forming ability in STS1-KO and dKO cells compared to wild type (wt) cells, particularly after serial replating. A careful analysis of the cells derived from methylcellulose culture revealed an increased proportion of immature (Mac1- CD48+ CD16/32-) cells in STS1-KO and dKO cells. Competitive repopulation assays showed an advantage for dKO cells when compared to wt, suggesting that the LT-HSC compartment is also affected. Even more pronounced were the differences in CFU-S assays (colony forming units spleen), displaying significantly more colonies of dKO compared to wt donor cells, functionally demonstrating a significantly increased ST-HSC / MPP population in dKO donors. A detailed analysis of the downstream signaling events demonstrated that loss of STS1 specifically causes an activated PI3-Kinase / AKT pathway.In summary, our data demonstrates that STS1 functions as a phosphatase of FLT3 and KIT and, using genetic mouse models, indicates a critical role in the maintenance and proliferation of long-term and short-term hematopoietic stem cells. This may also affect sensitivity to kinase inhibitors. Disclosures:No relevant conflicts of interest to declare.

The Short Lef-1 Isoform, Lacking The β-Catenin Binding Domain, Is Not Acting As a Dominant Negative Lef-1 Variant, But Is a Hematopoietic Active Protein With Unique DNA Binding Properties

Studies in the hematopoietic system of human and mice have demonstrated that the Wnt signaling pathway is essential for maintenance, activation and proliferation of normal hematopoietic stem cells. Lef-1 is a member of the Lef-1/T-cell-factor (Tcf) family of transcription factors regulated by the canonical Wnt signaling pathway and generally directly interacts with β-catenin in transcriptional complexes to induce expression of target genes, including the cell cycle regulators cyclin D1 and c-myc. We have previously shown that aberrant expression of Lef-1 perturbs normal hematopoietic stem cell (HSC) and progenitor function and induces acute myeloid leukemia in the murine bone marrow transplantation model and that it is a novel prognostic independent factor in patients with normal karyotype AML (Petropoulos et al. JEM, 2008; Metzeler et al., Blood 2012). Furthermore, we could recently demonstrate that shRNA mediated depletion of Lef-1 drastically compromises the function of long-term repopulating stem cells (Edmaier et al., Leukemia, in press), indicating a vital function of Lef-1 at the level of the most primitive stem cell compartment. So far, all these Lef-1 functions are ascribed to the full-length transcript of Lef-1, which acts as transcriptional mediator of Wnt signaling via its β-catenin binding domain, whereas the shorter Lef-1 isoform, deriving from an intronic promoter is thought to function as a dominant negative variant. To dissect the role of the ‘dominant negative' Lef-1 isoform we first retrovirally engineered primary murine bone marrow cells to express the N-terminal deleted Lef-1 (Lef-1d56), lacking the β-catenin binding domain in comparison to the full-length Lef-1 (Lef-1WT) and the empty retroviral EGFP control. Loss of binding to β-catenin was validated by Co-IP for Lef-1d56. Of note, Lef-1d56 induced a 2,05fold and significant increase at the level of clonogenic cells in vitro compared to the control (n=3; p<0,05) and did not differ substantially from the activity of Lef-1 WT. β-catenin binding did not impact Lef-1 activity at the level of the short-term repopulating stem cells as documented in the CFU-S assay with almost comparable CFU-S frequencies between the wild-type and the mutant Lef-1 (d56) (73 CFU-S/1x105 and 64 CFU-S/1x105 , respectively, compared to 38 CFU-S/1x105 for the EGFP control; p<0.05). In clear contrast, loss of β-catenin interaction reduced the CRU frequency dramatically (1:597.197(Lef-1 WT) and 1:1.675.238 (Lef-1 d56), respectively and 1: 1.233.152 for the EGFP control; p=0,066), indicating the necessity of Lef-1 to collaborate with β-catenin at the level of the long-term repopulating stem cell. Retroviral expression of Lef-1 d56 in murine bone marrow stem and progenitor cells for 48h induced a distinct gene expression profile and deregulated more genes than Lef-1WT compared to the EGFP control. Differentially expressed genes between Lef-1d56 and Lef-1WT comprised known hematopoietic factors such as like Gata2, Ets1 and genes associated to the G-protein coupled receptor protein signaling. ChIP-Seq in hematopoietic murine cells revealed unique binding sites for Lef-1d56 compared to Lef-1WT with binding of the Lef-1d56 to promoters of Wnt5a and CD81. Taken together, our data clearly indicate that loss of the β-catenin binding site does not convert Lef-1 into an overall dominant negative variant, but creates a ‘neomorphic' isoform with distinct biological and DNA binding properties. Analyses are ongoing which quantify the expression of LEF-1WT and the β-catenin domain lacking LEF-1 isoform in over 100 patients with normal karyotype AML in comparison to normal human stem and progenitor cells to test potential differences between normal and leukemic cells as well as associations of the two LEF-1 transcripts with the mutational status, gene expression and treatment outcome in this AML patient group. Disclosures:No relevant conflicts of interest to declare.

