Changes In Cell Surface Markers Research Articles

The proliferation/migration ability mediated by CD151/PI3K/AKT pathway determines the therapeutic effect of hUC-MSCs transplantation on rheumatoid arthritis

ABSTRACT Objective To elucidate the underlying mechanism by which the proliferation and migration abilities of human umbilical cord mesenchymal stem cells (hUC-MSCs) determine their therapeutic efficacy in rheumatoid arthritis treatment. Methods The DBA/1J mice were utilized to establish a collagen-induced RA (CIA) mouse model and to validate the therapeutic efficacy of hUC-MSCs transfected with CD151 siRNA. RNA-seq, QT-PCR and western blotting were utilized to evaluate the mRNA and protein levels of the PI3K/AKT pathway, respectively. Results IFN-γ significantly enhanced the proliferation and migration abilities of hUC-MSCs, up-regulating the expression of CD151, a gene related to cell proliferation and migration. Effective inhibition of this effect was achieved through CD151 siRNA treatment. However, IFN-γ did not affect hUC-MSCs differentiation or changes in cell surface markers. Additionally, transplantation of CD151-interfered hUC-MSCs (siRNA-CD151-hUC-MSCs) resulted in decreased colonization in the toes of CIA mice and worse therapeutic effects compared to empty vector treatment (siRNA-NC-hUC-MSCs). Conclusion IFN-γ facilitates the proliferation and migration of hUC-MSCs through the CD151/PI3K/AKT pathway. The therapeutic efficacy of siRNA-CD151-hUC-MSCs was found to be inferior to that of siRNA-NC-hUC-MSCs.

Staphylococcus aureus induces tolerance in human monocytes accompanied with expression changes of cell surface markers.

Exposure of human monocytes to lipopolysaccharide (LPS) or other pathogen-associated molecular pattern (PAMPs) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance (ET), associated with the pathogenesis of sepsis. In this study, we aimed to characterize the cellular state of human monocytes from healthy donors stimulated with Staphylococcus aureus in comparison to TLR2-specific ligands. We analyzed S. aureus induced gene expression changes after 2 and 24 hours by amplicon sequencing (RNA-AmpliSeq) and compared the pro-inflammatory response after 2 hours with the response in re-stimulation experiments. In parallel, glycoprotein expression changes in human monocytes after 24 hours of S. aureus stimulation were analyzed by proteomics and compared to stimulation experiments with TLR2 ligands Malp-2 and Pam3Cys and TLR4 ligand LPS. Finally, we analyzed peripheral blood monocytes of patients with S. aureus bloodstream infection for their ex vivo inflammatory responses towards S. aureus stimulation and their glycoprotein expression profiles. Our results demonstrate that monocytes from healthy donors stimulated with S. aureus and TLR ligands of Gram-positive bacteria entered the tolerant cell state after activation similar to LPS treatment. In particular reduced gene expression of pro-inflammatory cytokines (TNF, IL1β) and chemokines (CCL20, CCL3, CCL4, CXCL2, CXCL3 and CXCL8) could be demonstrated. Glycoprotein expression changes in monocytes tolerized by the different TLR agonists were highly similar while S. aureus-stimulated monocytes shared some of the PAMP-induced changes but also exhibited a distinct expression profile. 11 glycoproteins (CD44, CD274, DSC2, ICAM1, LAMP3, LILRB1, PTGS2, SLC1A3, CR1, FGL2, and HP) were similarly up- or downregulated in all four comparisons in the tolerant cell state. Monocytes from patients with S. aureus bacteremia revealed preserved pro-inflammatory responsiveness to S. aureus stimulation ex vivo, expressed increased CD44 mRNA but no other glycoprotein of the tolerance signature was differentially expressed.

Effects of LSD1 Inhibition with Img-7289 on the Leukemia Initiating Cell Population with Induction of Differentiation and Cell Death in Pediatric Relapsed/Refractory AML

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

Effect of bFGF on fibroblasts derived from the golden snub-nosed monkey.

