scholarly journals Phagocytic activity and oxygen metabolism of peripheral blood granulocytes from rabbits experimentally infected with Trichophyton mentagrophytes

2018 ◽  
Vol 62 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Katarzyna Wojcicka-Lorenowicz ◽  
Krzysztof Kostro ◽  
Urszula Lisiecka ◽  
Bolesław Gąsiorek
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Phagocytic Activity ◽  
Spontaneous Recovery ◽  
Trichophyton Mentagrophytes ◽  
Oxygen Metabolism ◽  
Experimental Conditions ◽  
The Mean ◽  
Activity Indices

AbstractIntroductionPhagocytic activity and oxygen metabolism of peripheral blood granulocytes from rabbits with experimental trichophytosis were assessed by flow cytometry.Material and MethodsVirulent species of T. mentagrophytes var. granulosum (Tm-K) isolated from rabbits with natural trichophytosis was used for experimental infection. The phagocytic activity of granulocytes was measured in whole blood by flow cytometry using the commercial Phagotest kit. Oxidative burst was measured in whole blood by flow cytometry using the commercial Bursttest kit.ResultsIt was found that rabbits were susceptible to infection with Trichophyton mentagrophytes under experimental conditions. The analysis of the phagocytic activity indices and oxygen metabolism of granulocytes in peripheral blood of infected rabbits showed that changes of the indices were connected with the progression and regression of the disease. A significant decrease in phagocytic activity and oxygen metabolism was observed during development of fungal lesions and it remained similar throughout the progress of the disease. The highest means of the percentage of activated and ingesting phagocytes and a significant increase in the mean fluorescence intensity (representing the number of ingested bacteria) were observed during spontaneous recovery. Therefore, the decrease or increase in the indices of phagocytic activity and oxygen metabolism of granulocytes from rabbits experimentally infected with T. mentagrophytes is somehow related to the progress of infection and suppressive activity of the fungus, whose elimination during recovery caused significant increases in investigated indices of non-specific cellular immunity.ConclusionThe results of the present investigation confirm that the mechanism of oxygen-dependent killing is crucial in infections caused by T. mentagrophytes.

Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2030-2030
Author(s):  
Nishant Tageja ◽  
Neha Korde ◽  
Constance Yuan ◽  
Kristen Cole ◽  
Jennifer Hsu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
High Risk ◽  
Tumor Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cell ◽  
Myeloma Cells ◽  
Cd34 Cells ◽  
The Mean

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Association of High Prevalence of Trophozoites in Peripheral Blood with Lower Antibody Response toP. falciparumInfected Erythrocytes among Asymptomatic Children in Sudan

2016 ◽  
Vol 2016 ◽  
pp. 1-4
Author(s):  
Sara N. Mohamed ◽  
Dina A. Hassan ◽  
Abdelrahim M. El Hussein ◽  
Ihssan M. Osman ◽  
Muntasir E. Ibrahim ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Falciparum ◽  
Peripheral Blood ◽  
Surface Antigens ◽  
Igg Antibodies ◽  
Autologous Plasma ◽  
Variant Surface Antigens ◽  
Asymptomatic Children ◽  
Infected Rbcs ◽  
The Mean

Background. The most prominent variant surface antigens (VSAs) ofPlasmodium falciparumare the var gene-encodedPlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) family, which serves as a parasite-sequestering ligand to endothelial cells. In this study we have examined the antibody reactivity of autologous plasma from symptomatic and asymptomatic malaria infected children against the infected erythrocytes’ surface antigens using flow cytometry.Methods. Ethidium-bromide-labelled erythrocytic mature forms ofP. falciparumparasites obtained from symptomatic and asymptomatic children were sequentially incubated with autologous plasma and fluorescein isothiocyanate-conjugated (FITC) antihuman IgG. Plasma antibody reactivity was detected by flow cytometry.Results. Asymptomatic children had more prevalence of trophozoites in peripheral blood (66%) compared to symptomatic children (16%),p=0.002. The mean percentage of infected RBCs reacting with autologous sera was 89.78 among symptomatic children compared to 79.62 among asymptomatic children (p=0.09). Moreover, the mean fluorescence intensity (MFI) in the asymptomatic was significantly higher compared to symptomatic children (pvalue = 0.040).Conclusion. Variant surface antigens onPlasmodium falciparuminfected RBCs from symptomatic malaria children tend to be better recognized by IgG antibodies. This may suggest a role of some IgG antibodies in severity of malaria.

