Polymorphonuclear leukocytes L-selectin expression decreases as they age in circulation

1997 ◽  
Vol 272 (1) ◽  
pp. H401-H408 ◽  
Author(s):  
S. F. Van Eeden ◽  
S. Bicknell ◽  
B. A. Walker ◽  
J. C. Hogg
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Life Span ◽  
Whole Blood ◽  
Half Life ◽  
Polymorphonuclear Leukocytes ◽  
In Vitro Studies ◽  
Double Immunolabeling

We recently reported that L-selectin expression increases on circulating polymorphonuclear leukocytes (PMN) during active bone marrow release, which suggests that older cells in the circulation have lower levels of L-selectin than those recently released from the bone marrow. The present study was designed to test the hypothesis that L-selectin expression reduces on PMN as they age in the circulation. In vitro studies using flow cytometry showed that PMN L-selectin decreased to 14.6 +/- 2.3% of baseline during a 24-h incubation at 37 degrees C. To test this hypothesis in vivo, rabbit PMN, labeled in vivo with 5'-bromo-2-deoxyuridine (PMN-brdU) in donor animals, were infused into recipient rabbits as whole blood (n = 5) and followed over 24 h in the circulation with a double immunolabeling technique. These results showed that the fraction of the L-selectin-negative PMN-BrdU in the circulation increased with time (P < 0.001), and nearly all of the PMNBrdU in the circulation were L-selectin negative after 24 h. Removal of L-selectin from the surface of PMN-BrdU with chymotrypsin before infusion did not change their rate of removal from the circulation (half-life or t 1/2 262 vs. 296 min, P = NS). We conclude that there is a continuous loss of L-selectin from PMN during their life span in the circulation, which supports the hypothesis that L-selectin expression decreases on PMN as they age.

Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽  
2017 ◽  
Vol 27 (1) ◽  
pp. 49-59 ◽  
Author(s):  
X Yang ◽  
J Yang ◽  
X Li ◽  
W Ma ◽  
H Zou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Lupus Nephritis ◽  
Bone Marrow Cells ◽  
Tfh Cells ◽  
Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

Effect of reticulocytosis on lactate dehydrogenase isoenzyme distribution in serum: in vivo and in vitro studies

1990 ◽  
Vol 36 (9) ◽  
pp. 1638-1641 ◽  
Author(s):  
S C Kazmierczak ◽  
W J Castellani ◽  
F Van Lente ◽  
E D Hodges ◽  
B Udis
Keyword(s):  
Flow Cytometry ◽  
Lactate Dehydrogenase ◽  
Cell Types ◽  
In Vitro Studies ◽  
Hemolytic Disease ◽  
Dehydrogenase Isoenzyme ◽  
Disease Analysis

Abstract We investigated the effect of reticulocytosis on the lactate dehydrogenase (LD; EC 1.1.1.27) isoenzyme LD1/LD2 ratio in patients with and without evidence of hemolytic disease. Analysis of sera from patients with reticulocytosis and in vivo hemolysis showed a mean LD1/LD2 ratio of 0.92 compared with a ratio of 0.69 in patients with in vivo hemolysis and normal reticulocyte counts. Determination of LD isoenzymes in erythrocyte lysate revealed significantly increased LD1/LD2 ratios for patients with marked reticulocytosis compared with those for patients with normal-to-minimal increases in reticulocytes. Finally, separation of mature erythrocytes and reticulocytes by flow cytometry revealed marked differences in the LD1/LD2 isoenzyme distribution between these two cell types. The ability of hemolysis to cause a "flipped" LD1/LD2 ratio is dependent on the proportion of the hemolyzed cells that are reticulocytes.

Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5256-5256
Author(s):  
Doug Cipkala ◽  
Kelly McQuown ◽  
Lindsay Hendey ◽  
Michael Boyer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Mouse Model ◽  
Scid Mouse ◽  
In Vitro Cytotoxicity ◽  
Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p&gt;0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p&gt;0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p&lt;0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

Enhanced Erythroblast Mobilization in Nix-Deficient Mice Confers Resistance to Phenylhydrazine-Induced Anemia Despite Accelerated Erythrocyte Turnover.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3659-3659
Author(s):  
Abhinav Diwan ◽  
Andrew G. Koesters ◽  
Amy M. Odley ◽  
Theodosia A. Kalfa ◽  
Gerald W. Dorn
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Half Life ◽  
Hematopoietic Cells ◽  
Dynamic Regulation ◽  
Genetic Ablation ◽  
Deficient Mice ◽  
Erythrocyte Fragility