Golden snub-nosed monkeys are endangered animals in China, and their cells have been demonstrated to be important as genetic resources and in applications for advancing biological research. Moreover, in primary research, basic fibroblast growth factor (bFGF) is used to promote the proliferation of fibroblasts to create abundant cells for cryopreservation. To further investigate the effect of bFGF on the efficiency of preservation of fibroblasts obtained from an endangered species, a fibroblast cell line was isolated from a dead golden snub-nosed monkey. Cell viability and mitochondrial membrane potential were assessed using CCK8 and JC-1 assay kits. The karyotype was analyzed by chromosomal microarray analysis, while RNA sequencing and gene expression analyses were performed to assess molecular changes in response to bFGF. Flow cytometry was used to characterize changes in cell surface markers in response to bFGF treatment. The results showed that cells maintained typical fibroblast morphology, while cell viability and mitochondrial membrane potential were not significantly affected between three and eight passages (p > 0.05). We also observed that the addition of bFGF promoted fibroblast proliferation and increased mitochondrial membrane potential. In addition, the bFGF treatment did not alter the normal karyotype of cells, downregulating fibroblast-associated genes and upregulating those associated with cell regulation, including those of the WNT, PI3K and MAPK pathways. The addition of bFGF also increased CD29, CD90, CD105, CD34 and CD44 expression while decreasing that of CD14 and HLA-DR at the protein level. Taken together, these results demonstrate that bFGF may upregulate the WNT, PI3K and MAPK pathways to promote cell proliferation while also increasing the expression of genes and surface markers associated with mesenchymal and hematopoietic cell linages.

Understanding regulatory B cell development with single cell analyses

There is very little research concerning human regulatory B cells and this may in part be due to their inconsistent responsesto immunosuppressive cytokines such as IL-10. The purpose of this critical review is to examine our current understandingof regulatory B cell development, such as time points of differentiation, and how in silico computer modelling can improvethis understanding. Specifically, bioinformatic analysis of the changes in cell surface markers and signalling moleculescan help guide our understanding of the timing of cell-fate decisions and regulatory B cell differentiation. Trackingregulatory B cell trajectory with bioinformatics and in silico methods may improve our understanding of their role in manyneurodegenerative diseases such as multiple sclerosis.

Understanding regulatory B cell development with single cell analyses

There is very little research concerning human regulatory B cells and this may in part be due to their inconsistent responsesto immunosuppressive cytokines such as IL-10. The purpose of this critical review is to examine our current understandingof regulatory B cell development, such as time points of differentiation, and how in silico computer modelling can improvethis understanding. Specifically, bioinformatic analysis of the changes in cell surface markers and signalling moleculescan help guide our understanding of the timing of cell-fate decisions and regulatory B cell differentiation. Trackingregulatory B cell trajectory with bioinformatics and in silico methods may improve our understanding of their role in manyneurodegenerative diseases such as multiple sclerosis.

Selective Uptake Into Drug Resistant Mammalian Cancer by Cell Penetrating Peptide-Mediated Delivery.

Research over the past decade has identified several of the key limiting features ofmultidrug resistance (MDR) incancer therapy applications, such as evolving glycoprotein receptors at the surface of the cell that limit therapeutic uptake, metabolic changes that lead to protection from multidrug resistant mediators which enhance degradation or efflux of therapeutics, and difficulty ensuring retention of intact and functional drugs once endocytosed. Nanoparticles have been demonstrated to be effective delivery vehicles for a plethora of therapeutic agents, and in the case of nucleic acid based agents, they provide protective advantages. Functionalizing cell penetrating peptides, also known as protein transduction domains, onto the surface of fluorescent quantum dots creates a labeled delivery package to investigate the nuances and difficulties of drug transport in MDR cancer cells for potential future clinical applications of diverse nanoparticle-based therapeutic delivery strategies. In this study, eight distinct cell penetrating peptides were used (CAAKA, HSV1-VP22, HIV-TAT, HIV-gp41, Ku-70, hCT(9-32), integrin-β3, and K-FGF) to examine the different cellular uptake profiles in cancer versus drug resistant melanoma (A375 & A375-R), mesothelioma (MSTO & MSTO-R), and glioma (rat 9L and 9L-R, and human U87 & LN18) cell lines. The results of this study demonstrate that cell penetrating peptide uptake varies with drug resistancestatusand cell type, likely due to changes in cell surface markers. This study provides insight into developing functional nanoplatform delivery systems in drug resistant cancer models.

