Formation Of Colony-forming Units Research Articles

Carvacrol Improves Vascular Function in Hypertensive Animals by Modulating Endothelial Progenitor Cells.

Carvacrol, a phenolic monoterpene, has diverse biological activities, highlighting its antioxidant and antihypertensive capacity. However, there is little evidence demonstrating its influence on vascular regeneration. Therefore, we evaluated the modulation of carvacrol on endothelial repair induced by endothelial progenitor cells (EPC) in hypertension. Twelve-week-old spontaneously hypertensive rats (SHR) were treated with a vehicle, carvacrol (50 or 100 mg/kg/day), or resveratrol (10 mg/kg/day) orally for four weeks. Wistar Kyoto (WKY) rats were used as the normotensive controls. Their systolic blood pressure (SBP) was measured weekly through the tail cuff. The EPCs were isolated from the bone marrow and peripherical circulation and were quantified by flow cytometry. The functionality of the EPC was evaluated after cultivation through the quantification of colony-forming units (CFU), evaluation of eNOS, intracellular detection of reactive oxygen species (ROS), and evaluation of senescence. The superior mesenteric artery was isolated to evaluate the quantification of ROS, CD34, and CD31. Treatment with carvacrol induced EPC migration, increased CFU formation and eNOS expression and activity, and reduced ROS and senescence. In addition, carvacrol reduced vascular ROS and increased CD31 and CD34 expression. This study showed that treatment with carvacrol improved the functionality of EPC, contributing to the reduction of endothelial dysfunction.

The Effect of Tafamidis on Circulating Endothelial Progenitor Cells in Patients with Transthyretin Cardiac Amyloidosis.

Endothelial microvascular dysfunction is a known mechanism of vascular pathology in cardiac amyloidosis (CA). Scientific evidence regarding the possible protective role of the amyloid transthyretin (ATTR) stabilizer, tafamidis, is lacking. Circulating endothelial progenitor cells (cEPCs) have an important role in the process of vascular repair. We aimed to examine the effect of tafamidis on cEPCs. Study population included patients with ATTR-CA. cEPCs were assessed using flow cytometry by the expression of CD34(+)/CD133(+) and vascular endothelial growth factor receptor (VEGFR)-2(+) and by the formation of colony-forming units (CFUs) and production of VEGF. Tests were repeated at pre-specified time-points up to 12months following the initiation of tafamidis. Included were 18 ATTR-CA patients at a median age of 77 (IQR 71, 85) years and male predominance (n = 15, 83%). Following the initiation of tafamidis and during 12months of drug treatment, there was a gradual increase in the levels of CD34(+)/VEGFR-2(+) (0.43 to 2.42% (IQR 1.53, 2.91)%, p = 0.002) and CD133(+)/VEGFR-2(+) (0.49 to 1.64% (IQR 0.97, 2.90)%, p = 0.004). Functionally, increase in EPCs-CFUs was microscopically evident following treatment with tafamidis (from 0.5 CFUs (IQR 0.0, 1.0) to 3.0 (IQR 1.3, 3.8) p < 0.001) with a concomitant increase in EPC's viability as demonstrated by an MTT assay (from 0.12 (IQR 0.03, 0.16) to 0.30 (IQR 0.18, 0.33), p < 0.001). VEGF levels increased following treatment (from 54 (IQR 52, 72) to 107 (IQR 62, 129) pg/ml, p = 0.039). Tafamidis induced the activation of the cEPCs pathway, possibly promoting endothelial repair in ATTR-CA.

The Effect of Proprotein Convertase Subtilisin Kexin Type 9 Inhibitors on Circulating Endothelial Progenitor Cells in Patients with Cardiovascular Disease.

