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Background: Recent years, the incidence and mortality of fungal infection has been on the rise in the patients with hematologic malignancies. This is mainly associated with antifungal resistance and the restricted number of available antifungal drugs. Candida species is one of the most prevalent pathogens in these immunodeficient patients. However, the study of azole resistance mechanisms of Candida has focused on C.albicans, C.glabrata, C.tropicalis. And few studies talked about resistance mechanisms of C.krusei, especially resistant to itraconazole. It was reported that the mutation or overexpression of 14¦Á-demethylases (encoded by ERG11) and upregulation of efflux transporters (encoded by ABC1 and ABC2) may be involved in azole resistance of C.krusei. Here, The purpose of the present study is to preliminarily explore the main molecular mechanisms responsible for Candida krusei clinical isolates to itraconazole, and may provide new sight into fungal infection therapy. Methods: The 14¦Á-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole- susceptible (S) C.krusei at the mRNA level. Results: We found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole- susceptible ones (P=0.015 and P=0.002 respectively). ABC2 gene mRNA levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P=0.007 in SDD compared with S, P=0.016 in SDD with R, and P Conclusions: There are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations were not found to be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates. Therefore, combination of azole and selective efflux transporter inhibitors may help reverse azole resistance and enhance antifungal effect. Figure 1. ABC2 relative gene expression levels in three groups of C.krusei clinical isolates. (A) Relative levels of ABC2 mRNA in all the C.krusei clinical isolates. ABC2 gene expression levels was quantified and normalized relative to the housekeeping gene, ACT1; S, itraconazole-susceptible; SDD, itraconazole-susceptibledose dependent; R, itraconazole-resistant. (B) Log10+3 fold increase of gene expression levels in three groups. (*P Disclosures No relevant conflicts of interest to declare.

A substantial portion of patients with inherited blood disorders such as beta thalassemia, or bone marrow failure syndromes such as aplastic anemia(AA), myelodysplastic syndromes(MDS) require frequent transfusions of red blood cells. Frequent blood transfusions may lead to the excess of plasma non-transferrin -bound iron(NTBI) and iron overload occurs, which will significantly injure bone marrow (BM) function as well as induce organ dysfunctions such as liver cirrhosis, diabetes and cardiac diseases. However, the exact mechanism behind this effect remains elusive and ideal treatment needs to be explored. In our preliminary studies, we have demonstrated free iron catalyzes oxidative damage to hematopoietic cells/ mesenchymal stem cells in vitro and suppresses hematopoiesis in iron overload patients (Zhao et al.,blood, 2010 abstract; Lu et al.,blood,2012 abstract; Lu et al., Eur J Haematol, 2013). Here we observed the hematopoiesis inhibitory effects of iron overload on the basis of estabalished iron overload mice model and preliminarily disscussed the mechanism. In this study, we first established an iron overload mice model by administering different doses(12.5mg/ml,25mg/ml,50mg/ml) iron dextran by intraperitoneal injection every three days for four weeks. To confirm the efficacy of the mice model, the BM, hepatic and splenic iron deposits were observed by morphological study and the labile iron pool level (LIP) of bone marrow mononuclear cells(BMMNCs) was detected using the calcein-AM fluorescent dye. It was found that iron deposits in BM cells of iron overload mice, liver and spleen were markedly increased and the BMMNCs LIP level was much higher than that of normal control mice. The above results showed that the iron-overloaded mice model has been established successfully. Next we observed whether iron overload (25mg/ml) could affect the hematopoiesis of BM. The colony-forming cell assay was performed by culturing BMMNCs in MethoCult M3434 methylcellulose medium to evaluate hematopoietic progenitor cells(HPCs) proliferation function. The competitive repopulation assay and single-cell colony cultures of sorted hematopoietic stem cells (HSCs,CD34-Lin- sca1+c-kit+cells,LSK+)were used to validate HSCs function. The counts of BMMNCs have no significant difference. However, It was found that hematopoietic colony-forming unit (CFU-E, BFU-E, CFU-GM and CFU-mix) was much lower than that of normal control(P We then explored the possible mechanism of this inhibitory effects. Our previous studies have shown that iron overload could elevated reactive oxygen species (ROS) levels of mesenchymal stem cells and HSCs in vitro. Similarly, the intracellular ROS levels were analyzed by a flow cytometer. It was found that ROS level in iron overload BM was increased by 3.32 folds in erythroid cells, 1.51 folds in granulocytes and 4.80 folds in LSK+ cells,respectively. And also, the expression of p53, p38MAPK and p16Ink4a mRNA remained significantly elevated, which indicated that ROS related signal pathway was involved in the deficient hematopoiesis of iron overload BM. In addition, we also observed the effects of iron overload on the mice with deficient hematopoiesis exposed to 4Gy total body irradiation(TBI), which was more similar to clinical pathological conditions such as AA or MDS. It was found that BM damage caused by iron overload was aggravated in pathological conditions (primary findings were not shown). In conclusion, our study confirmed that iron overload injures the hematopoiesis of BM by enhancing oxidative stress in mice, which would be helpful to further study on the mechanism and would provide an experimental basis to find new therapeutic targets for the treatment of iron overload in patients with hematopoietic dysfunction.Figure 1Results of hematopoietic colony forming unit of different groups(*P Disclosures: No relevant conflicts of interest to declare.