TLR2 stimulation impairs anti-inflammatory activity of M2-like macrophages, generating a chimeric M1/M2 phenotype

BackgroundToll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-κB, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation.MethodsHuman monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-γ/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-κB, IRF3/7, and MAPKs were detected by Western blotting.ResultsMonocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS.ConclusionsWe show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA.

Abstract B23: Detection of susceptibility to childhood Acute Lymphoblastic Leukemia (ALL)

Abstract Currently, there are no known methods to predict of susceptibility to, and means for prevent Acute Lymphoblastic Leukemia (ALL). We have evaluated and patented a group of proteins dubbed Protein X from a certain strain of Aspergillus Flavus (AF) and developed methods for screening and identifying totally asymptomatic patients in remission of ALL, including long term survivors of this disease, distinguishing them from normal controls. Subject to institutional approved consents/assents, 15-20 ml of blood was obtained from 40 cases of ALL in children and young adults, including long term survivors of ALL. Controls were normal individuals, sickle cell patients undergoing partial exchange transfusion and patients with solid tumors. Mononuclear leukocytes (MNL) of ALL patients in remission and controls were separated using Ficoll Paque Plus (GE Healthcare). Epstein Barr virus (EBV) was obtained commercially. Positive and negative controls for Protein X were aflatoxin and Mycocladus Corymbifera (MC). Avian leukosis virus (ALV) was used as control for EBV. MNL were co-incubated with Protein X ± EBV ± irradiation, for periods of 1-72 hours. Controls were treated identically with appropriate substitutions. Test and control MNLs were examined for genetic markers, NF-κB and cell surface markers (CSM) including CD10/CD19, CD34/CD19, and CD34/CD117. Changes were expressed as percentage of control. Using ELISA, plasmas were tested for antibodies against Protein X ± EBV time experiments reveled 72 hours was optimum for achieving results. Upon 72 hours exposure of MNL from ALL to Protein X ± EBV, cells from ALL patents in remission developed cell surface phenotypes typical of ALL. This was not seen in controls. Addition of EBV ± radiation to Protein X, enhanced these effects in MNL of ALL and not controls. Changes were statistically significant and clearly separated ALL from controls. Evaluation of NF-κB revealed enhancement in ALL and not controls. Aflatoxin indiscriminately induced cell surface marker changes in both, normal and ALL, while ALV and supernatant of MC had no effect. ELISA, using Protein X ± EBV, distinguished ALL from controls. Gene array and biomarkers confirmed transformation to leukemic cell markers upon exposure to Protein X in cells from ALL patients but not controls. These studies reveal, in vitro, upon exposure to Protein X, unlike normal controls, MNL from ALL patients in remission develop cell surface phenotypes and genetic markers typical of ALL. These techniques have potential for screening for ALL and may have implications for etiology of ALL and its prevention. Citation Format: Cameron K. Tebbi. Detection of susceptibility to childhood Acute Lymphoblastic Leukemia (ALL). [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr B23.

Do IL-3/GM-CSF effect on the myofibroblastic differentiation of human adipose derived stromal cells?