Imatinib mesylate has an inhibitory effect on hematopoiesis in a mice model

Imatinib mesylate (Gleevec or STI-571), a tyrosine kinase inhibitor, can potently block both platelet-derived growth factor (PDGF) receptor-α and β by inhibiting the receptor tyrosine kinase activity. Imatinib mesylate has been successfully used to treat patients with chronic myeloid leukemia (CML) and Ph positive acute lymphoid leukemia (ALL). However, a significant number of patients with imatinib mesylate treatment have developed neutropenia, thrombocytopenia and anemia (van Deventer, Hall et al. 2002). Our previous studies have demonstrated that PDGF/PDGFR plays an important role on thrombopoiesis, and blockage of PDGFR by imatinib inhibited the downstream signal pathway, which led to reduce of megakaryocyte production (Ye, et al. Haematologica, 2010). This may partially explain the imatinib-related thrombocytopenia. However, the role of imatinib on normal hematopoiesis remains to be investigated. In the present study, we identified the expression of PDGFR-β on the human megakaryoblastic cells: Meg-01, CHRF-288-11, Dami and M-07e, human erythromyeloblastoid leukemia cells: K-562 and human myeloblastic cells: HL-60 by RT-PCR, western blot and flow cytometry. We then demonstrated the effect of imatinib on normal hematopoiesis in the 8 wk old C57BL/6 mice model. Gleevec (25 mg/kg/day) dissolved in distilled water or water only were given orally for up to 2 wks. 15ul whole blood was taken from postorbital weekly to perform blood cell counts. The Gleevec -treated group showed a reduction of platelet count as compared to the control at day 14 (543.3±71.95×109/L vs. 330.0±62.23×109/L, n=5, P<0.05). Similarly, Gleevec caused a significant decrease in the number of white blood cells on day 14 (9.667×±0.8511×109/L vs. 6.080±0.8015×109/L, n=5, P<0.05). While no significant differences were found on red blood cell count between Gleevec and control groups. The mice bone marrow was harvested from each group to perform CFU assays. Gleevec significantly inhibited the formation of bone marrow CFU-MK (15.33±1.764 vs 3.333±0.6667, n=3), and CFU-GM(13.50±2.784 vs 6.333±0.7265, n=3) compared with the control. There were no significant differences found in CFU-GEMM and CFU/BFU-E formation between the Gleevec and control treatment. Our studies suggested that PDGFR were expressed on hematopoietic cells. Imatinib mesylate, an inhibitor of PDGFR, not only played an important role in malignant hematopoiesis, but also in normal hematopoiesis. Administration of Gleevec reduced white blood cells and megakaryocytes/platelets production, which implied its potential therapeutic role in treating myeloproliferative diseases, such as Essential Thrombocytosis. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Intracellular Survival of Multi Drug Resistant Clinical Isolates of Mycobacterium tuberculosis in Macrophages by Curcumin

Curcuma longa commonly known as turmeric has been used in Indian Ayurvedic medicine as a constituent to treat various disorders. It is by now clear that principal curcuminoid of turmeric; curcumin, a yellow pigment, is responsi- ble for these beneficiary activities. The aim of the present study was to evaluate anti-mycobacterial effect of curcumin (CMN) on intracellular growth of MDR clinical isolates of Mycobacterium tuberculosis (MTB). Curcumin was evaluated for its efficacy to inhibit the intracellular growth of MTB H37Rv and two MDR clinical isolates in Raw 264.7 cell line us- ing CFU assay. Resazurin microtiter plate assay (REMA) was used to evaluate its direct anti-mycobacterial activity. Curcumin, though did not show direct anti-mycobacterial activity against three MTB strains, exhibited dose dependent in- hibition of intracellular growth for MTB H37Rv as well as two MDR clinical isolates. These results suggest that CMN could be a potential candidate for future, novel adjunctive anti-TB therapy.

