scholarly journals Monocyte-Derived Macrophages Are Impaired in Myelodysplastic Syndrome

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Yu Han ◽  
Huaquan Wang ◽  
Zonghong Shao
Keyword(s):  
Myelodysplastic Syndrome ◽  
Ex Vivo ◽  
Regulatory Protein ◽  
Control Group ◽  
Phagocytic Capacity ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Increased Risk ◽  
Macrophage Colony

Background. The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic stem cell diseases characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Macrophages are innate immune cells that ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. We analyzed the role of macrophages from MDS patients in vitro. Methods. Macrophages were induced from peripheral blood of patients with MDS via granulocyte macrophage colony-stimulating factor (GM-CSF). Phagocytic capacity of macrophages was measured with carboxyfluorescein succinimidyl ester and fluorescent microspheres. CD206 and signal regulatory protein alpha (SIRPα) on macrophages were detected by flow cytometry. Inducible nitric oxide synthase (iNOS) was measured by ELISA method. Results. Compared with normal control group, the number of monocytes increased in MDS patients. However, the monocytes showed impaired ability to induce macrophages and the number of macrophages induced from MDS samples was lower. Further, we demonstrated that the ex vivo phagocytic function of macrophages from MDS patients was impaired and levels of reorganization receptors CD206 and SIRPα were lower. Levels of iNOS secreted by macrophages in MDS were increased. Conclusions. Monocyte-derived macrophages are impaired in myelodysplastic syndromes.

scholarly journals Monocyte-Derived Macrophages Are Impaired in Myelodysplastic Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5522-5522
Author(s):  
Yu Han ◽  
Huaquan Wang ◽  
Zonghong Shao
Keyword(s):  
Myelodysplastic Syndrome ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Regulatory Protein ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Increased Risk ◽  
Macrophage Colony

Abstract The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic stem cell diseases characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Macrophages are innate immune cells that ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. Tumor-associated macrophages (TAMs) play an important role in the pathophysiology of human malignancies. They support growth of cancer cells by promoting angiogenesis, inhibiting tumor cell apoptosis and anti-tumor immune reactions. In this study, we analyzed the role of macrophages from MDS patients in vitro. Macrophages were induced from peripheral blood of patients with MDS via granulocyte macrophage colony-stimulating factor (GM-CSF). Compared with that in the normal control group, the number of monocytes increased in MDS patients. However, the monocytes showed impaired ability to induce macrophages and the number of macrophages induced from MDS samples was lower. Further, we demonstrated that the ex vivo phagocytic function of macrophages from MDS patients was impaired and levels of reorganization receptors CD206 and signal regulatory protein alpha (SIRPα) were lower. Levels of inducible nitric oxide synthase(iNOS) secreted by macrophages in MDS were increased. In conclusion, monocyte-derived macrophages are impaired in myelodysplastic syndromes. Disclosures No relevant conflicts of interest to declare.

Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1068-1068
Author(s):  
Naoko Takebe ◽  
Thomas MacVittie ◽  
Xiangfei Cheng ◽  
Ann M. Farese ◽  
Emily Welty ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4101-4101
Author(s):  
Wen-ming Chen ◽  
Zi-xing Chen ◽  
Jian-nong Cen ◽  
Jun He ◽  
Xiao-li Jiao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Biological Characteristics ◽  
Bone Marrow Mononuclear Cell ◽  
Feeder Layer ◽  
Rt Pcr ◽  
Osteoblastic Cell Line ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract It was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). By using human fetal osteoblastic cell line hFOB1.19 as a model of control, we investigated the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured and checked for their morphology, immunophenotype, CFU-F forming capacity and the expression of hematopoietic cytokines. A feeder layer was prepared by osteoblasts induced from 3rd generation of cultured MSCs and treated with mitomycin C. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to co-culture in vitro without exogenous cytokines. FCM revealed that both MSCs and hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed mRNA of SCF, IL-6, IL-11, SDF-1, GM-CSF and G-CSF. MSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and CFU-F forming capacity were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS could sustain GM-CFC survival for at least 3 weeks. The CFU-GM yield from cells in upper layer of co-culture was not different from those of control in hematopoiesis supportive experiments in vitro (P>0.05). RT-PCR clearly demonstrated that the cultured BM-MSCs from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSCs differentiated toward osteoblasts, the expression of G-CSF could be detected, whereas GM-CSF remained undetectable. The same expression profile of above cytokines were also seen in osteoblasts induced from BM-MSCs of MDS patients. In conclusion, osteoblasts may play a pivotal role in hematopoiesis. The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support survival of GM-CFC hematopoietic progenitor cells in vitro, according to their expression of multiple cytokines. These findings suggested that the osteoblasts derived from MDS patients may not be involved in the malignant process.