BackgroundCapsular contracture is an incurable complication after silicone-based implant surgery. Myofibroblast is the predominant cell in the contracted capsule. We hypothesized that human adipose derive stromal cells (hASCs) together with fibroblast may show a similar phenotypic characteristics of myofibroblast after the treatment of inflammatory cytokines in vitro. Materials and methodsInterleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) were treated in the culture of hASCs and HDFs. Lyn peptide inhibitor was applied as an inhibitor. The changes of cell surface markers (CD105, CD73, CD34, CD45, CD31, CD325 and CD146) were assessed. The expression of various cytokines related to wound contraction were tested such as TGF-β, α-SMA, HGF, FGF, ENT-1, and TSP-1. Myo-D, α-SMA, and glial fibrillary acidic protein (GFAP) were evaluated by blotting and immunocytochemical staining. The collagen-gel contraction assay was performed for the functional contraction of myofibroblastic phenotype. ResultsThe expression of α-SMA, Myo-D and GFAP after the treatment of IL-3/GM-CSF showed similar results in hASCs and HDFs. Enhanced expression of TGF- β was observed in HDFs and the increase of ENT-1 and TSP-1 was significant in hASCs. Collagen-gel with HDFs contracted significantly within 24h after the treatment of IL-3/GM-CSF, and the contraction was inhibited by Lyn peptide inhibitor. But in hASCs, the gel-contraction was not significant. ConclusionIL-3/ GM-CSF effected on the myofibroblastic differentiation of hASCs as well as it did on HDFs. But hASCs did not show the phenotypic gel-contraction within 24h.

Stromal Cells Act as Guardians for Endothelial Progenitors by Reducing Their Immunogenicity After Co-Transplantation.

Regeneration of injured tissues requires effective therapeutic strategies supporting vasculogenesis. The lack of instantly available autologous cell sources and immunogenicity of allogeneic endothelial (progenitor) cells limits clinical progress. Based on the immunosuppressive potency of mesenchymal stem/progenitor cells (MSCs), we investigated whether crosstalk between endothelial colony-forming progenitor cells (ECFCs) and MSCs during vasculogenesis could lower allogeneic T cell responses against ECFCs allowing long-term engraftment in vivo. Immunodeficient mice received subcutaneous grafts containing human ECFCs alone, or pairs of human ECFCs/MSCs from the same umbilical cord (UC) to study vasculogenesis in the presence of human leukocyte antigen (HLA)-mismatched human peripheral blood mononuclear cells (PBMCs). In vitro, cell surface marker changes due to interferon gamma (IFNγ) stimulation during ECFC/MSC coculture were determined and further effects on allostimulated T cell proliferation and cytotoxic lysis were measured. IFNγ-induced HLA-DR expression on ECFCs and MSCs, but both cell types had significantly less HLA-DR in cocultures. ECFC-induced T cell proliferation was abolished after MSC coculture as a result of HLA-DR downregulation and indolamin-2,3-dioxygenase activation. Additionally, allospecific CD8+ T cell-mediated lysis of ECFCs was reduced in cocultures. ECFC/MSC coapplication in immunodeficient mice not only promoted the generation of improved blood vessel architecture after 6 weeks, but also reduced intragraft immune cell infiltration and endothelial HLA-DR expression following PBMC reconstitution. Crosstalk between UC-derived ECFCs and MSCs after combined transplantation can lower the risk of ECFC rejection, thus enabling their coapplication for therapeutic vasculogenesis. Stem Cells 2017;35:1233-1245.

Ex Vivo High-Throughput Flow Cytometry Screening Identifies Subsets of Responders to Differentiation Agents in Individual AML Patient Samples