Circulating endothelial progenitor cells (cEPCs) are vital to vascular repair by re-endothelialization. We aimed to explore the effect of proprotein convertase subtilisin kexin type 9 inhibitors (PCSK9i) on cEPCs hypothesizing a possible pleiotropic effect. Patients with cardiovascular disease (CVD) were sampled for cEPCs at baseline and following the initiation of PCSK9i. cEPCs were assessed using flow cytometry by the expression of CD34(+)/CD133(+) and vascular endothelial growth factor receptor (VEGFR)-2(+), and by the formation of colony-forming units (CFUs) and production of VEGF. Our cohort included 26 patients (median age 68 (IQR 63, 73) years; 69% male). Following 3months of treatment with PCSK9i and a decline in low-density lipoprotein cholesterol levels (153 (IQR 116, 176) to 56 (IQR 28, 72) mg/dl), p < 0.001), there was an increase in CD34(+)/CD133(+) and VEGFR-2(+) cell levels (0.98% (IQR 0.37, 1.55) to 1.43% (IQR 0.90, 4.51), p = 0.002 and 0.66% (IQR 0.22, 0.99) to 1.53% (IQR 0.73, 2.70), p = 0.05, respectively). Functionally, increase in EPCs-CFUs was microscopically evident following treatment with PCSK9i (1 CFUs (IQR 0.0, 1.0) to 2.5 (IQR 1.5, 3), p < 0.001) with a concomitant increase in EPC's viability as demonstrated by an MTT assay (0.15 (IQR 0.11, 0.19) to 0.21 (IQR 0.18, 0.23), p < 0.001). VEGF levels increased following PCSK9i treatment (57 (IQR 18, 24) to 105 (IQR 43, 245), p = 0.006). Patients with CVD treated with PCSK9i demonstrate higher levels of active cEPCs, reflecting the promotion of endothelial repair. These findings may represent a novel mechanism of action of PCSK9i.

Effect of short-term in vitro antibiotic administration into the human umbilical cord tissue on cfu count and mesenchymal stem cell immunophenotype

Mesenchymal stem cells play important roles in regenerative and reparative medicine applications. Their differentiation and proliferation abilities both in the human body and in the laboratory, environment have been revealed through many clinical and pre-clinical trials. Umbilical cord tissue is an important source of mesenchymal stem cells and has become a popular option in cord blood banking, especially in the last decade. As done for the cord blood, the steps of obtaining, transferring, processing, and cryopreserving umbilical cord tissue are defined by standard procedures. However, there is limited information about the microbiological contamination rate of tissues and their decontamination before the procedure. This study aimed to assess the effect of in vitro short-term antibiotic treatment applied to the umbilical cord tissue obtained via superficial ethanol disinfection immediately after birth on the formation of colony-forming units (CFUs) and immunophenotype of the mesenchymal stem cells. For this purpose, tissues obtained from 10 individuals were evaluated in three different groups that included the control group and groups of tissues with 5- and 10-min antibiotic treatment. There was no significant difference among the groups in the expression levels of CD45, CD34, CD73, CD105, and CD44, which are the markers used in immunophenotyping. Similarly, no difference was found in CFU formation either. The present study has revealed for the first time that an antibiotic preparation used in in vitro cell culture for cord tissue banking can be used safely in tissue preparation and banking stages. Our results show that short-term in vitro antibiotic administration can contribute to the standard formation during decontamination/disinfection stages in cord tissue banking.

Cryopreservation of lipoaspirates: in vitro measurement of the viability of adipose‐derived stem cell and lipid peroxidation

As the storage time of the fat tissue passes by, lipid peroxidation and creation of by‐products may take place. The objective of this study was to evaluate the cell viability and functional changes of adipose‐derived stem cells (ADSCs) in the cryopreserved lipoaspirates at different temperatures in accordance with lipid peroxidation. Lipoaspirates acquired from liposuction were divided into four different temperature groups and stored at 4°C, −20°C, −80°C, and −196°C. After isolating ADSC from each sample, gross cell morphology and cell viability were compared with doubling time and colony‐forming unit (CFU) formation ability. Acid value, that is, thiobarbituric acid value was measured to assess lipid peroxidation. No viable ADSC was observed in −20°C and −196°C samples for past 1 week and a superior number of the live cells were detected in the 4°C group compared with the −80°C group. However, the persistence of cell division and CFU formation after 1 week was only observed in adipocytes stored at −80°C. Lipid peroxidation mainly occurred at 4°C and −20°C storage samples. If the lipoaspirates were planned to be cryopreserved, it is advised to store at −80°C. However, the number of actually functional ADSCs is very low. Furthermore, even in the cryopreserved status, continuous lipid peroxidation and by‐product creation took place, suggesting shorter preservation period as possible in the clinics.