Cytokine Treatment of CD34+ Cord Blood Cells with G-CSF, GM-CSF, or SCF During 48 Hours of Ex Vivo Culture Alters CD26/DPPIV Peptidase Activity and Subsequent Engraftment Into NSG Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1460-1460
Author(s):  
Laura A Paganessi ◽  
Lydia Luy Tan ◽  
Sucheta Jagan ◽  
Robin Frank ◽  
Antonio M. Jimenez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Gm Csf ◽  
Cytokine Treatment ◽  
Ex Vivo Culture ◽  
Inhibitor Treatment

Abstract Abstract 1460 Many patients with hematologic malignancies choose hematopoietic stem cell transplantation (HSCT) as a treatment option. The most common source of Hematopoietic Stem and Progenitor Cells (HSC/HPC) for adult recipients is mobilized Peripheral Blood (mobPB). Limited quantities of HSC/HPC obtainable from an umbilical cord restricts its use for adult recipients. Ex vivo treatment of umbilical cord blood (CB) with cytokines and growth factors is being used to expand the population of cord blood HSC/HPCs in hopes of obtaining higher numbers of transplantable CB cells. In addition, cytokines and growth factors are often utilized post-transplant in an attempt to improve the rate of immune reconstitution. It has been previously reported that granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-colony-stimulating factor (GM-CSF) up-regulate CD26 (dipeptidyl peptidase IV/DPPIV) activity on freshly isolated CD34+ CB cells within 18 hours of culture [Christopherson, et al. Exp Hematol 2006]. Separate studies have demonstrated that treatment of uncultured CD34+ CB cells with the CD26 inhibitor Diprotin A increases transplant efficiency into immunodeficient mice [Christopherson, et al. Stem Cells Dev. 2007]. We evaluated here the in vitro and in vivo effects of CD26 inhibitor treatment on previously frozen CB CD34+ cells cultured ex vivo with G-CSF, GM-CSF or SCF for 48 hours. We examined CD26 expression by multivariate flow cytometry, CD26 activity using the established chromogenic CD26 substrate, Gly-Pro-p-nitroanilide (Gly-Pro-pNA), and SDF-1α induced migration and adhesion. In vivo, we examined long-term engraftment in NSG (NOD/SCID/IL2Rγnull) immunodeficient mice. After 48 hours of culture with cytokine treatment we observed altered CD26 expression on CD34+ CB cells. There was both an increase in the percentage of CD26+ cells and the mean fluorescence intensity (MFI) of CD26. Additionally, CD26 activity was 1.20, 1.59, 1.58, and 1.65 fold greater after ex vivo culture in untreated, G-CSF, GM-CSF and SCF treated CB CD34+ cells respectively compared to the CD26 activity prior to culture. The increase in CD26 activity as a result of treatment with G-CSF (p≤ 0.01), GM-CSF (p≤ 0.05) or SCF (p≤ 0.01) was significantly higher than the CD26 activity measured in the untreated cells following 48 hours of culture. Post-culture treatment with the CD26 inhibitor, Diprotin A, significantly improved SDF-1α induced migration and adhesion of cultured CD34+ CB cells in vitro, particularly in G-CSF treated cells (p≤ 0.05). Diprotin A treatment of CD34+ CB cells previously treated with G-CSF also significantly increased the long-term in vivo engraftment of stem and progenitor (CD34+CD38-, p=0.032), monocyte (CD14+, p=0.015), and megakaryocyte/platelet (CD61+, p=0.020) cells in the bone marrow of NSG mice. CD26 has been previously shown to cleave SDF-1 (stromal cell-derived factor 1/CXCL12). After cleavage, SDF-1 retains its ability to bind to its receptor (CXCR4) but no longer signals. SDF-1 is a powerful chemoattractant and has been shown to be important in mobilization, homing, and engraftment of HSCs and HPCs. This study demonstrates the influence of ex vivo culture and the effect of cytokine treatment on CD26 activity and subsequent biologic function related to HSCT. All three cytokines studied caused a significant increase in enzymatic activity at 48 hours compared to untreated cells. The up-regulation of CD26 protein expression caused by cytokine treatment for 48 hours, in particular G-CSF, had a significant impact on SDF-1 stimulated migration and adhesion. This was demonstrated in vitro by the improvement in cell function after CD26 inhibitor treatment and in vivo by the improved engraftment seen in the G-CSF treated cells with CD26 inhibitor treatment. These experiments suggest that combining CD26 inhibitor treatment following culture with G-CSF treatment during culture has the greatest overall benefit in engraftment outcome. By increasing our understanding of the effects of exogenous cytokines during culture on trafficking, ex vivo expanded CB has the potential to become a more effective means of not only increasing numbers of CB HSC/HPCs but also engraftment outcomes. This would ultimately allow expanded cord blood to become a more viable option for HSCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2203-2210 ◽  
Author(s):  
GP Bagnara ◽  
G Zauli ◽  
L Vitale ◽  
P Rosito ◽  
V Vecchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Diamond Blackfan Anemia ◽  
Clear Explanation ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte- conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation.