Background:Prognoses for acute promyelocytic leukemia (APL) patients improved drastically upon the introduction of differentiation therapy with all-trans-retinoic acid (ATRA) in combination with conventional chemotherapy. Unfortunately, this therapeutic approach has not translated to other genetic subtypes of acute myeloid leukemia (AML) where patients demonstrate marked heterogeneity to differentiating agents. To provide improved detection of drug-induced differentiation in AML patients, we have developed a high-throughput, flow cytometry-based personalized medicine platform.Methods:Total white blood cells were isolated from each patient sample by red cell lysis, plated in serum-free media in 384-well format and incubated with drugs for 3 days. Viable cells remaining after each drug treatment were identified and quantified using cell surface marker expression, cell membrane integrity, and morphology (FSC/SSC) to determine the compound's efficacy and specificity against the blast population. Changes in cell surface marker expression and shifts in morphology indicative of blast differentiation were also evaluated with each compound. As a control for ex vivo differentiation, two APL patient samples were treated ex vivo with ATRA and we observed the blasts gaining CD66b expression indicating granulocytic differentiation.Results:A refractory AML patient was identified whose leukemic blasts exhibited a strong differentiating response to dexamethasone treatment ex vivo. This resulted in loss of CD34 expression (a marker of immature blast cells), gain of CD163 expression (a marker of monocytic/macrophage maturation) and a significant change in cellular size and granularity. After being enrolled in a clinical trial (REB: 13-6962-C) the patient was treated based on the assay for 1 week (40 mg/day) with dexamethasone. Post-treatment samples from the peripheral blood and bone marrow of the patient exhibited the same morphological and cell surface marker changes predicted by the ex vivo assay. The CD163+ cells in the patient also gained additional markers of myeloid differentiation (CD11b, CD14, CD16). After additional cytarabine and fludarabine treatment, the patient remains in remission 4 months post-treatment.Conclusions:Following this initial study, we have continued to identify subgroups of both AML and Myelodysplastic Syndrome patients where blasts differentiate in response to dexamethasone, calcitriol, ATRA or other known differentiating agents using unique cell surface markers of monocytic and myeloid maturation. Flow cytometry expression changes correlated with changes in morphology as observed by May-Grunwald Giemsa staining. In the patient described above this included an increase in cytoplasm and vacuoles consistent with monocytic/macrophage differentiation, which positively correlates with CD163 expression. We aim to apply our assay towards the identification of subgroups of AML patients who respond to differentiation therapies and develop clinical trials to combine differentiating agents with chemotherapy. This approach has the potential to extend the clinical success of APL differentiation therapy to AML patients. DisclosuresPrashad:Notable Labs: Employment, Equity Ownership. Western:Notable Labs: Consultancy. Biondi:Notable Labs: Employment. Shah:Notable Labs: Employment. Liu:Notable Labs: Employment, Equity Ownership. Nguyen:Notable Labs: Employment, Equity Ownership. Warnock:Notable Labs: Employment, Equity Ownership. Quinzio:Notable Labs: Employment, Equity Ownership. De Silva:Notable Labs: Employment, Equity Ownership. Schimmer:Novartis: Honoraria. Heiser:Notable Labs: Employment, Equity Ownership.

An ultra scale-down analysis of the recovery by dead-end centrifugation of human cells for therapy.

An ultra scale-down method is described to determine the response of cells to recovery by dead-end (batch) centrifugation under commercially defined manufacturing conditions. The key variables studied are the cell suspension hold time prior to centrifugation, the relative centrifugal force (RCF), time of centrifugation, cell pellet resuspension velocities, and number of resuspension passes. The cell critical quality attributes studied are the cell membrane integrity and the presence of selected surface markers. Greater hold times and higher RCF values for longer spin times all led to the increased loss of cell membrane integrity. However, this loss was found to occur during intense cell resuspension rather than the preceding centrifugation stage. Controlled resuspension at low stress conditions below a possible critical stress point led to essentially complete cell recovery even at conditions of extreme centrifugation (e.g., RCF of 10000 g for 30 mins) and long (∼2 h) holding times before centrifugation. The susceptibility to cell loss during resuspension under conditions of high stress depended on cell type and the age of cells before centrifugation and the level of matrix crosslinking within the cell pellet as determined by the presence of detachment enzymes or possibly the nature of the resuspension medium. Changes in cell surface markers were significant in some cases but to a lower extent than loss of cell membrane integrity. Biotechnol. Bioeng. 2015;112: 997–1011. © 2014 Wiley Periodicals, Inc.