Cytokine-induced killer (CIK) cells are the effective kind of immunocytes participated in biotherapy of tumors and CD3+CD56+ cells display a major role in cytolytic activity against tumors. Recombinant human interleukin-2 (rhIL-2) is one of the most necessary cytokines during induction of CIK cells, however, some extra kinds of cells eapecially regulatory T (Treg) cells may expand during the induction of CIK cells because of the presence of IL-2. As Treg cells may exert a negative effect on function of CIK cells through cell-to-cell contact and some cytokines secreted by Treg cells, so what we need to consider is how to reduce Treg cells in the culture system of CIK cells. Some studies have confirmed that IL-21, which is belonged to a subset of cytokines where the receptors share the common cytokine receptor γ chain, could express an inhibitory influence on proliferation in Treg cells and our previous study has also confirmed its enhancement on proliferation and function of CIK cells. Here we hypothesize that IL-21 not only promotes the proliferation and function of CD3+CD56+ CIK cells, but also depressed Treg cells in the culure system of CIK cells and then result in the enhanced anti-leukemia effect. In this study, we first detected whether Treg cells existed in the culture system of CIK cells. Firstly, CIK cells were obaind from umbilical cord blood mononuclear cells (CBMCs) by the sequential addition of interferon-γ, anti-CD3 antibody , and rh-IL-2. Then the immunophenotype of Treg cells was detected by the flow cytometry through CD4-FITC and Foxp3-PE antibodies double staining. It was found that a certain proportion of Treg cells at about (10.24±1.42)% consisted in the culture system of CIK cells. The second step in this study was to explore the effect of IL-21 acting on CD3+CD56+ CIK cells. Firstly, CD3+CD56+ CIK cells were separated from the culture cells mentioned above by positive selection using Fluorescence-activated Cell Sorter. Cells were then stimulated with IL-21 for a defined period of time and subjected to CCK-8 and LDH assays to measure cellular viability and cytotoxicity against to leukemia cell line—K562 cells, respectively. The CCK-8 assay results showed that OD values in the cells without IL-21 stimulation was 1.08 which was much lower than that in the cells with IL-21 stimulaiton at 1.45 (P Next, we investigated the effect of IL-21 on both Treg and CIK cells in the culture system. The culture system of CIK cells was established as mentioned above, and then part of these cells were stimulated with IL-21 for 72 hours. Immunophenotype of Treg and CIK cells was detected by flow cytometry. It was found that cells stimulated by IL-21 showed a decreased proportion of CD4+Foxp3+ Treg cells at (1.48±0.06)% compared with the cells without IL-21 stimulation at (10.24±1.42)% (P In conclusion, our findings indicate that IL-21 could promote the proliferative and cytotoxic activity of CD3+CD56+ CIK cells, meanwhile it also could suppress the viability of Treg cells in the CIK cells culture system. So a conclusion can be summarized that IL-21 could enhance anti-leukemia effect not only by promoting CD3+CD56+ CIK cells but also by depressing Treg cells derived from umbilical cord blood in vitro. Disclosures: No relevant conflicts of interest to declare.

Abstract 1059 Adoptive transfer of activated T and NK cells has had significant clinical benefits in certain tumor models. Cytokine induced killer (CIK) cells are a group of cells that possess both T and NK cell like recognition of target cells. These cells are generated after extensive ex vivo manipulation of PBMCs. Maintenance of not only CIK cells but other activated effector T and NK cells in culture is vital for their effective transfer and development following adoptive immunotherapy. IL-21 is the newest member of the common γ chain family which has been shown to increase cytotoxic factors and cytokine secretion in immune cells without over stimulation. Such qualities make IL-21 a suitable agent in immunotherapy of tumors. IL-21 has shown effective antitumor function and is currently going clinical trials for tumors such as renal cell carcinoma, melanoma and lymphoma. Our previous experiment showed that like in T cells and NK cells, IL-21 significantly improves the cytotoxicity of CIK cell on K562 cells and primary leukemic cells from patients. Although proliferation of cells in a CIK cell pool was not observed we found that it helped maintain and grow the CD3+ and CD56+ phenotype. Our present experiment aims to explain the mechanism through which IL-21 promotes CIK cells survival and cell cytotoxicity. In our experiment, blood from healthy donors was collected and PBMCs were transformed into CIK cells following 14 days of culture using appropriate methods. The cells were then stimulated with IL-21 for a defined period of time and subjected to MTT assays to measure cellular viability and cytotoxicity to K562 cells. To elucidate the mechanism of action of IL-21, CIK cells were checked for the level of mRNA expression of perforin, Granzyme B, FasL, INF-γ, TNF-α,Granzyme A,NKG2D, TNF-β using RT-PCR. Furthermore the expression of significantly important cytotoxic factors and cytokines was measured through flow cytometry and ELISA. Western blot was performed to check the involvement of JAK/STAT pathway