Anti-apoptotic effect of Astragalus Polysaccharide on myeloid cells

This study was aimed to assess the effect of Astragalus Polysaccharide (ASPS) on in-vitro hematopoiesis. CFU-GM assays were used to determine the effect of ASPS and thrombopoietin (TPO) on granulocytic-monocyte progenitor cells. The CFU assays were also used to investigate the effect of ASPS on the proliferation of HL-60 cells.HL-60 cells were cultured with serum-free RPMI 1640 medium and treated with or without of different concentrations of ASPS. After 72 h incubation, the number of cells were counted.In addition, the caspase-3 and JC-1 expression was determined by flow cytometry with Annexin V/PI double staining. The results showed that ASPS (100, 200 µg/ml) and TPO (100 ng/ml) significantly promoted CFU-GM formation in vitro. Various concentrations of ASPS and TPO also promoted the colony formation of HL-60 cells, the largest effect of ASPS was observed at a concentration of 100 µg/ml. There were no synergistic effects between TPO and ASPS on cellular proliferation. The results also showed that ASPS significantly protected HL-60 cells from apoptosis in condition of serum-free medium culture, suppressed caspase 3 activation, and reduced the cell apoptosis. It is concluded that ASPS can significantly promote the formation of bone marrow CFU-GM and the proliferation of HL-60 cells, the optimal concentration of ASPS is at 100 µg/ml. In the absence of serum inducing apoptosis, ASPS also significantly reduced the apoptosis of HL-60 cells via suppressing the activation of caspase-3.

Expression and promoter methylation changes of the P15INK4b during ex vivo cord blood CD34+ cell expansion following co-culture with mesenchymal stromal cells

BackgroundBecause of the insufficient number of cord blood hematopoietic stem cells (CB-HSC), expansion of these cells seems to be important for clinical application in adults. Cell cycle inhibitors are important regulators in normal hematopoietic regeneration. In this study, mRNA expression and promoter methylation status of p15INK4b were evaluated during CB-HSC ex vivo expansion using cytokines and in co-culture system with a mesenchymal stem cells (MSCs) feeder layer.MethodsEx vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with an MSCs feeder layer, cytokines without a MSCs feeder layer, and co-culture with MSCs without cytokines. After expansion, measuring the total number of cells, CD34+ cells, and CFU assay was performed. Methylation status of the p15INK4b gene promoter was analyzed using methylation-specific polymerase chain reaction and p15 mRNA expression was evaluated by real-time reverse transcriptase polymerase chain reaction.ResultsMaximum CB-HSC expansion was observed on day 10 of expansion. The data showed that after 10 days, p15 mRNA expression in the expanded cells in all the three culture conditions was higher than in CD34+ fresh cells (P < 0.01). p15 gene promoter of expanded CD34+ cells remained in an unmethylated form just like fresh CD34+ cells in all the three culture conditions at days 5, 10, and 14 of culture.ConclusionsExpression of p15INK4b in HSCs was not decreased during ex vivo expansion. Also, no methylation of p15 promoter was observed, otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.

Murine Model of Disseminated Fusariosis: Evaluation of the Fungal Burden by Traditional CFU and Quantitative PCR

Systemic disease is the most severe clinical form of fusariosis, and the treatment involves a challenge due to the refractory response to antifungals. Treatment for murine Fusarium solani infection has been described in models that employ CFU quantitation in organs as a parameter of therapeutic efficacy. However, CFU counts do not precisely reproduce the amount of cells for filamentous fungi such as F. solani. In this study, we developed a murine model of disseminated fusariosis and compared the fungal burden with two methods: CFU and quantitative PCR. ICR and BALB/c mice received an intravenous injection of 1×10(7) conidia of F. solani per mouse. On days 2, 5, 7, and 9, mice from each mice strain were killed. The spleen and kidneys of each animal were removed and evaluated by qPCR and CFU determinations. Results from CFU assay indicated that the spleen and kidneys had almost the same fungal burden in both BALB/c and ICR mice during the days of the evaluation. In the qPCR assay, the spleen and kidney of each mouse strain had increased fungal burden in each determination throughout the entire experiment. The fungal load determined by the qPCR assay was significantly greater than that determined from CFU measurements of tissue. qPCR could be considered as a tool for quantitative evaluation of fungal burden in experimental disseminated F. solani infection.