Clinical Predictors of Abnormal Peripheral Blood Lymphocytoses Diagnosed by Flow Cytometry: An Algorithmic Approach That Can Be Applied in Routine Clinical Practice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3932-3932
Author(s):  
Jared M. Andrews ◽  
Mitchel T. Holm ◽  
Jerome B. Myers
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Lymphocyte Count ◽  
Flow Cytometric Analysis ◽  
Clinical History ◽  
Lymphocytic Leukemia ◽  
Western World ◽  
Abnormal Phenotype ◽  
Flow Cytometric ◽  
The Mean

Abstract Background Elevated peripheral blood lymphocyte counts in adults can occur in benign reactive conditions as well as malignant disease processes. Chronic lymphocytic leukemia (CLL) is the most common adult hematologic malignancy of the western world affecting the middle aged and elderly. Less commonly B, T, and Natural Killer (NK) cell leukemia / lymphomas may also present with lymphocytosis. Flow cytometry has greatly improved the ability to detect low levels of abnormal lymphocyte populations in peripheral blood. It is, however, a relatively expensive test and clinical guidelines for its appropriate usage are not well defined. Methods We conducted a retrospective review of peripheral blood lymphocytoses that were submitted for flow cytometric analysis at Madigan Army Medical Center, Tacoma, WA from 2002 – 2004. Under laboratory protocol, all patients ≥ 50 years of age with an absolute lymphocyte count (ALC) of > 4 X 109 Cells/L had a peripheral smear evaluated by both a hematology technician and pathologist. Specimens determined to warrant flow cytometric analysis based on review of clinical history, prior lab values, degree of lymphocytosis, and morphology were either recommended for flow cytometry in a comment; or sent directly for analysis with the clinician’s approval. We reviewed complete blood counts (CBCs), previous flow cytometry results, as well as bone marrow and electronic clinical history. All patients with previous diagnoses of lymphoproliferative disorders (LPDs) or ALC < 4 X 109 Cells/L were excluded. Results Approximately 7,300 CBC specimens/month (3,400 from patients ≥ 50 years of age) were performed. Of these, an average of 44 specimens/month had a lymphocytosis of ≥ 4 X 109 Cells/L, from approximately 28 different patients. From this group 71 flow cytometric cases (an average of 2/month) were performed over the 2 year period. 42 cases (59%) had an abnormal phenotype. 27 had a phenotype consistent with CLL, and the other 15 were a mixture of LPDs involving B and T-lymphocytes as well as NK cells. Comparing normal phenotype to abnormal phenotype showed statistically significant differences between the mean age (n-60.4 ±7.5, abn-69.8±8.7), ALC (n-4.9±0.8, abn-9.2±8.1), and relative lymphocyte count (RLC) (n-43.9±7.5%, abn-59.3±8.8%). Conclusion Absolute lymphocyte counts ≥ 4 X 109 Cells/L in adults ≥ 50 years of age represent approximately 1% of the CBCs performed in our laboratory. Review of these cases by a pathologist is logistically feasible due to the low incidence. Our method of reviewing for morphology, clinical history, and past lymphocyte counts with comments to the ordering clinician yielded a high incidence of abnormal phenotype diagnoses when evaluated by flow cytometric analysis (59%). Age, ALC, and relative lymphocyte counts are variables that can be used to develop guidelines for determining the appropriateness of flow cytometric analysis. Patients < 52.4 years of age fall below two standards of deviation from the mean age of the abnormal phenotype group. The standard of deviation for mean ALC is very small (4.9±0.8), which indicates that counts > two standards of deviation above the mean, or 6.5 X 109 Cells/L, would correlate strongly with an abnormal phenotype. The same conclusion could be made with a RLC > 58.9%. In conclusion, patients ≥ 50 years of age with an ALC > 6.5 X 109 Cells/L or a RLC > 58.9% are likely to have a lymphoproliferative disorder and flow cytometric analysis is indicated.

