GMP-Grade Large-Scale Expansion of Bone-Marrow- (BM) Derived Human Mesenchymal Stem/Stroma Cells (MSC): Comparison of Efficacy of Different Expansion Systems and Role of Cytokines/Chemokines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 337-337
Author(s):  
Markus Rojewski ◽  
Natalie Fekete ◽  
Daniel Fuerst ◽  
Philippe Bourin ◽  
Ramin Lotfi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Large Scale ◽  
Buffy Coat ◽  
Single Step ◽  
Adherent Cells ◽  
Transfusion Medicine ◽  
Healthy Donors ◽  
Medium Exchange ◽  
Low Levels

Abstract Abstract 337 Background: MSC cells can differentiate into different tissues and exhibit non-HLA-restricted immunosuppressive properties. They are promising candidates for cellular therapy. Therapeutic use requires large-scale GMP-grade expansion of MSC. Several protocols have been published. Here we systematically compare different expansion procedures with particular emphasis on role of cytokines/chemokines in the expansion medium. Materials and Methods: Bone marrow (BM) was obtained by aspiration from the iliac crest of healthy donors after informed consent and IRB approval. BM aspirate (anticoagulated with heparin (500 U/ml)) was incubated without manipulation in 5-chamber stacks (CellStacks; Corning) in a medium free of animal components (α-MEM (Lonza) with 10% human platelet lysate (hPL)). In the single-step protocol 1.2×104 MNC/ cm2 were seeded. Non-adherent cells were washed off after 72–96 hrs. Partial medium exchange (40%) was performed twice a week (wk). After 11 days MSC were harvested by incubation with recombinant trypsin (TrypZean, Lonza). In the two-step protocol 5×104 leukocytes/cm2 were seeded in 2-chamber stacks. Non-adherent cells were removed after 72–96 hours and complete medium exchange was performed twice/wk. Cells were harvested after 10 days and the harvest was seeded in a 2nd culture at a density of 0.4×104 MSC/cm2. This 2nd culture was harvested after 5 days. Cytokines/chemokines in hPL and in culture medium during the course of expansion was measured by Milliplex MAP Kit (Millipore Corp). Surface marker expression was measured on FACSAria and FACScan. Results: Higher number of MSC could be achieved in cultures with hPL compared to fetal calf serum. hPL was equally effective in supporting MSC proliferation if prepared from apheresis platelet concentrates (PC), buffy coat-derived pooled PC in plasma or pooled PC in additive solution. hPL contained large amounts of PDGF-AB/BB (790 ng/ml; mean of 3 batches of hPL from buffy coat-derived pooled PC), PDGF-AA (266 ng/ml), RANTES (2706 ng/ml), sCD40L (27 ng/ml), GRO (11 ng/ml), sVCAM (2511 ng/ml), sICAM (188 ng/ml). During culture, sCD40L declined rapidly to very low levels. Concentration of PDGF-AA, RANTES and sICAM remained almost stable. In contrast, PDGF-AB/BB declined to low levels (<0.007 ng/ml) in MSC expansion culture whereas concentration remained stable under the same conditions in the absence of MSC. Decline was associated with MSC numbers in the expansion. BM samples from healthy donors (n=4) were split in order to perform paired comparison of single-step vs. two-step expansion protocol. In the single-step protocol 16.3×103±5.8×103 MSC/μl BM seeded were harvested after 11±0 days. In the two-step protocol 12.0×103±4.4×103 MSC/μl BM were harvested after 10 days at the end of passage 0 and 104.0×103±60.4×103 after 5±1 days at the end of passage 1. The overall consumption of medium in the single-step protocol was substantially higher than in the two-step protocol. Phenotype of MSC from the two culture systems did not significantly differ regarding standard markers (positive for CD73, CD90, CD105, HLA-class I; neg. for CD45, CD3, CD34, HLA-DR). However, in passaging experiments we could demonstrate that proportion of MSC positive for CD49a, CD71, CCR4/CD194, CD349 and MSCA-1 decreased whereas proportion of cells positive for c-kit/CD117, CCR3/CD193, CXCR4 and CD200 increased. Conclusion: hPL-based system allows efficient expansion of MSC up to a total number >1×109 MSC from a 15 ml BM aspirate in 2–3 wks with only one passaging step. hPL is a rich source of cytokines, some of which (PDGF-AB/BB) seem to be consumed during expansion and arrive at very low concentrations at the end of the expansion culture. A two-step system provides higher number of MSC per BM cells seeded and requires less medium/culture vessels. Phenotype and differentiation capacity does not differ between single- or two-step culture. However, further passaging goes along with substantial changes of the phenotype. Previous conflicting results regarding chemokine expression of MSC might be due to differences in ex-vivo culture period. Given that chemokine receptor expression affects in-vivo behaviour of cells, MSC harvested after initial expansion (passage 0 or 1) substantially differ from older cells, emphasizing the need to highly standardize all parameters of expansion. (Supported by EU 7th Framework Programme, Projects CASCADE and REBORNE). Disclosures: Fekete: Institute of Clinical Transfusion Medicine and Immunogenetics: Employment. Fuerst:Institute of Clinical Transfusion Medicine and Immunogenetics: Employment. Schrezenmeier:Institute of Clinical Transfusion Medicine and Immunogenetics: Employment.