Determination of In Vitro Activities of Solithromycin at Different pHs and Its Intracellular Activity against Clinical Isolates of Neisseria gonorrhoeae from a Laboratory Collection

We evaluated the activity of solithromycin against 196 clinical gonococcal isolates collected at the Public Health Ontario Laboratories, Toronto, Canada, including isolates with different levels of azithromycin resistance, as well as the role of pH in MIC determinations using pH-adjusted agar plates (pH range, 5.6 to 7.6). In vitro invasion assays were performed using monolayers of HeLa epithelial cells and clinical gonococci displaying different azithromycin MICs; infected cultures were treated with solithromycin, and its intracellular activity was determined by CFU assays after 3 and 20 h of exposure. Solithromycin displayed a MIC50 and MIC90 of 0.0625 and 0.125 μg/ml, respectively, making its activity at least 4-fold higher than that of azithromycin. Clinical isolates with elevated MICs for azithromycin (MICs of ≥2,048 μg/ml and 4 to 8 μg/ml) showed solithromycin MIC values of 8 and 0.5 μg/ml, respectively. In contrast to azithromycin, solithromycin MICs were not significantly affected by acidic pHs, suggesting more stability at lower pH. Moreover, when intracellular Neisseria gonorrhoeae isolates were incubated with solithromycin at 4 times, 1 times, and one-fourth of the MIC, the exposure to solithromycin resulted in the progressive loss of viability of most isolates over time. The intracellular activity of solithromycin, combined with the low MICs to this agent, indicates that it may be an attractive option for gonorrhea treatment if clinical trials in development reveal that this drug can be used safely in adult indications, especially when multidrug-resistant clinical isolates are now emerging.

AB0064 Spontaneous adipogenesis in bone marrow derived mesenchymal stromal cells after chronic treatment with glucocorticoids

BackgroundMesenchymal stromal cells (MSCs) are a promising therapeutic tool for many inflammatory autoimmune diseases because of their immunosuppressive potential. MSCs autologous transplantation showed less therapeutic efficacy respect to the allogenic...

Abstract 4352: Down regulation of retinoblastoma protein expressionimpedes the antileukemic activity of erufosine.

Abstract Erufosine is a new antineoplastic agent from the class of alkylphosphocholines, which interferes with AKT phosphorylation, the related signal transduction and confers apoptosis. An improved understanding of its mode of action is important for its potential clinical application. The present study demonstrates that erufosine's antileukemic efficacy is related to the level of cellular retinoblastoma (Rb) protein expression. Stable Rb-knockdown in SKW-3 leukemia T-cells was induced by lentiviral delivery of constructs expressing short-hairpin-RNA species with 21 or 27 bp lengths. The cytotoxicity of erufosine was tested via MTT proliferation test on thus engineered cell populations with reduced Rb expression levels. Likewise, the clonogenicity of these engineered cell populations was determined by CFU assay before and after treatment with erufosine. The effects on cell cycle were investigated by FACS analysis. The expression of signal transduction factors was examined by RT-PCR and immunoblot. The Rb protein expression levels in the shRNA-transduced cells ranged from 1%- 36% of wild type or nonsense control. Low Rb expression correlated with significantly diminished antiproliferative and anticlonogenic activities of erufosine (p&amp;lt;0.05) at concentrations near and above the IC50. Erufosine induced a G2 arrest, and complete Rb-deficiency aggravated this condition. With regard to proteins involved in cell cycle and apoptosis induction, Rb-deficiency reduced the expression of cyclin D3, that of the subsequently regulated kinase Cdk 4 and activated caspases 3 and 9, followed by PARP-cleavage. In partial variance, erufosine reduced cyclin D3 at RNA and protein levels, but affected not or only slightly the expression of its partner kinase Cdk 4. Rb-knockdown impaired the cytotoxic effect of erufosine significantly and contributed to the formation of a resistant cell fraction. It is concluded that Rb is a main mediator of erufosine's antileukemic activity and this suggests that the effect of the drug in patients might be predicted by determining the Rb expression status. Citation Format: Maya M. Zaharieva, Milen Kirilov, Minquang Chai, Stefan M. Berger, Spiro M. Konstantinov, Martin R. Berger. Down regulation of retinoblastoma protein expressionimpedes the antileukemic activity of erufosine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4352. doi:10.1158/1538-7445.AM2013-4352