Abstract 3604: Investigation of the role of CSF-1 and its receptor in the bone marrow stroma microenvironment in acute myeloid leukemia

Abstract Supporting cells in the bone marrow microenvironment are an essential source of growth factors and cytokines that drive both normal hematopoeisis and leukemogenesis. Acute myeloid leukemia (AML) is a heterogeneous and growth factor-dependent disease of the bone marrow. The human macrophage colony stimulating factor (M-CSF, CSF-1) is a cytokine produced by supporting cells in the bone marrow that promotes the differentiation and maturation of monocytes and macrophages from myeloid precursors. Since CSF-1 is an essential factor in myelopoeisis, it has important implications in AML, a disease characterized by the increased proliferation of immature myeloid blast cells. Moreover CSF-1 and CSF-1R are highly expressed in some AML cases, lending further support for their investigation. CSF-1 exists as a full-length secreted (variant 1 or v1) and membrane-bound (variant 3 or v3) isoform. The membrane-bound v3 isoform is the predominant form in the bone marrow. CSF-1 binds to the colony stimulating factor-1 receptor (CSF-1R), a class III receptor tyrosine kinase found on mononuclear phagocytes. CSF-1R activation leads to stimulation of survival, proliferation and differentiation pathways. In this study, the effects of the CSF-1/CSF-1R interaction were investigated in the context of the bone marrow microenvironment in an in-vitro co-culture system. In this model, human leukemic cells (patient samples and cell lines) were grown in co-culture with mouse stromal cells expressing human CSF-1. The cellular and molecular responses arising from the interaction between leukemic cells expressing CSF-1R and stromal cells producing CSF-1 were examined. AML cells cultured with CSF-1-expressing stroma displayed better long-term (2-5 weeks) growth and survival than cells cultured with non-CSF-1-expressing stroma. There was a 2-fold increase in the number of cells on CSF-1 v3 stroma compared to empty vector (ev) control stroma after 5 weeks of co-culture. AML cells were maintained on CSF-1 v3 stroma 1.5-1.75 fold longer than on ev stroma (survival in days). Co-culture of AML cells with CSF-1-expressing stroma led to a decrease in CSF-1R expression on the surface of AML cells, accompanied by an increase in CD34 and decrease in CD38 expression. CSF-1-expressing stroma led to increases in phosphorylation of Akt and p70S6K, effectors of the PI3k/Akt pathway, in AML cells. Analysis of cytokine production in AML-stromal cell co-cultures revealed an increase in the secretion of IL-3 and IL-6 from stromal cells and an increase in IL-8 and vascular endothelial growth factor (VEGF) from AML cells. These findings suggest that CSF-1 presented by stromal cells mediates the growth and survival of AML cells through changes in cell surface markers, molecular signaling effectors and cytokine secretion. This work has helped identify targets of interest that may be exploited for therapeutic purposes in AML-stromal cell interactions. Citation Format: Ayesha Rashid, Mohammed Fateen, Rhe Juan, Rob C. Laister, Mark Minden. Investigation of the role of CSF-1 and its receptor in the bone marrow stroma microenvironment in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3604. doi:10.1158/1538-7445.AM2014-3604

PiggyBac Transposon Mediated Reprogramming and Flow Cytometry Analysis of CD44 and ICAM1 Cell-Surface Marker Changes.

Generation of iPSCs is inefficient and the molecular mechanisms underlying reprogramming are not well understood. While several studies have demonstrated that reprogramming is not entirely a random process and contains predictable stepwise changes, varying degrees of cellular heterogeneity that arise in different reprogramming systems can obscure the process. Among several reprogramming systems available, delivery of polycistronic reprogramming factor expression cassettes with piggyBac transposon into mouse embryonic fibroblasts (MEFs) is one of the simplest and most robust reprogramming approaches that provide a low background of partially reprogrammed cells. Using two novel cell surface markers, ICAM1 and CD44, clear cell population changes undergoing reprogramming can be observed over a time course upon induction of the reprogramming factors. Consequently, this technique allows for easy identification of factors that enhance or delay reprogramming, and can be a useful strategy in elucidating key mechanisms for efficient generation of iPSCs.

Does plerixafor destroy the hematopoietic progenitor cell/mesenchymal stromal cell niche?