96 GRANULOCYTE–MACROPHAGE COLONY-STIMULATING FACTOR INCREASES DEVELOPMENTAL POTENTIAL OF PORCINE EMBRYOS IN VITRO

2012 ◽  
Vol 24 (1) ◽  
pp. 160
Author(s):  
K. Lee ◽  
J. Teson ◽  
L. Spate ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

There have been significant improvements in the culture of porcine embryos in vitro; however, it is still suboptimal. Improvements in porcine embryo culture would benefit utilisation of porcine embryos for a variety of purposes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be expressed in the female reproductive tract and the level of its expression is high between conception and implantation. Previous studies show supplementing GM-CSF in embryo culture promotes embryonic development in human and bovine embryos. The aim of this study was to investigate the effect of GM-CSF on the culture of porcine embryos derived from somatic cell nuclear transfer (SCNT) and IVF. Different concentrations of recombinant porcine GM-CSF (0, 2, 10 ng mL–1) were introduced into Porcine Zygote Medium 3 from Day 1 to 6. Frequencies of cleaved embryos and blastocyst formation were recorded and analysed by using ANOVA following arcsin transformation. Total cell number in blastocysts from each group were counted and compared by using the Student's t-test. Differences at P < 0.05 were considered significant. A total of 563 SCNT embryos from 6 different donor cell lines on 11 different days were produced for the study. Incubation of SCNT embryos with GM-CSF did not affect the frequency of cleaved embryos. Frequencies of cleaved embryos in control (0 ng mL–1), 2 ng mL–1 GM-CSF and 10 ng mL–1 GM-CSF were 64.2%, 68.1% and 65.0%, respectively. Interestingly, both concentrations of GM-CSF significantly increased the frequency of blastocyst formation as compared with the control. In 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, 30.8% and 32.3% of embryos reached blastocyst respectively, whereas only 22.4% of embryos reached blastocyst in the control group. A significant increase in total cell number in blastocysts was observed when GM-CSF was introduced into embryo culture. An average of 28.8 ± 0.9 cells was recorded in the control group, whereas 31.9 ± 1.1 and 31.8 ± 1.1 were observed in 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, respectively. Similar effects were observed when GM-CSF was introduced to the culture of IVF embryos. For IVF study, 525 embryos were generated on 10 different days and embryos cultured in the presence of GM-CSF tended to show higher blastocyst formation (P = 0.1). Frequencies of blastocyst per cleaved in the 3 groups were 55.7% (control), 65.7% (2 ng mL–1 GM-CSF) and 66.7% (10 ng mL–1 GM-CSF). In addition, culture of IVF embryos with GM-CSF significantly increased total cell number in Day 6 blastocysts. Total cell number in blastocysts in 2 ng mL–1 GM-CSF (34.2 ± 0.8) and 10 ng mL–1 GM-CSF (34.4 ± 1.2) were significantly higher compared with control (27.3 ± 1.2). Our results indicate that introducing GM-CSF into embryo culture media can increase the quality of blastocyst stage embryos. An increase in the frequency of blastocyst formation and total cell number in blastocysts suggests that GM-CSF can be used to produce better-quality embryos in vitro. Currently, effects of GM-CSF on implantation of SCNT embryos are under investigation. Further studies would elucidate the specific mechanism of GM-CSF on porcine embryos.