Abstract Steady-state and dynamic regulation of erythrocyte production occurs by altering the balance of cell-survival versus apoptosis signaling in maturing erythroblasts. Previously, the pro-apoptotic factor Nix was identified as a critical death signal in normal erythropoietic homeostasis, acting in opposition to erythroblast-survival signaling by erythropoietin and Bcl-xl. However, the role of Nix in stress-erythropoiesis is not known. Here, by comparing the consequences of erythropoietin administration, acute phenylhydrazine-induced anemia, and aging in wild-type and Nix-deficient mice, we show that complete absence of Nix, or its genetic ablation specifically in hematopoietic cells, mimics the effects of erythropoietin (Epo). Both Nix ablation and Epo treatment increase early erythroblasts in spleen and bone marrow and increase the number of circulating reticulocytes, while maintaining a pool of mature erythroblasts as an “erythropoietic reserve”. As compared with WT, Nix null mice develop polycythemia more rapidly after Epo treatment, consistent with enhanced sensitivity to erythropoietin observed in vitro. After phenylhydrazine administration, anemia in Nix-deficient mice is less severe and recovers more rapidly than in WT mice, despite lower endogenous Epo levels. Anemic stress depletes mature erythroblasts in both WT and Nix null mice, but Nix null mice with basal erythroblastosis are resistant to anemic stress. These findings show that Nix null mice have greatly expanded erythroblast reserve and respond normally to Epo- and anemia-stimulated induction of erythropoiesis. However, the hematocrits of young adult Nix null mice are not elevated, and these mice paradoxically develop anemia as they age with decreased hemoglobin content (10g/dl) and hematocrit (36%; at 80±3 weeks of age) compared to WT mice (13g/dl and 46%; 82±5 weeks of age), inspite of persistent erythoblastosis observed in the bone marrow and spleen. Nix null erythrocytes, which are macrocytic and exhibit membrane abnormalities typically seen in immature cells or with accelerated erythropoiesis, demonstrate shorter life span with a half life of 5.2±0.6 days in the peripheral circulation by in vivo biotin labeling (as compared with a half life of 11.7±0.9 days in WT), and increased osmotic fragility as compared with normal erythrocytes. This suggests that production and release of large numbers of reticulocytes in Nix null mice can decrease erythrocyte survival. To rule out a non-hematopoietic consequence of Nix ablation that contributes to or causes increased erythrocyte fragility and in vivo consumption, such as primary hypersplenism, we undertook Tie2-Cre mediated conditional Nix gene ablation. Nixfl/fl + Tie2-Cre mice (hematopoietic-cell specific Nix null) develop erythroblastosis with splenomegaly, reticulocytosis, absence of polycythemia and increased erythrocyte fragility; suggesting that erythroblastosis and accelerated erythrocyte turnover are a primary consequence of Nix ablation in hematopoietic cells. Hence, dis-inhibition of erythropoietin-mediated erythroblast survival pathways by Nix ablation enhances steady-state and stress-mediated erythropoiesis.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3090-3090 ◽  
Author(s):  
Folashade Otegbeye ◽  
Nathan Mackowski ◽  
Evelyn Ojo ◽  
Marcos De Lima ◽  
David N. Wald
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

scholarly journals Enhancement of the biologic effects of interleukin-3 in vivo by anti- interleukin-3 antibodies

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1133-1141
Author(s):  
AT Jones ◽  
HJ Ziltener
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Half Life ◽  
Low Dose ◽  
Interleukin 3 ◽  
Concurrent Use ◽  
Biologic Effects ◽  
Total Body

Interleukin-3 (IL-3) has been shown to be a promising agent in the stimulation of bone marrow regeneration following myeloablative therapy. The biologic half-life of this agent is very short (5 to 15 minutes), which limits the effectiveness of low-dose therapy. Here we show that the biologic effects of low-dose IL-3 in mice may be enhanced by concurrent use of polyclonal anti-IL-3 antibodies. The biologic effects of IL-3 in vivo were enhanced dramatically by the combination of the cytokine and polyclonal rabbit anti-IL-3 antibodies, which recognized a peptide comprising the first 29 amino acids of the IL-3 molecule. Enhancing effects were not apparent in vitro, where weak neutralizing properties were observed for these antibodies. The mechanism of this enhancement by the antibody appears to be via a ninefold reduction in the total-body clearance of the cytokine in vivo. The apparent volumes of distribution for IL-3 and for the IL-3/antibody complex were surprisingly similar and exceeded the expected intravascular volume. The prolonged biologic half-life of IL-3 was reproducibly associated with a threefold to fivefold increase in splenic mast-cell precursors over levels observed in mice treated with IL-3 alone; increases in the numbers of mature mucosal-type mast cells in the spleen, but not in the jejunum or lung; increases in IL-3- dependent colony-forming unit-cell in the spleen; and an apparent redistribution of mast cells away from the bone marrow. These experiments demonstrate that antibodies to a cytokine can enhance the biologic activity of that cytokine in vivo.