Hyperbaric Oxygen Therapy Improves Post-Transplant Umbilical Cord Blood Engraftment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4663-4663
Author(s):  
Omar S. Aljitawi ◽  
Xiao Yinghua ◽  
Tara Lin ◽  
Jeff Eskew ◽  
Nikhil Parelkar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
B Cell ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Hyperbaric Oxygen Therapy ◽  
Oxygen Therapy ◽  
Cell Engraftment ◽  
The Mean

Abstract Abstract 4663 Background: Delayed engraftment following umbilical cord blood (UCB) transplantation is associated with increased transplant-related mortality. Earlier, we have reported that hyperbaric oxygen therapy (HBO) might have altered early UCB engraftment kinetics in a transplant animal model. Herein, we report use of HBO to improve long-term UCB engraftment. Methods: Six-to eight-week old NSG mice were sublethally irradiated 24 hours prior to CD34 selected UCB cell transplant. The irradiated mice were separated into a control group (where mice remained under normoxic conditions) and the HBO group (where mice received two hours of HBO therapy; 100% oxygen at 2.5 atmospheres absolute). Six hours after starting HBO therapy, both groups received 1×105 CD34 selected UCB cells via tail vein injection. The infused CD34 selected cells were transduced with a lentivirus carrying luciferase gene for in vivo imaging. Mice (n=4) from each treatment group were sacrificed after 3 and 4 months to assess long-term engraftment. Engraftment was measured as assessed by flow cytometry. Myeloid, B-cell, and T-cell subset engraftment was determined by flow cytometry. Results: At 3 and 4 months post-transplant, the mean percentage of human CD45 expressing cells in peripheral blood of HBO mice was significantly higher than controls (p=0.03 and p=.008 respectively). The mean percentage of human CD45 expression was higher in bone marrow of HBO mice at 3 months and 4 months, but only reached statistical significance at 4-month time point (p=0.04). At 3 months, the mean percentage of human CD19 expressing cells was significantly higher in peripheral blood of HBO mice (p=0.03) while no differences were noted between the two groups in terms of CD33 or CD3 expression. At 4 months, the mean percentage of human CD19 expressing cells and human CD3 expressing cells was significantly higher in peripheral blood of HBO mice (p=0.009 and p=0.0009, respectively) while no differences were noted between the two groups in terms of CD33 expression. Conclusions: HBO therapy given prior to UCB cell infusion significantly improved UCB's long-term engraftment. At 3 months, engraftment was skewed toward B-cell engraftment, while more balanced engraftment was noted at 4 months. Future studies are planned to confirm these findings and to determine the underlying mechanisms by which HBO improved UCB CD34 cell engraftment. Disclosures: No relevant conflicts of interest to declare.