scholarly journals Cytokine Receptor Expression on Hematopoietic Stem and Progenitor Cells

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 65-71 ◽  
Author(s):  
William J. McKinstry ◽  
Chung-Leung Li ◽  
John E.J. Rasko ◽  
Nicos A. Nicola ◽  
Gregory R. Johnson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Population ◽  
Progenitor Cell ◽  
Cell Populations ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Hematopoietic stem and progenitor cell populations were obtained by fluorescence activated cell sorting of murine bone marrow (BM) cells into Rhodamine-123lo lineage−Ly6A/E+ c-kit+ (primitive stem cells highly enriched for long-term BM repopulating activity), Rhodamine-123med/hi lineage− Ly6A/E+ c-kit+ (mature stem cells highly enriched for short-term BM repopulating activity and day 13 spleen colony-forming activity) and lineage− Ly6A/E− c-kit+ (enriched for in vitro colony forming cells) populations. Neither stem cell population responds to single cytokines in vitro and each requires the synergistic action of two or more cytokines for proliferation, whereas the progenitor cell population proliferates in response to single cytokines. Since each of these cell populations was sorted as c-kit+, they express receptors for stem cell factor. Cell populations were also analyzed by autoradiography for their ability to specifically bind iodinated cytokines and this revealed that both stem cell populations expressed receptors for interleukin-1α (IL-1α), IL-3, IL-6, and granulocyte colony-stimulating factor (G-CSF ), but lacked receptors for macrophage colony-stimulating factor (M-CSF ), granulocyte-macrophage colony stimulating factor (GM-CSF ), and leukemia inhibitory factor (LIF ). Cells within the progenitor cell population specifically bound IL-3, GM-CSF, G-CSF, IL-6, and IL-1α, whereas no receptors were detected for M-CSF and LIF. Within each cell population examined, heterogeneity was observed in the percentage of cells labeled and the number of receptors per cell. These results suggest that stem cell populations can be further subdivided according to their cytokine receptor profile and it will be of interest to determine if such subpopulations have distinctive functional properties.