Astragalus Polysaccharide Promotes Hematopoiesis in an Irradiated Mouse Model and Reduces Apoptosis of Hematopoietic Cells

Background: Huangqi (the dried root of Radix Astragali) is a notable Chinese herb that has been used to promote hematopoiesis for centuries. Astragalus polysaccharide (ASPS), a bioactive constituent extracted from Radix Astragali, may play an essential role for hematopoiesis. The objective of this study is to investigate the hematopoietic effects of ASPS in mouse models and explore underlying mechanism, which provides scientific basis for future applications. Methods: An irradiation-induced myelosuppressive mouse model and colony-forming unit (CFU) assays were used to determine the in vivo hematopoietic effects of ASPS. The anti-apoptotic effects of ASPS were also examined by using annexin V, caspase-3, and JC-1 assays with flow cytometry in HL-60 cells. Results: In the myelosuppressed mouse model, ASPS enhanced the recovery of platelet and red blood cell at day 21, and white blood cell at day 14. ASPS also promoted the CFU formation of all lineages (MK, GM, E, GEMM and F). Morphological study of bone marrow revealed that as compared to the control group, tri-lineage hematopoiesis was preserved in the ASPS- and TPO-treated group. Overall cellularity (MTC/area) of ASPS-treated group was similar to that of TPO. Furthermore, in-vitro study showed that 100 μg/ml of ASPS had maximum effect on CFU formation. ASPS also reduced apoptosis via inhibiting mitochondrial/Caspase-3 signal pathway. Conclusions: ASPS may promote hematopoiesis in irradiated myelosuppressive mice by enhancing hematopoietic proliferation and inhibiting apoptosis of hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

The impact of thermal cycling on Staphylococcus aureus biofilm growth on stainless steel and titanium orthopaedic plates

BackgroundOrthopaedic implant infections are difficult to eradicate because bacteria adhering to implant surfaces inhibit the ability of the immune system and antibiotics to combat these infections. Thermal cycling is a temperature modulation process that improves performance and longevity of materials through molecular structural reorientation, thereby increasing surface uniformity. Thermal cycling may change material surface properties that reduce the ability for bacteria to adhere to the surface of orthopaedic implants. This study aims to determine whether thermal cycling of orthopaedic implants can reduce bacterial growth.MethodsIn a randomized, blinded in-vitro study, titanium and stainless steel plates treated with thermal cycling were compared to controls. Twenty-seven treated and twenty-seven untreated plates were covered with 10 ml tryptic soy broth containing ~ 105 colony forming units (CFU)/ml of bioluminescent Staphylococcus aureus (S. aureus)Xen29 and incubated at 37 °C for 14d. Quantity and viability of bacteria were characterized using bioluminescence imaging, live/dead staining and determination of CFUs.ResultsSignificantly fewer CFUs grow on treated stainless steel plates compared to controls (p = 0.0088). Similar findings were seen in titanium plates (p = 0.0048) following removal of an outlier. No differences were evident in live/dead staining using confocal microscopy, or in metabolic activity determined using bioluminescence imaging (stainless steel plates: p = 0.70; titanium plates: p = 0.26).ConclusionThis study shows a reduction in CFUs formation on thermal cycled plates in-vitro. Further in-vivo studies are necessary to investigate the influence of thermal cycling on bacterial adhesion during bone healing. Thermal cycling has demonstrated improved wear and strength, with reductions in fatigue and load to failure. The added ability to reduce bacterial adhesions demonstrates another potential benefit of thermal cycling in orthopaedics, representing an opportunity to reduce complications following fracture fixation or arthroplasty.

Selection, assessment of virulence to Alphitobius diaperinus, and Pr1 enzyme production of Beauveria bassiana (Bals.) Vuill. isolates cultured at stress temperatures

The entomopathogenic fungus Beauveria bassiana is a promising agent for use in insect control. Its pathogenic activity, as well as other factors such as temperature that can interfere with its development, should be assessed, thus, establishing the foundations for B. bassiana use in biological control programs. The objective of this study was to select and induce tolerance of B. bassiana isolates to high and low temperatures and to assess their virulence before and after exposure to those temperatures. A pre-selection test was performed, in which the tolerance of isolates to stress temperatures was tested and compared to the ideal growth temperature of 25 °C for this organism. For the isolates/temperature combinations resulting in growth, conidia germination and colony-forming units (CFUs) were assessed. The isolates Unioeste 4 and Unioeste 40 exhibited &gt;95% germinated conidia at 16 and 31 °C. Thereafter, they underwent four consecutive passages at maximum and minimum tolerated temperatures (10 and 37 °C). A significant difference in germination was observed between the two isolates at all temperatures tested. More CFUs were observed for Unioeste 4 compared to Unioeste 40 at all temperatures, and in the case of the latter, there was no difference in CFU formation at 10 and 25 °C. For both isolates, decreased vegetative growth was observed at 37 °C. Recovery of virulence was observed in both isolates, as determined by insect mortality. No relationship was observed between production of the enzyme Pr1 and the virulence of the isolates.