Rapid whole blood assay using flow cytometry for measuring phagocytic activity of chicken leukocytes

2019 ◽  
Vol 207 ◽  
pp. 53-61 ◽  
Author(s):  
Mohammed Naghizadeh ◽  
Frederik T. Larsen ◽  
Eva Wattrang ◽  
Liselotte R. Norup ◽  
Tina S. Dalgaard
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Phagocytic Activity ◽  
Whole Blood Assay ◽  
Blood Assay

Cat carotid body oxygen metabolism and chemoreception described by a two-cytochrome model

1986 ◽  
Vol 250 (2) ◽  
pp. H202-H207 ◽  
Author(s):  
P. K. Nair ◽  
D. G. Buerk ◽  
W. J. Whalen
Keyword(s):  
Oxygen Metabolism ◽  
Disappearance Rate ◽  
Nerve Activity ◽  
Experimental Conditions ◽  
Carotid Bodies ◽  
Tissue Po2 ◽  
O2 Concentration ◽  
Consumption Rates ◽  
The Mean ◽  
Increased Activity

We have analyzed O2 disappearance curves (DCs) in cat carotid bodies (CBs) measured with our O2 microelectrode, after stopping flow of either blood (108 DCs in 12 cats) or a hemoglobin-free (Locke) perfusion solution (35 DCs in 6 cats). Prior to occlusion, the mean tissue PO2 levels were 74.5 +/- 2.8 (SE) Torr in blood-perfused CBs and 103.4 +/- 2.6 in Locke-perfused CBs. The O2 consumption rates (VO2) determined from the initial 3 s of the DCs were 1.46 +/- 0.08 and 1.50 +/- 0.10 (SE) ml O2 . 100 g-1 . min-1, respectively, for the blood-perfused and Locke-perfused CBs. The change in total sinus nerve activity from the CB was also measured following stopped flow. The nerve activity began to increase immediately, providing further evidence that classic hypoxia is not the mechanism of chemoreceptor discharge. However, about two-thirds of the increased activity in blood-perfused CBs occurred after tissue PO2 levels fell below 20 Torr. As the CB tissue PO2 decreased, the O2 disappearance rate (-dPO2/dt) also decreased for both experimental conditions, indicating that the CB VO2 varies with O2 concentration. The increase in nerve discharge and O2 disappearance rate can be interpreted by a two-cytochrome model for O2 metabolism, with both high and low affinities.

scholarly journals CD11b Regulates Fungal Outgrowth but Not Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3690-3690
Author(s):  
Daniel Teschner ◽  
Anna Cholaszczyńska ◽  
Frederic Ries ◽  
Hendrik Beckert ◽  
Matthias Theobald ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Phagocytic Activity ◽  
Invasive Pulmonary Aspergillosis ◽  
Fungal Culture ◽  
Integrin Receptors ◽  
Pulmonary Aspergillosis ◽  
Adaptive Immune ◽  
Travel Grant