Tissue homeostasis is dependent on replacement of damaged and aged cells, and stem cells are the primary source of new cell generation. Most tissue and organs of the body contain stromal cells that are part of the stem cell niche and are essential for the maintenance, proliferation and differentiation of stem cells. Hematopoiesis is a well-characterized tissue system in which interactions between hematopoietic stem cells (HSCs) and stromal cells in the bone marrow (BM) have been clearly established (1e3). The BM environment is subject to complex regulation that is critical for hematopoiesis. Many studies have demonstrated the production of both stimulating and inhibitory factors by BM stromal cells (mesenchymal stromal cells, MSCs) (4e6). However, the full potential of MSCs and their role in control of the HSC proliferation and differentiation remains unclear. Critical to sustained hematopoiesis is themaintenance of stem cell quiescence. Without a niche in which a stem cell pool can reside unstimulated by proliferative and differentiation signals, hematopoiesis cannot be sustained life-long. There is evidence that HSC quiescence is at least in part regulated by direct contact between HSCs and MSCs. This contact is mediated through chemokine receptor 4 (CXCR4) on HSCs engaging stromal cellederived factor (SDF)-1 on MSCs (7). With the advent of plerixafor—a CXCR4 antagonist used to mobilize HSCs for stem cell transplantation—it is pertinent to ask how this agent interferes with HSC/MSC interactions. In this issue of Cytotherapy, Ludwig et al. (8) cocultured HSCs and MSCs with and without plerixafor, studying cell division kinetics and stem cell surface marker changes to explore the relationship between HSCs and MSCs and the role of the CXCR4/SDF-1 axis. They found that co-culture of

AGE-RELATED CHANGES IN CELL SURFACE MARKERS IN BALB/C MICE: A FLOW CYTOMETRIC ANALYSIS

Event Abstract Back to Event AGE-RELATED CHANGES IN CELL SURFACE MARKERS IN BALB/C MICE: A FLOW CYTOMETRIC ANALYSIS EMINE YAVUZ1, SUHEYLA HASGUR1 and EMIN U. BAGRIACIK1* 1 GAZI UNIVERSITY SCHOOL OF MEDICINE, IMMUNOLOGY, Turkey Introduction: Age-related phenotypic alterations have been indentified in lymphocytes. However, the effect of aging on lymphocyte surface markers in BALB/c mice was not studied inclusively and systematically in a single study. The aim of this study is to analyze and identify the cell surface markers of lymphocyte populations from various lymphoid organs of young and aged mice. Material and Methods: Spleen, lymph nodes, mesenteric lymph nodes, thymus, and intraepithelial T lymphocytes were isolated from BALB/c mice. Flow cytometric analysis of lymphocytes was performed by using cell surface receptor spesific labelled antibodies. Results: The CD4:CD8 ratio has declined with age in thymus. In spleen, while the number of CD4+CD45R+, CD4+CD28+, CD4+CTLA4+, and CD8+CD28+, NKpan+ cell populations have increased with age, TCRαβ+ and CD62L+ cell counts have decreased. In lymph nodes, CD4+CTLA4+, CD8+CD45RB+, CD8+CD45R+, CD11b+ , and CD11c+ cell numbers have increased with age but the number of CD8+CD28+, CD4+CD45RB+ cells have declined. Partially different results was seen in mesenteric lymph nodes: CD4+CD8-, CD4+CD25+, CD8+CD25+, CD8+CD28+, CD62L+, and CD11b+ cell counts were less in older mice. In intestinal intraepithelial lymphocytes, the number of TCRγδ+ cells has decreased with increasing age while CD4+CD8α+ cell count has increased. Discussion: The changes in cell surface markers of T cells of lymphoid organs and mucosal lymphoid tissues in BALB/c mice are crucial during aging. The number of innate immune system cells also differs between young and old BALB/c mice. These results might be important to understand the physiological process of immune senescence and immune regulatory mechanisms of aging immune system. Keywords: Immune senescence, Balbc mice, Flow Cytometry, Lymphocytes, IEL Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: YAVUZ E, HASGUR S and BAGRIACIK EU (2013). AGE-RELATED CHANGES IN CELL SURFACE MARKERS IN BALB/C MICE: A FLOW CYTOMETRIC ANALYSIS. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00199 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 10 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. EMIN U BAGRIACIK, GAZI UNIVERSITY SCHOOL OF MEDICINE, IMMUNOLOGY, ANKARA, Turkey, eubagriacik@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers EMINE YAVUZ SUHEYLA HASGUR EMIN U BAGRIACIK Google EMINE YAVUZ SUHEYLA HASGUR EMIN U BAGRIACIK Google Scholar EMINE YAVUZ SUHEYLA HASGUR EMIN U BAGRIACIK PubMed EMINE YAVUZ SUHEYLA HASGUR EMIN U BAGRIACIK Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Predominance of IL-10 and TGF-β production from the mouse macrophage cell line, RAW264.7, in response to crude antigens from Clonorchis sinensis