Effects of Three Volatile Oxylipins on Colony Development in Two Species of Fungi and on Drosophila Larval Metamorphosis.

The aim of this study is to investigate the effects of three volatile oxylipins on colony development in two fungi and on Drosophila larval metamorphosis. Using an airborne exposure technique, three common and volatile oxylipins (1-octen-3-ol, (E)-2-hexenal, and 1-hexanol) were compared for their effects on spore germination and colony growth in Aspergillus niger and Penicillium chrysogenum, as well as for their effects on the morphogenesis of larvae of Drosophila melanogaster. Conidia of both A. niger and P. chrysogenum plated in the presence of low concentrations (50ppm) of these three volatile organic compounds (VOCs) formed fewer colony-forming units (CFUs) and exhibited reduced radial growth of colonies as compared to controls. When A. niger and P. chrysogenum spores were germinated in the presence of the enantiomers of 1-octen-3-ol, (R)-(-)-1-octen-3-ol had the greatest impact on colony morphology (decreased sporulation and colony diameter), while (S)-(+)-1-octen-3-ol and the racemic form yielded similar morphological changes but to a lesser extent. In addition, Drosophila larvae exposed to vapors of these oxylipins exhibited serious delays in metamorphosis and toxic effects on pupae and adult stages. Low concentration of these three VOCs can significantly inhibit the formation of CFUs and the growth of fungi. (R)-(-)-1-octen-3-ol imposed the greatest impact on fungal morphology compared to (S)-(+)-1-octen-3-ol and the racemic form. The three volatile oxylipins could also delay the metamorphosis of Drosophila and impose toxic effects on its pupae and adult stages.

MicroRNAs and Metabolites in Serum Change after Chemotherapy: Impact on Hematopoietic Stem and Progenitor Cells.

Hematopoietic regeneration after high dose chemotherapy necessitates activation of the stem cell pool. There is evidence that serum taken after chemotherapy comprises factors stimulating proliferation and self-renewal of CD34+ hematopoietic stem and progenitor cells (HSPCs) – however, the nature of these feedback signals is yet unclear. Here, we addressed the question if specific microRNAs (miRNAs) or metabolites are affected after high dose chemotherapy. Serum taken from the same patients before and after chemotherapy was supplemented for in vitro cultivation of HSPCs. Serum taken after chemotherapy significantly enhanced HSPC proliferation, better maintained a CD34+ immunophenotype, and stimulated colony forming units. Microarray analysis revealed that 23 miRNAs changed in serum after chemotherapy – particularly, miRNA-320c and miRNA-1275 were down-regulated whereas miRNA-3663-3p was up-regulated. miRNA-320c was exemplarily inhibited by an antagomiR, which seemed to increase proliferation. Metabolomic profiling demonstrated that 44 metabolites were less abundant, whereas three (including 2-hydroxybutyrate and taurocholenate sulphate) increased in serum upon chemotherapy. Nine of these metabolites were subsequently tested for effects on HSPCs in vitro, but none of them exerted a clear concentration dependent effect on proliferation, immunophenotype and colony forming unit formation. Taken together, serum profiles of miRNAs and metabolites changed after chemotherapy. Rather than individually, these factors may act in concert to recruit HSPCs into action for hematopoietic regeneration.

Interleukin-10 inhibits autonomous myelopoiesis in patients with myelofibrosis.