following stimulation. We found that IL-21 doesn't enhance in vitro proliferation of CIK cells, but does increase the number of cells expressing the CD3+/CD56+ phenotype. IL-21 can also significantly increase the cytotoxic potential of CIK cells to K562 cells. It does so with significantly increased production of perforin which increased almost 2 folds from (0.5592±0.1457) to (0.9831±0.1265); Granzyme B also by almost 2 folds from (0.4084±0.1589) to (0.7319±0.1639) and FasL which increased by almost 2 folds from (0.4015±0.2842) to (0.7381±0.2568). Increase in secretion of cytokines such as INF-γ was observed from (25.8±6.1)ng/L to (56.0±2.3)ng/L; and TNF-α from (5.64±0.61)ug/L to (15.14±0.93)ug/L while no significant difference was observed in the expression of Granzyme A,TNF-β and NKG2D. Measurement of IL-21R receptor on CIK cell surface following IL-21 stimulation caused a more than two folds increase in expression of IL-21R from 1.88% to 4.25%. We further affirm that JAK/STAT is actively involved in IL-21 signalling. STAT3 and STAT5b could be potential signalling mechanisms taking part in IL-21 enhanced cytotoxic potential of CIK cells. Using this information we have concluded that increased expression of perforin, Granzyme B, FasL, IFN-γ and TNF-α plays a significant role in IL-21 enhanced cytotoxic potential of CIK cells and STAT-3 and STAT-5b signaling pathway are involved in the processes. Our data indicate that IL-21 is a potent enhancer of antitumor function of CIK cells. As CIK cells and IL-21 have both been shown to increase patient survival or tumor free periods in certain hematological malignancies using them in conjunction might be therapeutically more beneficial. Disclosures: No relevant conflicts of interest to declare.

Abstract 2097 Iron is a useful component of cytochromes, oxygen-binding molecules and some enzymes due to its capacity to accept and donate electrons readily. However, excessive iron accumulation can damage tissues and cells by catalyzing the conversion of superoxide and hydrogen peroxide to free radical species that can attack cellular membranes, proteins and DNA. Recent multiple data revealed that iron chelation therapy was effective in treating cytopenia in iron overload disease, which supported the idea that iron overload affected hematopoiesis in bone marrow(BM). Based on these findings, We demonstrated that iron overload suppressed hematopoiesis by inhibiting hematopoietic stem/progenitor cells and the effects could be restored by iron chelation or anti-oxidants(Zhao et al., Blood, 2010, 116:4247a). However, it is unclear whether iron overload can impair BM hematopoiesis by injuring the microenvironment. As an important component of the BM microenvironment, Mesenchymal stem cells (MSCs) secrete a large amount of cytokines and extracellular matrix protein which provides a favorable platform for the localization, self-renewal, and differentiation of hematopoietic stem cells. Here we hypothesize that iron overload impairs BM microenvironment by affecting the function and survival of MSCs which is mediated by ROS. In this study we first established an iron overload model of MSCs by adding ferric ammonium citrae (FAC) to the culture medium. To confirm this model, the labile iron pool (LIP) level of MSCs was detected using the calcein-AM method. We found that the LIP of MSCs was significantly higher than control and reached the highest level when cultured at 400μmol/L FAC for 12h. Next we analyzed whether iron overload can affect proliferation, apoptosis and function of MSCs by the following experiments. Firstly, the proliferation of MSCs was evaluated using population doubling time (DT). Under iron overload, the population doubling time (DT) of MSCs was 24.43± 2.72 hours, which was signifcantly longer than control(16.03± 2.31 hours; P=0.015). However, the difference wasn't significant after two passages (P=0.936). Possible explanation could be that the injury to MSCs is reversible following decreased concentration of iron after passaging. Secondly, the apoptosis of MSCs altered by iron overload was measured by staining Annexin V/PI, and we found the apoptosis rate was higher in the iron overload group(12.75±0.32%) than control (3.63±0.80%)(P We then explored the possible mechanism that may take part in this process. It has already been reported that iron overload may result in the generation of reactive oxygen species (ROS). Similarly, we found that ROS level of MSCs could be positively correlated with the concentration of FAC and reached its highest level when cultured at 400μmol/L FAC for 12h. Finally, Western blot analysis of whole cell lysates from umbilical derived MSC using antibodies recognizing known ROS-related signaling pathways revealed robust increases in phospho-p38, p53 in response to FAC compared with control, with inhibition of these signaling pathways noted in response to NAC or GSH at suitable dose, suggesting that antioxidant can inhibit ROS-induced signaling pathway in iron overload. In conclusion, Our finding indicates that iron overload can injure hematopoiesis by enhancing oxidative stress in MSC. Our data further suggests creatively that antioxidant and cytotherapy may be an effective method in curing deficient hematopoiesis in iron overload. Disclosures: No relevant conflicts of interest to declare.