STIMULATION OF BONE MARROW CELLS AND BONE FORMATION BY NACRE. IN VIVO AND IN VITRO STUDIES

Bioceramics ◽  
1999 ◽  
Author(s):  
M. Lamghari ◽  
S. Berland ◽  
A. Laurent ◽  
H. Huet ◽  
M.J. Almeida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Bone Marrow Cells ◽  
In Vitro Studies ◽  
Stimulation Of ◽  
Marrow Cells

Acidosis in Human Whole Blood Does Not Necessarily Impair the Coagulation and the Effect of RFVIIa.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3930-3930
Author(s):  
Dorthe Viuff ◽  
Marianne Kjalke ◽  
Vivian Lind ◽  
Egon Persson ◽  
Mirella Ezban
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Whole Blood ◽  
Annexin A5 ◽  
In Vivo Models ◽  
Human Whole Blood ◽  
Major Interest ◽  
R Value

Abstract Introduction: Acidosis is associated with high mortality in trauma patients. Therefore there is a major interest in generating acidosis models in vitro and in vivo to determine the effect of acidosis on coagulation and to develop treatments. The aim of this study was to examine the effect of acidosis induction in human whole blood using HCl versus Hepes and to analyze the subsequent effect of rFVIIa (NovoSeven®). Materials and Methods: Native human whole blood was obtained from healthy volunteers (n=6) and pH was adjusted to 6.8 using 1 M HCl or 1 M Hepes (pH 6.8). Coagulation was triggered with kaolin or tissue factor (TF, Innovin, final dilution 1:42500) and measured by thrombelastography (TEG, Haemoscope®). Furthermore, the effect of rFVIIa (25nM ∼ 90 mcg/kg) was measured. The TEG parameters R (sec), angle (deg) and maximum amplitude (MA, mm) were recorded and presented as mean±SD. A shorter R and greater angle and MA values are indicative of a more robust clot formation. Statistical analysis was performed by a two-way ANOVA-model. Platelet function was analyzed by platelet aggregation using Multiplate (Dynabyte Medical). Exposure of P-selectin, negatively charged phospholipids (annexin A5 binding) and induction of the active conformation of the fibrinogen receptor GPIIb/IIIa (PAC-1 binding) on platelets after TRAP-stimulation of whole blood was analyzed using a FACS Canto flow cytometer (BD). Results: TEG, platelet aggregation and flow cytometry indicated that lowering the pH to 6.8 by HCl affected the blood significantly different than when pH was lowered by addition of Hepes. HCl-treated blood triggered with either kaolin or TF showed a significantly decreased R value (378±45 or 661±130 vs 539±98 or 888±353 in untreated controls), significantly decreased MA (52±6 or 51±9 vs 66±8 or 62±13) and decreased angle (50±7 or 36±10 vs control 57±10 or 44±19, not significant). Hepes-treated blood triggered with kaolin showed no difference in R (458±52), angle (64±4) and MA (58±9) compared to untreated controls, whereas blood triggered with TF showed significantly shortened R-value (461±91) and enhanced angle (63±5) compared to untreated controls. Hepes treatment had no effect on MA (64±12). rFVIIa significantly shortened R irrespective of the acidosis inducer or clot trigger(HCl/kaolin 283±34, HCl/TF 307±52; Hepes/kaolin 363±32, Hepes/TF 313±46). Although the other TEG parameters were also improved, the effect was only significant when blood was treated with HCl and clotting initiated with TF (angle 48±11, MA 56±10). HCl-induced acidosis abolished platelet aggregation, whereas Hepes-induced acidosis did not alter platelet aggregation compared to normal blood. Flow cytometry showed that platelets from HCl-treated blood were pre-activated as evidence by expression of P-selectin on 70% of the platelets, annexin A5 binding to 14% of the platelets and PAC-1 binding to 62% of the platelets before stimulation. TRAP-stimulation increased P-selectin expression, and PAC1 and Annexin A5 binding to platelets in HCl-treated blood. In contrast, Hepes-treatment did not pre-activate the platelets and the increase in P-selectin expression, and annexin A5 and PAC-1 binding after TRAP-stimulation was as seen for control blood. Conclusion: The method used to lower pH in human blood strongly influences the functionality of the platelets and coagulation factors independent of the final pH. It is therefore important in experimental in vitro and in vivo models to be aware of these dramatically different effects in order to draw correct conclusions.