Parasitic helminths are well-known to have the ability to modulate host immune responses. In this study, we investigated the fundamental immunoregulatory mechanism of the liver fluke Clonorchis sinensis (C. sinensis) using a murine macrophage RAW 264.7 (RAW) cell line and mouse bone marrow derived macrophages (BMDMs). We found that C. sinensis crude antigen (CA) is able to differentiate macrophage RAW cells into dendritic-like cells that can be detected by morphological observations. In addition, CA induces prominent secretion of anti-inflammatory cytokines such as IL-10 and TGF-β; however, we did not observe changes in cell surface markers that are involved in antigen recognition, antigen presentation, and T cell activation. Additionally, CA treatment induced ERK and JNK phosphorylation, and unexpectedly, elevated levels of IL-10 and TGF-β were inhibited by the addition of an ERK-specific inhibitor. Taken together, these data demonstrate that CA from C. sinensis may modulate host immune responses by upregulating anti-inflammatory cytokines via the regulation of ERK.

The Deficiency of PGI 2 Signaling Induces STAT6-independent Allergic Airway Inflammation

S A T U R D A Y 209 Peripheral Blood Natural Killer Cells from Asthmatic Subjects Respond Differently to Stimulation with Poly(I:C) and Diesel Exhaust Particles C. V. E. Chehrazi, K. M. Horvath, I. Jaspers; University of North Carolina, Chapel Hill, NC. RATIONALE: It has been suggested that NK cells in asthmatic and healthy subjects differ, but it is unknown whether these differences alter NK responses to virus and contribute to increased asthmatic susceptibility to viral infections. Data from our lab suggest that diesel exhaust exposure enhances virally-induced exacerbation of allergic inflammation, yet the role of NK cells in these responses is unknown; therefore, we examined the response of asthmatic peripheral NK cells to polyriboinosinic acid-polyribocytidylic acid [poly(I:C)a mimetic of virus-derived dsRNA] and diesel exhaust particles (DEP). METHODS: NK cells were isolated from the blood of asthmatic subjects and subsequently incubated with vehicle, poly(I:C), DEP, or poly (I:C)+DEP. Flow cytometry and cytokine analysis were performed to evaluate changes in cell surface markers and mediator profiles, respectively. RESULTS: As compared to vehicle control, poly(I:C) and DEP stimulation significantly augmented surface expression of CD16, which mediates antibody-dependent cell-mediated cytotoxicity. In contrast, only DEP stimulation significantly enhanced surface expression of CXCR3, the receptor for CXCL10, which is expressed by virally-infected cells and promotes NK chemotaxis. Analysis of cytokines and other cell surface receptors will be performed. CONCLUSIONS: Stimulation with poly(I:C) and DEP alters the expression of cell surface markers involved in NK activity. Whereas NK cytotoxicity appears enhanced by stimulation with dsRNA, NK chemotactic potential appears enhanced by DEP, suggesting that the NK response of asthmatics may be altered by air pollution. These novel findings offer insight into potential mechanisms by which pollutant exposure amplifies asthmatic morbidity due to viral infection.

B‐lymphocyte biology

B‐lymphocyte biology