Abstract Background and Aims: In immunosuppressed individuals Aspergillus (A.) fumigatus is a frequent cause of invasive pulmonary aspergillosis (IPA) which is highly associated with relevant morbidity and mortality. Moreover, it often occurs in patients suffering from leukocyte-adhesion deficiency type 1 (LAD1) which is triggered by a functional loss of CD18 in ß2 integrin receptors as these receptors consist of an alpha subunit (CD11a-CD11d) and CD18 as the common beta subunit. ß2 integrin receptors are differentially expressed by leukocytes, and are required for cell-cell interaction, transendothelial migration, uptake of opsonized pathogens, and cell signaling processes. Here, we asked for the importance of CD11b/CD18 also termed MAC-1 which is required for phagocytosis of opsonized A. fumigatus conidia by polymorphonuclear neutrophils (PMN) for control of pulmonary A. fumigatus infection. Methods: We used a murine IPA model (C57BL/6) and challenged CD11b deficient (CD11b-/-) or wild type (WT) mice with A. fumigatus conidia intratracheally. Afterwards, some mice were sacrificed 24h after infection. In these mice PMN recruitment and cytokine patterns were examined by analyzing bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) using flow cytometry, cytospin analysis, and cytometric bead array. Additionally, pulmonary fungal clearance and inflammation in murine lungs were analyzed by fungal culture assays and histopathologic examination using a scoring system. Furthermore, survival was studied with neutropenic animals serving as positive controls. To determine PMNs phagocytic activity and fungal killing capacity ex vivo, PMN were purified from bone marrow of CD11b-/- or WT mice by magnetic cell sorting using Ly6G specific antibodies. Afterwards, isolated PMN were stimulated with pacific blue-labeled tomato red-fluorescent modified A. fumigatus conidia and analyzed by flow cytometry. Results: We found that lung homogenates from CD11b-/- mice obtained 24h after infection showed an enhanced fungal burden as compared to lungs from WT mice. In contrast, lung tissue from infected CD11b-/- mice displayed impaired pulmonary inflammation as assessed by Hematoxilin-Eosin (HE) staining. Furthermore, the number of mucus-producing cells in bronchi of A. fumigatus infected CD11b-/- mice was decreased compared to cells in bronchi of WT mice. However, we observed markedly higher numbers of PMN in BALF of infected CD11b-/- mice compared to corresponding WT mice samples. In contrast, BALF derived from infected CD11b-/- mice contained lower levels of three different proinflammatory cytokines (TNF-α, IL-1α, IL-1β) compared to BALF from WT mice while levels of other cytokines and chemokines (IL-5, IL-6, IL-10, GM-CSF, CXCL1, CCL2) were comparable. In contrast, we measured higher levels of the chemokine CCL5 known as a relevant chemoattractant in innate and adaptive immune cells in BALF obtained from CD11b-/- mice. Interestingly, numbers of PMN, lymphocytes, and monocytes in the peripheral blood of A. fumigatus infected mice did not differ in a genotype-dependent manner. However, CD11b-/- mice PMN showed lower phagocytic activity than WT PMN, indicating an impaired capability to clear A. fumigatus. Nevertheless, CD11b-/- mice were finally characterized by similar long-term survival as WT mice after IPA induction while the PMN-depleted control mice died as expected. Conclusions: Our study demonstrates, that CD11b deficiency on myeloid cells affects the early course of IPA. This may be due to the importance of MAC-1 for PMN effector functions, and their interplay with DC and other leukocytes. Further work is necessary to elucidate the long-term course of IPA in CD11b-/- mice with regard to the interplay of PMN with dendritic cells, the efficacy of adaptive immune responses, and the potential pulmonary overexpression of CCL5. Beyond patients with LAD1 syndrome, we suggest our findings as being clinically relevant in other immunocompromised patients suffering from severe opportunistic infections. Disclosures Radsak: Celgene: Honoraria, Other: Travel grant; Jazz Pharmaceuticals: Other: Travel grant; TEVA: Consultancy; Gilead: Other: Travel grant; Takeda: Consultancy; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Honoraria, Other: Travel grant.

Phospho-Specific Flow Cytometry of Fixed Whole Blood During AML Clinical Trials to Monitor In Vivo FLT3 Inhibition in Leukemic Blasts,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3508-3508
Author(s):  
Alexander E Perl ◽  
Grace R Jeschke ◽  
Takashi Sato ◽  
Shiro Akinaga ◽  
Niranjan S. Rao ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Flow Cytometry ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Initial Dose ◽  
Research Funding ◽  
Ex Vivo ◽  
Blood Samples ◽  
Flt3 Inhibitors