Multiplex Analysis of Serum Cytokine Levels in Waldenström Macroglobulinemia Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2616-2616
Author(s):  
Sherine F. Elsawa ◽  
Anne J. Novak ◽  
Steven C. Ziesmer ◽  
Thomas E. Witzig ◽  
Vincent Rajkumar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Elevated Serum ◽  
Fold Increase ◽  
Serum Levels ◽  
Healthy Controls ◽  
Β2 Microglobulin ◽  
Healthy Donors ◽  
Cytokine Levels

Abstract Waldenström macroglobulinemia (WM) is a monoclonal B cell disorder characterized by a circulating monoclonal IgM protein that may lead to serum hyperviscosity in association with an infiltration of lymphoplasmacytic cells into the bone marrow. Although proinflammatory and chemotactic cytokines can profoundly affect tumor cells and the tumor microenvironment, and many cytokines have been shown to have potent therapeutic efficacy in preclinical cancer models, the role of cytokine networks in WM is not fully understood. In this study, we used a high-throughput xMAP multiplex immunobead assay technology (Luminex Corp., Austin, TX) to simultaneously test 30 cytokines, chemokines, angiogenic factors as well as growth factors and soluble receptors in the sera of WM patients and compared them with other B cell malignancies including IgM monoclonal gammopathy of undetermined significance (MGUS), follicular lymphoma, chronic lymphocytic leukemia (CLL) as well as healthy controls. Using a Mann-Whitney U test to analyze the differences between the groups, 15 of the 30 cytokines tested had significantly different levels in WM compared to healthy controls. Of those 15 cytokines, 11 were elevated in WM patients and 4 were decreased. Cytokines were grouped into 3 groups; those with &lt; 2-fold difference, 2–8 fold difference and those having &gt; 8-fold difference in their cytokine levels compared to healthy donors. There was a greater than 8-fold increase in the serum levels of Rantes, G-CSF and IL-2R (p&lt;0.0001) in WM patients. Furthermore, 3 cytokines had between 2–8-fold increase in WM patients including IL-4 (p&lt;0.0001), IL-6 (p&lt;0.0019) and IP-10 (p&lt;0.0006). Five cytokines had statistically elevated levels in WM patients compared to healthy controls, however the fold increase was &lt; 2 including HGF (p&lt;0.0185), IL-10 (p&lt;0.0002), MIP-1α (P&lt;0.0484), IL-2 (P&lt;0.0130) and IL-12 (P&lt;0.0155). Of the cytokines that had significantly lower levels in the sera of WM patients, IL-8 (p&lt;0.0001) and EGF (p&lt;0.0001) were &gt; 8-fold decreased, MCP-1 (p&lt;0.0001) was 2–8 fold lower and Eotaxin (p&lt;0.0004) was &lt; 2-fold lower in WM patients. All of the cytokines that had the greatest fold difference (&gt; 8-fold) in WM patients compared to healthy donors also differed significantly from the MGUS patients. Rantes, G-CSF, IL-2R and EGF had significantly different levels compared to other B cell malignancies. We tested for a correlation between the cytokines that had &gt; 2-fold difference between the WM group and control group with clinical features of the disease and found the cytokines IL-6 and IL-2R had a significant correlation with β2-microglobulin levels (p&lt;0.01). We analyzed cytokine levels in the bone marrow plasma of the same patients and found that high levels of IL-2R in the bone marrow microenvironment significantly correlated with anemia and elevated serum β2-microglobulin (p&lt;0.01). In conclusion, we have simultaneously analyzed sera from WM patients for 30 cytokines and found the most significantly elevated cytokines are Rantes, G-CSF and IL-2R and the most significantly downregulated cytokines are IL-8 and EGF. Furthermore, we found that elevated serum levels of IL-6 and IL-2R correlated with β2-microglobulin levels, a measure of disease activity. Further analysis of the biological role of these cytokines in WM may offer insight into disease pathogenesis and provide a basis for novel targeted therapies.