GM-CSF, via PU.1, regulates alveolar macrophage FcγR-mediated phagocytosis and the IL-18/IFN-γ–mediated molecular connection between innate and adaptive immunity in the lung

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 4193-4200 ◽  
Author(s):  
Pierre-Yves Berclaz ◽  
Yoko Shibata ◽  
Jeffrey A. Whitsett ◽  
Bruce C. Trapnell
Keyword(s):  
Ex Vivo ◽  
Lung Infection ◽  
Surfactant Protein ◽  
Interferon Γ ◽  
Adenoviral Infection ◽  
Gm Csf ◽  
Ifn Γ ◽  
Macrophage Colony

Severely impaired pulmonary microbial clearance was observed in granulocyte-macrophage colony-stimulating factor (GM-CSF)–deficient mice. To determine mechanisms by which GM-CSF mediates lung host defense, FcγR-mediated phagocytosis (opsonophagocytosis) by alveolar macrophages (AMs) was assessed in GM-CSF–sufficient (GM+/+) and –deficient (GM−/−) mice and in GM−/− mice expressing GM-CSF only in the lungs from a surfactant protein C (SPC) promoter (SPC-GM+/+/GM−/−). Opsonophagocytosis by GM−/− AMs was severely impaired and was restored by pulmonary GM-CSF expression in vivo or by PU.1 expression in vitro. Defective opsonophagocytosis by GM−/− AMs was associated with decreased FcγR expression. Because interferon-γ (IFN-γ) augments macrophage FcγR levels, the role of GM-CSF/PU.1 in the regulation of AM FcγR expression by IFN-γ was assessed during adenoviral lung infection. Adenoviral infection stimulated IFN-γ production and augmented FcγR levels on AMs in GM-CSF–expressing but not GM−/− mice. However, IFN-γ exposure ex vivo stimulated FcγR expression on GM−/− AMs. Because interleukin-18 (IL-18) and IL-12 stimulate IFN-γ production during adenoviral infection, their role in GM-CSF/PU.1 regulation of IFN-γ–augmented FcγR expression on AMs was assessed. Adenoviral infection stimulated IL-18 and IL-12 production in GM-CSF–expressing mice, but both were markedly reduced or absent in GM−/−mice. IL-18 expression by GM−/− AMs was severely impaired and was restored by pulmonary GM-CSF expression in vivo or by PU.1 expression in vitro. Pulmonary administration of IL-18 in GM−/− mice stimulated IFN-γ production and restored FcγR expression on AMs. These results show that GM-CSF, via PU.1, regulates constitutive AM FcγR expression and opsonophagocytosis and is required for the IFN-γ–dependent regulation of AM FcγR expression, enabling AMs to release IL-18/IL-12 during lung infection.