The spontaneous formation of colony-forming units granulocyte/macrophage (CFU-GM) in semisolid cultures has been shown to be due to the endogenous release of cytokines and/or to the hypersensitivity of cells against growth factors. We have reported that increased autonomous CFU-GM growth is an in vitro characteristic of myelofibrosis (MF) which may reflect aberrant hematopoiesis in vivo. Because of its cytokine synthesis-inhibiting action, we speculated that interleukin-10 (IL-10) may inhibit pathological overproduction of myeloid cells in MF by suppression of autonomous myelopoiesis. In this study, IL-10 significantly inhibited autonomous CFU-GM formation in vitro from peripheral blood mononuclear cells (PB MNC) in 10 of 11 patients with MF tested. In all patients, there was a mean inhibition of 69% ranging from 35% to 100%. Suppression of autonomous CFU-GM formation by IL-10 was dose dependent and reversible by the addition of anti-IL-10 antibodies. Our results indicate that IL-10 is a potentially useful molecule to affect aberrant myelopoiesis in patients with MF.

Proteasome Inhibitor Treatment in Multiple Myeloma Can Mobilize Hematopoietic Stem Cells in the Absence of G-CSF

▪IntroductionProteasome inhibitor (PI) use in patients (pts) with multiple myeloma (MM) has been associated with increased hematopoietic stem/progenitor cell (HSPC) collection yields in both induction and autologous stem cell collection settings (Niesvizky et al., 2013). Animal models have confirmed this observation (Ghobadi et al., 2012). The mechanism remains unclear, but there is suggestion PI treatment affects pathways associated with HSPC anchoring and migration (Niesvizky et al., 2013). The effect of PIs on HSPC migration in the absence of filgastrim (G-CSF) stimulation remains unknown. We sought to characterize the molecular mechanisms of HSPC mobilization in a cohort of pts undergoing active PI treatment.MethodsMM pts undergoing treatment with PIs were consented to obtain peripheral blood (PB) under IRB approval. Pts were eligible if they had symptomatic MM and were undergoing treatment with a PI. Pts receiving alkylating chemotherapy (such as cyclophosphamide) in combination with a PI were excluded. Pts were enrolled on the first day of a new cycle containing a PI. PB was drawn prior to administration of the PI (T0) and just prior to the next dose of PI, 24 or 72 hours later (T1), depending on whether the pt was receiving carfilzomib or bortezomib, respectively. PB mononuclear cells were collected and purified with Ficoll-Paque, viably frozen in CS-10 freezing medium and stored in liquid nitrogen. Serum samples were collected after a 1:2 dilution with PBS and stored at -80oF. Cells were later thawed to perform multiparameter flow cytometry and colony forming unit (CFU) assays. Multiparameter flow cytometry was performed using a BD LSR-II and analyzed using FloJo V9.0 software. Cells were gated on CD45dim SSC-lo characteristics. HSPCs were defined as CD34+/CD133+. Pts were stratified into 3 groups (>2, 1-2, <1) based on fold change in peripheral HSPCs from baseline T0. Expression of surface markers including CD38, CD184, CD202b, CD25, CD90 and CD31 within the HSPC population, were analyzed. Serum protein concentrations were analyzed using ELISAs.ResultsTwenty-three pts consented and collected at the 2 prespecified time points. Six pts (26%) increased the percentage of peripheral HSPCs>2 fold. Nine (39%) and 8 (35%) pts increased the percentage of HSPCs 1-2 fold and <1 fold over T0 percentage, respectively. There were no statistical differences within the 3 groups, in baseline characteristics, prior chemotherapy, use of IMIDs, or radiation exposure history. There was a significant positive correlation between peripheral HSPC fold change and CFU formation p=0.003 indicating the mobilized HSPC population’s capacity to form progeny. Furthermore, there was a significant negative correlation between fold change of HSPCs and CD90 expression on CD34+ CD133+ CD38- stem cell populations at T1 p=0.032. To determine changes in serum proteins as a result of PI treatment that could contribute to HSPC mobilization we evaluated TGF-ß levels in 13 pt plasma samples. Two pts from the>2fold group were available and revealed TGF-ß levels increase 67.24 pg/mL compared to a decrease of 17.67 pg/mL in 5 pts in the <1fold group trending towards significance p=0.094. Baseline levels of TGF-ß in the two groups,>2fold and <1fold were 18.1 pg/mL and 30.1 pg/mL respectively, which was not significant.DiscussionObservations have noted increased HSPC yields in animal models and MM pts after treatment with PIs in both induction and mobilizing regimens (Ghobadi et al., 2012; Niesvizky et al., 2013). Here we demonstrate that treatment with PIs is associated with increases in peripheral HSPC percentages in approximately 2/3 of MM pts despite the lack of concurrent G-CSF. Decreased CD90 has previously been observed in peripherally mobilized HSPC products and, similar to TGF-ß, plays a role in regulation of Rhokinase GTPase pathways known to affect migration and adherence of many different cell types (Tsuchiya et al., 1997; Kim et al., 2006; Wen et al., 2013; Kim et al., 2014). Our study shows a correlation between decreased CD90 expression and fold increase of peripheral HSPCs. We also found an increase in TGF-ß serum levels after treatment in the>2fold group compared to the <1fold group, which may approach statistical significance with more sampling. These findings may help understand the failure to collect adequate HSPCs in a subset of MM pts and could highlight new pathways to disrupt and improve HSPC mobilization regimens. DisclosuresNiesvizky:Onyx Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau; Millennium: The Takeda Oncology Company: Consultancy, Research Funding, Speakers Bureau. Mark:Onyx: Research Funding, Speakers Bureau; Millennium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau. Rossi:Celgene: Speakers Bureau.