Abstract Abstract 3508 Although first-generation FLT3 inhibitors may have had limited anti-leukemic effects due to suboptimal target inhibition, newer drugs such as AC220 and KW-2449 have substantially greater in vitro potency and bioavailability. Ex vivo assays such as the plasma inhibition assay (PIA) are useful to estimate free drug bioavailability, but direct confirmation of biochemical FLT3 inhibition in leukemic blasts in vivo has proven more challenging to employ systematically for drug development. Here we report the development of a fixed whole blood intracellular flow cytometry platform to measure real-time signal inhibition during a clinical trial of the second-generation FLT3 inhibitor KW-2449. Methods: Anticoagulated blood samples were aliquoted into FACS tubes within four hours of collection; a subset was exposed to signaling inhibitors (KW-2449, rapamycin × 30 min.) or activators (phorbol ester/PMA or FLT3 ligand/FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed, red cells were lysed with the permeabilizing agent triton X-100, and specimens were stored at −20C in glycerol medium. Subjects' samples from all time points were simultaneously thawed, denatured with ice-cold methanol, and stained with a single cocktail of antibodies. Blasts were identified by CD45 and side scatter (SSC) and confirmed by multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Positive gates for phospho-proteins were created by comparing blasts in stimulated and unstimulated conditions and/or autofluorescence (FMO) controls. Results: Despite adequate controls, flow demonstrated limited changes in FLT3-ITD+ blasts' pSTAT5 signal following either FL stimulation or ex vivo KW-2449 treatment of these peripheral blood primary samples. This contrasted with the FLT3-ITD+ cell line Molm14, in which FLT3 inhibition reduced pSTAT5. However, the PI3K/AKT/mTOR downstream target ribosomal protein S6 (S6) was consistently observed to be constitutively phosphorylated in both Molm14 cells and peripheral blood FLT3-ITD+ AML blasts. pS6 in all FLT3-ITD+ samples markedly augmented with ex vivo FL, and decreased following ex vivo KW-2449 treatment. We therefore serially monitored S6 phosphorylation during therapy on a phase 1/2 trial of KW-2449. In this clinical trial, subjects were treated with KW-2449 every 6–8 hours, due to the drug's relatively short half life. 10 subjects (9 FLT3-ITD+, 1 FLT3-WT) provided serial blood samples for analysis. All FLT3-ITD+ subjects had blasts identifiable by morphology and immunophenotype. Samples with as few as 500 blasts/uL were informative for pS6. In all cases, blasts showed dynamic changes in pS6 in response to ex vivo FL. As previously described using intracellular flow cytometry, pS6 in primary AML samples was heterogeneous, and, at basal state, frequently only demonstrable in a subset of blasts. We observed constitutive S6 phosphorylation in 8/9 subjects' leukemic cells. The mean percentage of blasts with constitutive pS6 was 21% (median 7%, range 5–70%). To directly quantify FLT3 kinase inhibition in vivo, we serially monitored pS6 in blasts by flow prior to and following their initial oral KW-2449 dose. In 8/8 patients with baseline constitutive S6 phosphorylation, blood obtained two hours following the initial dose showed marked reduction in the percentage of pS6+ blasts to a mean of 3.8% (median 1.3% range 0.1 to 20%). This reflected an 83% mean reduction in the percentage of pS6+ blasts. PIA was performed in 8/9 of FLT3-ITD+ subjects and confirmed that potent FLT3-inhibitory concentrations were present 2 hours after a single dose of KW-2449 (mean reduction from baseline of 79% for pFLT3 and 88% for pSTAT5). Two subjects' samples were followed serially by flow cytometry throughout the dosing interval. One showed sustained inhibition (consistent with concurrent PIA), while in the other, pS6 returned to baseline within 4–6 hours of the initial dose (concurrent PIA not done). Summary: We confirm that PI3K/AKT/mTOR is a major downstream pathway of FLT3 signaling in primary AML samples. We further demonstrate the feasibility of intracellular flow cytometry for S6 phosphorylation to monitor the biochemical efficacy of FLT3 inhibitors in patients. Studies are underway to correlate biochemical FLT3 inhibition by flow cytometry with clinical response/resistance to KW-2449 and other FLT3 inhibitors. Disclosures: Sato: Kyowa Hakko Kirin Co., LTD: Employment. Akinaga:Kyowa Hakko Kirin Co., LTD: Employment. Rao:Kyowa Hakko Kirin Co., LTD: Employment. Levis:Kyowa Hakko Kirin Co., LTD: Research Funding; Ambit Biosciences: Consultancy. Carroll:Kyowa Hakko Kirin Co., LTD: Research Funding.

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