Terminal Differentiation of FLT3/ITD AML Blasts In Patients Treated with the FLT3 Inhibitor AC220

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 660-660
Author(s):  
Mark J. Levis ◽  
Amy Sexauer ◽  
Trivikram Rajkhowa ◽  
Donald Small ◽  
Michael J. Borowitz
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Peripheral Blood ◽  
Terminal Differentiation ◽  
Flt3 Inhibitor ◽  
Microscopic Evaluation ◽  
Pre Treatment

Abstract Abstract 660 AML is characterized by abnormal proliferation of myeloid cells that have a block in differentiation. FLT3/ITD mutations are relatively common in AML, and previous in vitro studies have demonstrated that signaling from ITD-mutated FLT3 blocks myeloid differentiation through repression of CEBP/a. As part of an ongoing phase 2 trial, we treated 6 patients with FLT3/ITD AML who were refractory to either primary induction therapy or salvage therapy after relapse with the highly potent and selective FLT3 inhibitor AC220. At the start of therapy, all 6 patients had circulating blasts (mean 9864 blasts/mm3; median 2970) and the median blast percentage in the bone marrow was 71.5%. Western blotting revealed a high level of sustained in vivo FLT3 inhibition in all patients. By Day 8, no patient had detectable blasts in the peripheral blood. After 14 days of treatment with AC220, all 6 patients displayed striking differentiation to the myelocyte stage within the bone marrow. By light microscopic evaluation of bone marrow aspirates, myelocytes (promyelocytes, myelocytes, and metamyelocytes) increased from a median of 10.5% pre-treatment to 52% after 2 weeks. Most patients were neutropenic on Day 1 of treatment (mean 574, median 560 neutrophils/mm3), but rose to a mean of 3275 neutrophils/mm3 after 4–8 weeks of treatment (median time to peak 34 days). By Day 28 of treatment, marrows were most often still hypercellular, but consisted primarily of fully differentiated neutrophils. Marrow blasts were markedly reduced or absent by Day 28 in all 6 cases (mean 2.3%, median 1.5%). In all 6 patients the FLT3/ITD mutation originally detected at the beginning of treatment was present in the marrow and peripheral blood despite the absence of circulating blasts after the first week of therapy. The FLT3 mutant allelic ratio did not change between pre-therapy and Day 28. Neutrophils were isolated to homogeneity (confirmed by cytospin) from peripheral blood by double ficoll density centrifugation. Using genomic DNA obtained from these purified neutrophils, we confirmed by PCR that the FLT3/ITD mutation was present, at a similar ratio as compared with the pre-treatment blasts. However, there was no detectable expression of FLT3 either by RNA (quantitative PCR) or protein (western blotting and flow cytometry) in these neutrophils. The isolated neutrophils morphologically resemble normal neutrophils by light microscopy, and by flow cytometry they express the differentiation antigen CD15 and CD11b, and have lost expression of immature markers such as cKIT and CD34. Stimulation of these neutrophils by endotoxin results in normal respiratory burst activity, as measured by reduction of nitroblue tetrazolium. They also express lactoferrin and MMP-9, proteins typically expressed in mature neutrophils. Clinically, lung nodules and fever occurred in 3 of the 6 patients within 14 days of the peak neutrophil count. They were not treated with steroids, but rather with antibiotics, and in all cases resolved. Other patients on this trial have developed Sweet's syndrome during the neutrophil surge. CEBPa transcript levels in Molm14 cells (an AML cell line with a FLT3/ITD mutation) rose 3–5-fold over baseline following treatment with AC220. This is consistent with our previously published findings, and suggests at least one mechanism for the observed release of the differentiation block observed in the AC220-treated patients. These clinical and correlative laboratory results suggest that effective, sustained in vivo FLT3 inhibition in AML patients with FLT3/ITD mutations induces terminal differentiation in blasts in many ways similar to that seen with all trans retinoic acid in acute promyelocytic leukemia. Furthermore, these findings demonstrate the direct link between the growth factor receptor pathway and control of differentiation, and provide new insight into mechanisms of leukemogenesis. Disclosures: Levis: Ambit Biosciences, Inc: Consultancy.

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