IL-4 and IL-13 differentially regulate TLR-induced eosinophil-basophil differentiation of cord blood CD34+ progenitor cells.

Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent allergic disease development. We have previously shown that fewer lipopolysaccharide (LPS)-stimulated eosinophil-basophil (Eo/B) colonies grow from cord blood (CB) of high-atopic risk infants, compared to low-atopic risk infants. In the present study, we investigated whether a surrogate ex vivo TH2 milieu (i.e., either IL-4 or IL-13) could represent an underlying mechanism to explain our previous findings. CB CD34+ cells from healthy donors were cultured with IL-4 or IL-13 (in combination with LPS) and assessed for Eo/B differentiation using methylcellulose cultures and flow cytometry for related intracellular signalling pathways. Pharmacological inhibitors were added to the methylcellulose cultures to determine the effect of blocking intracellular signalling in CB CD34+ cells in relation to Eo/B colony forming unit (CFU) formation. Stimulation of CD34+ cells with IL-4, but not IL-13, reduced Eo/B CFU formation in the presence of LPS; this was found to be dependent on IL-4Rα and not IL-13Rα1. Additionally, IL-4 reduced the expression of ERK 1/2 after LPS stimulation, which was recovered by inhibition of IL-4Rα. While IL-13 did not have an inhibitory effect on ERK 1/2 expression, inhibition of ERK 1/2 significantly reduced Eo/B CFU formation. Thus, the responsiveness of CB CD34+ progenitor cells to LPS is differentially regulated by the TH2 cytokines, IL-4 and IL-13. This may have implications for in utero interactions between placental-derived pro-allergic cytokines and neonatal progenitor cells influencing Eo/B-mediated inflammatory responses in early life.

The effects of thymic stromal lymphopoietin and IL-3 on human eosinophil-basophil lineage commitment: Relevance to atopic sensitization.

An important immunopathological hallmark of allergic disease is tissue eosinophilic and basophilic inflammation, a phenomenon which originates from hemopoietic progenitors (HP). The fate of HP is determined by local inflammatory cytokines that permit “in situ hemopoiesis,” which leads to the accumulation of eosinophils and basophils (Eo/B). Given that recent evidence supports a critical immunomodulatory role for thymic stromal lymphopoietin (TSLP) in allergic inflammation, as well as TSLP effects on CD34+ progenitor cytokine and chemokine secretion, we investigated the role of TSLP in mediating eosinophilo- and basophilopoiesis, the mechanisms involved, and the association of these processes with atopic sensitisation. In the studies presented herein, we demonstrate a direct role for TSLP in Eo/B differentiation from human peripheral blood CD34+ cells. In the presence of IL-3, TSLP significantly promoted the formation of Eo/B colony forming units (CFU) (including both eosinophils and basophils) from human HP (HHP), which was dependent on TSLP–TSLPR interactions. IL-3/TSLP-stimulated HHP actively secreted an array of cytokines/chemokines, key among which was TNFα, which, together with IL-3, enhanced surface expression of TSLPR. Moreover, pre-stimulation of HHP with IL-3/TNFα further promoted TSLP-dependent Eo/B CFU formation. HHP isolated from atopic individuals were functionally and phenotypically more responsive to TSLP than those from nonatopic individuals. This is the first study to demonstrate enhanced TSLP-mediated hemopoiesis ex vivo in relation to clinical atopic status. The capacity of HHP to participate in TSLP-driven allergic inflammation points to the potential importance of “in situ hemopoiesis” in allergic inflammation initiated at the epithelial surface.