Abstract 5448: Genome-wide ChIP-seq of C/EBPε reveals networks controlling myelopoiesis .

Abstract CAAT/enhancer-binding protein epsilon (C/EBPε) is known to be a critical transcription factor mediating terminal differentiation of granulocytes and a target to boost killing of bacteria by the innate immune system. However, our data found that ∼9 month old C/EBPε knockout (KO) mice developed a novel phenotype similar to the myeloproliferative disorder. In bone marrow (BM) of C/EBPε KO mice, colonies of granulocyte-macrophage progenitor (CFU-GM) were increased (143±21 vs. 59±13, P&amp;lt;0.01) while erythroid progenitors (BFU-E) were decreased (8±2 vs.14±4, P&amp;lt;0.01) as compared to age- and sex- matched wild type controls. Flow data showed that the percentage of BM immature myeloid (Mac1+Gr1int) were much higher (48%±3% vs. 9%±5%, P&amp;lt;0.01) while Ter119+ erythrocytes were less (13%±4% vs. 47%±1%, P&amp;lt;0.01) than control. In addition, the ratio of spleen/ body weight in C/EBPε KO mice was augmented (0.81%±0.16% vs. 0.25%±0.03%, P&amp;lt;0.01) and spleen H&amp;E staining showed extramedullary hemopoiesis. Compared to spleen samples in control mice, CFU-GM (97±16 vs. 9±2, P&amp;lt;0.01), BFU-E (20±5 vs. 4±1, P&amp;lt;0.01) and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitors (CFU-GEMM, 11±3 vs. 2±1, P&amp;lt;0.01) in C/EBPε KO mice were significantly increased. Consistent with spleen CFU assay, the percentage of Mac1+Gr1int immature myeloid cells (18%±8% vs. 3%±1%, P&amp;lt;0.05) and Ter119+ erythrocytes (38%±1% vs. 12%±2%, P&amp;lt;0.01) were raised in KO than in control mice by flow cytometry analysis. Taken together, C/EBPε deletion in mice blocks myeloid differentiation and impairs erythoid development in BM as well as leads to extramedullary hemopoiesis in spleen. To investigate regulatory networks controlled by C/EBPε, we performed genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq) in wild type and C/EBPε KO mice BM using N- and C-terminal C/EBPε antibodies. We have identified ∼15000 significant C/EBPε binding sites and as with the majority of transcription factors, C/EBPε targets predominantly distal intergenic and intronic regulatory regions. However, 15% of significant binding sites are localized within gene promoters and some of them are known to play a critical role in hematopoietic lineage differentiation such as PU.1, RUNX1 and SCL. Interestingly, C/EBPε also binds known distal regulatory regions as the PU.1 enhancer, and our data has highlighted novel potential intergenic and intronic enhancers for RUNX1. The ChIP-PCR results of these transcription factors are consistent with ChIP-seq. In summary, our study suggests that C/EBPε can have a profound effect on myeloid development by regulating key genes and its deregulation can produce a myeloproliferative abnormality. Citation Format: Tong Yin, Touati Benoukraf, Phillip H. Koeffler. Genome-wide ChIP-seq of C/EBPε reveals networks controlling myelopoiesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2013-5448 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Stemvision™: a bench-top instrument for auto-mated and standardized counting of all hematopoietic colony types in CFU assays of human bone marrow, cord blood and mobilized peripheral blood cells

Stemvision™: a bench-top instrument for auto-mated and standardized counting of all hematopoietic colony types in CFU assays of human bone marrow, cord blood and mobilized peripheral blood cells