scholarly journals Evaluation of a Bifunctional Sirpα-CD123 Fusion Antibody for the Elimination of Acute Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2544-2544 ◽  
Author(s):  
Siret Tahk ◽  
Saskia Schmitt ◽  
Christian Peter Augsberger ◽  
Binje Vick ◽  
Laia Pascual Ponce ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Regulatory Protein ◽  
Target Antigen ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Background: Despite considerable advances in the development of novel strategies for the treatment of acute myeloid leukemia (AML) the relapse rate is still high with only limited treatment options. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate outgrowth of the disease, highlighting the need of targeting LSCs to improve overall survival. Immunotherapies represent a promising strategy to target chemotherapy-resistant LSCs in AML. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows only a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen (Haubner et al, Leukemia 2019). CD47, known as a marker of self, is also highly expressed on LSCs as immune escape mechanism. CD47 transmits a "don't eat me" signal upon its interaction with the myeloid-specific signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs and provide AML patients a possibility for prolonged relapse-free survival, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123 positive AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity. Methods: The bifunctional SIRPα-CD123 antibody was generated by fusing the endogenous extracellular domain of SIRPα, which functions as the CD47 blocking domain, to an CD123 antibody CD123. We assessed the selective binding of the bifunctional antibody to CD123+CD47+ AML-derived cells and the ability to block CD47 on CD123+ cells in vitro. Furthermore, the biological activity of the SIRPα-CD123 antibody was examined using the AML-derived cell line MOLM-13, patient-derived xenografted (PDX) AML cells as well as primary cells from patients with newly diagnosed or relapsed AML. Results: We engrafted the endogenous SIRPα V-like domain to an antibody targeting CD123, which improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody (MFI ratioCD123 = 2.46 0.25 vs MFI ratioSIRPα-CD123 = 4.44 0.60). The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity (EC50CD123 = 38.5 pM vs EC50SIRPα-CD123 = 10.1 pM, n = 9). Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells ex vivo. Further, an improved ex vivo cytotoxicity towards AML PDX cells was observed with the SIRPα-CD123 antibody (% lysis at 100 nM: 14.27 5.40 vs 42.94 10.21 for CD123 and SIRPα-CD123 antibodies respectively, n = 3). With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ positive cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in allogenic as well as autologous settings (% phagocytosis, normalized to isotype control and maximum phagocytosis in an autologous setting: 20.11 4.59 vs 90.37 6.22, n = 5 for CD123 and SIRPα-CD123 antibodies, respectively). We were further able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity. Taken together, our in vitro data supports the elimination of the CD123+CD47+ positive AML LSC compartment by a synergistic effect of avidity-dependent binding to CD123 and CD47 and the simultaneous inhibition of the innate immune CD47-SIRPα signaling pathway. Conclusions: The SIRPα-CD123 is a bifunctional antibody with the potential to deplete CD123+CD47+ AML LSCs by a dual mode of action mechanism resulting in NK cell dependent cytotoxicity and macrophage-mediated phagocytosis. By combining a high affinity binding to CD123+ cells and a low affinity CD47 blockade that is restricted to CD123+ cancer cells we effectively minimize the risk for CD47-related on-target off-leukemia toxicity. The results of our in vitro assays using AML cell lines are consistent with the data from PDX and primary AML samples and support further preclinical testing of the SIRPα-CD123 antibody in vivo. Disclosures Subklewe: Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Oxford Biotherapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Janssen: Consultancy.

scholarly journals Chronic Exposure to HIV-Derived Protein Tat Impairs Endothelial Function via Indirect Alteration in Fat Mass and Nox1-Mediated Mechanisms in Mice

2021 ◽  
Vol 22 (20) ◽  
pp. 10977
Author(s):  
Laszlo Kovacs ◽  
Thiago Bruder-Nascimento ◽  
Lindsey Greene ◽  
Simone Kennard ◽  
Eric J. Belin de Chantemèle
Keyword(s):  
Body Weight ◽  
Nadph Oxidase ◽  
Endothelial Function ◽  
Fat Mass ◽  
Vascular Function ◽  
Ex Vivo ◽  
Regulatory Protein ◽  
Increased Risk ◽  
Nadph Oxidase 1

People living with human immunodeficiency virus (HIV) (PLWH) have increased risk for atherosclerosis-related cardiovascular disease (CVD), the main cause of death in this population. Notwithstanding, the mechanisms of HIV-associated vascular pathogenesis are not fully elucidated. Therefore, we sought to determine whether HIV-regulatory protein Tat mediates HIV-induced endothelial dysfunction via NADPH oxidase 1 (Nox1)-dependent mechanisms. Body weight, fat mass, leptin levels, expression of reactive oxygen species (ROS)-producing enzymes and vascular function were assessed in C57BL/6 male mice treated with Tat for 3 days and 4 weeks. Aortic rings and human endothelial cells were also treated with Tat for 2–24 h in ex vivo and in vitro settings. Chronic (4 weeks) but not acute (3 days and 2–24 h) treatment with Tat decreased body weight, fat mass, and leptin levels and increased the expression of Nox1 and its coactivator NADPH oxidase Activator 1 (NoxA1). This was associated with impaired endothelium-dependent vasorelaxation. Importantly, specific inhibition of Nox1 with GKT771 and chronic leptin infusion restored endothelial function in Tat-treated mice. These data rule out direct effects of HIV-Tat on endothelial function and imply the contribution of reductions in adipose mass and leptin production which likely explain upregulated expression of Nox1 and NoxA1. The Nox1 and leptin system may provide potential targets to improve vascular function in HIV infection-associated CVD.

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