Toll‐like receptor‐mediated eosinophil–basophil differentiation: autocrine signalling by granulocyte–macrophage colony‐stimulating factor in cord blood haematopoietic progenitors

Eosinophils are multi-functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil-basophil (Eo/B) differentiation-inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll-like receptor (TLR)-mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony-forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34-enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive, as opposed to interleukin-5-responsive, Eo/B CFU, which also correlated with significant increases in CD34(+) cell GM-CSFRα expression. Functionally, CB CD34(+) cells secrete abundant amounts of GM-CSF following LPS stimulation, via a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK-dependent GM-CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.

APS From the Root of Angelica Sinesis Promotes Megakaryocytopoiesis and Thrombopoiesis Through PI3k/Akt Pathway

AbstractAbstract 4648We have demonstrated the thrombopoietic effects of Chinese Medicine Dang Gui Bu Xue Tong (DBT), a formula composed of Radix Angelicae Sinensis and Radix Astragali in a thrombocypenic mouse model (Yang M et al, J Ethnopharm 2009). In this study, we further test the effect of polysaccharide (APS) from the root of Radix Angelicae Sinensis (APS) in in-vivo thrombopoiesis and in in-vitro megakaryocytopoiesis. A myelosuppression and thrombocytopenia mouse model was treated with APS (10 mg/kg/day), thrombopoietin (TPO) and saline as control. Peripheral blood cells from APS, TPO and vehicle-treated samples were then counted in different time-points. Using the colony-forming unit (CFU) assays, we determined the effects of APS on the hematopoietic stem/progenitor cells and megakaryocytic lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were also conducted in megakaryocytic cell line M-07e. Lastly, the effects of APS on cells treated with Ly294002, a Phosphatidylinositol 3-Kinse inhibitor and the effect of APS on the phosphorylation of AKT were further studied in megakaryocytic cells. Our results showed that APS significantly enhanced the platelets and other blood cells recovery and CFU formation in this model. Morphological examination of bone marrows showed that APS treatment significantly increased the recovery of the megakaryocytic series and other hematopoietic progenitor cells. We observed the antiapoptotic effects of APS on M-07e cells. Addition of Ly294002 alone increases the percentage of cells undergoing apoptosis. In contrast, additional of APS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the phosphorylation of AKT. Our studies demonstrate that APS promotes platelet recovery in the mouse model and has anti-apoptosis effect in megakaryocytic cells. This effect is likely to be mediated by the PI3K/AKT pathway. Disclosures:No relevant conflicts of interest to declare.

Endothelial progenitor cell response to antiproliferative drug exposure

BackgroundVascular stent coverage by endothelial cells, derived from endothelial progenitor cells (EPC) is considered a surrogate for healing. However, the effects of antiproliferative drugs used in current drug-eluting stents (DES) on EPC proliferative and antithrombotic function remains poorly defined. Method and resultsHerein, we studied and compared the in vitro and in vivo effects of four antiproliferative drugs – paclitaxel, sirolimus, everolimus, and zotarolimus on several EPC properties including colony forming units (CFU), cell proliferation, apoptosis, antithrombotic and prothrombotic gene expression and nitric oxide (NO) as well as prostacyclin (PGI2) release. We also examined EPC migration and adhesion under flow conditions. We find that whereas all antiproliferative agents inhibited EPC proliferation and caused cell apoptosis, only paclitaxel and sirolimus reduced CFU formation. Paclitaxel treatment also resulted in the greatest down-regulation of antithrombotic gene expression and up-regulation of prothrombotic gene expression. NO release, migration, and adhesion of EPC under shear stress were inhibited by all antiproliferative drugs, most notably by paclitaxel and sirolimus. ConclusionsThese results indicate that antiproliferative drugs on DES, particularly paclitaxel, impair the proliferative and antithrombotic functions of EPC, and thereby could contribute to incomplete vascular healing and increase the risk of stent thrombosis.

Abstract A158: Therapeutic strategies for targeting histone methyltransferase DOT1L in MLL rearranged leukemia.

Abstract MLL fusion proteins activate target genes in part via recruitment of the histone-modifying enzyme, DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase with selective activity for histone H3 lysine 79 (H3K79). The resulting hypermethylation of H3K79 at the HOX and MEIS1 loci appears to be pivotal for leukemogenesis in both AML and ALL. These findings emphasize the central role of DOT1L in leukemogenesis and strongly suggest that pharmacologic inhibition of DOT1L could be of therapeutic benefit for a number of ALL and AML subtypes. Applying a biochemical screening approach, two classes of small molecule DOT1L substrate competitive inhibitors were discovered. They selectively inhibit the cell growth in leukemic cells with MLL-translocation, in comparison with leukemic cells without MLL translocation, resulting in a dose and time dependent decrease of cellular H3K79 methylation, decrease in expression of MLL-target genes HOXA9 and MEIS1, inducing G0/G1 cell cycle arrest, an increase in expression of differentiation markers and death by apoptosis. Although the DOT1L inhibitors showed no toxicity on normal bone marrow, knockout studies show that DOT1L deficiency is associated with chromosomal instability and fatal progressive anemia. This suggested that developing therapeutic strategies other than global inhibition of DOT1L activity may be necessary. Therefore we characterized the protein-protein interaction (PPI) between DOT1L and ENL/AF9 and assessed its importance on MLL fusion protein-mediated transformation. We found that ENL and AF9 C-terminal domains bind to DOT1L with Kd of 238 nM and 111 nM respectively. We have mapped the binding site in DOT1L and found that only 10 amino acid of DOT1L are involved in interaction with ENL and AF9. Synthetic DOT1L 10mer peptide binds ENL/AF9 and disrupts the interaction between DOT1L/ENL and DOT1L/AF9. Alanine scanning mutagenesis studies showed that four hydrophobic residues are detrimental for the binding of DOT1L to both proteins ENL/AF9. To examine the critical role of the DOT1L-AF9 interaction in transformation by MLL fusion protein, colony forming unit (CFU) assay was performed using MLL-AF9 transformed cells lacking endogenous DOT1L. The colony forming potential of the MLL-immortalized cells was completely abolished by introducing DOT1L construct lacking 10 amino acid AF9 interacting residues (DOT1L (Δ10aa)), while the wild type DOT1L construct was able to restore CFU formation. Importantly global levels of H3K79 methylation were equivalent, but only the DOT1L (Δ10aa) construct failed to restore CFU formation. These results demonstrate that DOT1L interaction with MLL-AF9 in addition to H3K79 methylation is required for transformation of MLL-AF9. This suggests that selective disruption of this PPI is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL-fusion protein associated leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A158.

In vitro effects of perifosine, bortezomib and lenalidomide against hematopoietic progenitor cells from healthy donors

The novel AKT inhibitor perifosine possesses myelopoiesis-stimulating effects in rodents. We studied the in vitro effects of the novel agents perifosine, bortezomib and lenalidomide in addition to adriamycin against normal human hematopoietic progenitor cells (HPC) using different clonogenic and non-clonogenic assays. All agents inhibited colony-forming unit (CFU) formation, perifosine inhibiting mainly CFU-granulocyte/macrophage formation and the other agents burst-forming unit-erythroid formation. Perifosine combined with lenalidomide or adriamycin tended to act antagonistically in suppressing CFU formation. Despite their inhibition of CFU formation, perifosine, bortezomib and lenalidomide induced only slight or moderate cytotoxicity in CD34(+) selected HPC, as assessed using different assays such as flow cytometry-based detection of activated caspases and immunohistochemistry studies (e.g., Ki-67 staining). In contrast to its myelopoiesis-stimulating effects in rodents, perifosine--like bortezomib and lenalidomide--suppresses the clonogenic potential of HPC from healthy donors in vitro and thus probably plays no role in preventing neutropenia or in shorting its duration after intensive chemotherapy. However, all these novel agents typically induce only slight or moderate suppression of the clonogenic potential or loss of viability of normal HPC at clinically achievable plasma concentrations, assuming that hematoxicity is manageable and functional HPC can be collected after treatment with these compounds.

BAS/BSCR52 Investigating the importance of heparan sulphate in determining endothelial progenitor cell function

The increased risk of cardiovascular disease with age may, in part, be due to a decline in the function of endothelial progenitor cells (EPCs). Cell surface heparan sulphate proteoglycans (HSPGs)...