Aberrant Bone Marrow Perivascular Niches are Involved in Multiple Myeloma Progression: Role of Notch–Delta Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2800-2800
Author(s):  
Sara Lamorte ◽  
Marta Costa ◽  
Giovanni Camussi ◽  
Sergio Dias
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Pcr Analysis ◽  
Adherent Cells ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Lower Chamber

Abstract Abstract 2800 Poster Board II-776 Bone marrow (BM) angiogenesis is implicated in Multiple Myeloma (MM) progression. In this study, we tested the hypothesis that MM progression occurs when aberrant BM perivascular niches are established. We isolated BM endothelial cells derived from MM patients (MM-BMECs) from BM aspirates using anti-CD31Ab coupled to magnetic beads. FACS analysis showed that of all the cell lines isolated were endothelial: more than 95% expressed Ulex Europaeus Agglutinin-1 and Factor VIII and were negative for monocyte-macrophage (CD14) and plasma cell markers (CD38). To test the hypothesis that in MM patients BM perivascular niches are aberrant we analyzed how MM-BMECs modulate hematopoietic stem cells (HSCs) properties using a BM microvascular endothelial cell line isolated from a healthy donor (BMECs) as control. We co-cultured cord blood cells CD34+ HSCs in the presence of MM-BMECs or BMECs feeder layer and we analyzed the ability of MM-BMECs compared with BMECs to modulate HSCs adhesion, chemotaxis and apoptosis. The results show that MM-BMECs promote CD34+ HSCs adhesion, recruitment and protect them from apoptosis. In detail, we showed that after 24h of co-culture there was a significant increase in the number of adherent HSCs on MM-BMECs than on BMECs: 43±9% versus 25±6%. Moreover, when HSCs were cultured for 48 hours in 1% of serum in the presence of MM-BMECs they were less sensitive to apoptosis (9±11% of Annexin V+ cells) than HSCs cultured in the presence of BMECs (14±1% of Annexin V+ cells) or without a feeder layer, as control (17±3% of Annexin V+ cells). For the migration assay a transwell chamber system, in which the upper and the lower chambers were separated by 5-μm pore-size filter, was used. BMECs, MM-BMECs or nothing was plated in the lower chamber, while HSCs were seeded into the upper chamber. Both chambers were loaded with unsupplemented EBM-2 plus 2% of serum. Cell migration was studied over a 6-8 hours period and evaluated as number of cells migrated into the lower chamber. The results showed a significantly greater migration of HSCs in the presence of MM-BMECs than BMECs: 12±2% versus 5±1% of migrated cells. Taken together, these data showed that MM-BMECs promoted HSCs migration, adhesion and survival. Next we evaluated how MM-BMECs modulate the hemopoiesis recovery after irradiation in a NOD-SCID mouse model. When injected into sub-lethally irradiated (3 Grey) NOD-SCID mice MM-BMECs were detected in the BM integrated within the murine BM vessels and promoted hematopoietic recovery. In detail, MM-BMECs provided signals favoring the commitment towards lymphoid lineage. In fact, 7 days after injection, the BM of mice injected with MM-BMECs showed an increase in the percentage of lymphoblast (2.7%), compared with mice injected with BMECs or PBS, as control (respectively, 1.5% and 1.4%); followed, 14 days after injection, by a significant increase in the percentage of peripheral blood lymphocytes in mice injected with MM-BMECs (75±6%) versus mice injected with BMECS and PBS (respectively 60±0.5% and 47±7%). Since MM is a plasma cells disorder and the Notch-Delta pathway has been shown to play a central role in regulating HSCs properties, including the decisions of HSCs to undergo T- or B-cell differentiation, we investigated the involvement of this pathway in MM-BMECs and HSCs interaction. As determined by FACS and RT-PCR analysis, MM-BMECs, compared to BMECs, over expressed Delta-like Notch ligand 4 (DII4). Thus, we investigated the role of DII4 in the MM-BMECs/BMECs-HSCs adhesion. The first results showed that the expression of DII4 by MM-BMECs is necessary to promote HSCs adhesion. In fact, using a blocking antibody against DII4 (AbαDII4) at 50ug/ml there was an impairment in HSCs adhesion to MM-BMECs (43±9% versus 24±2% of adherent cells without and with AbαDII4 treatment), but not on BMECs (25±6% versus 26±1.4% of adherent cells without and with AbαDII4 treatment). Ongoing experiments are focusing on the role of DII4 in the modulation of HSCs proliferation, protection against apoptosis and in vitro-in vivo B commitment by MM-BMECs. Taken together, all these data suggest that BMECs in MM may function as “aberrant perivascular niches”, modulating HSCs properties. This aberrant phenotype could be due to an alteration of the Notch-Delta pathway in BMECs that favors malignant clonal growth by protecting it from apoptosis, favoring migration, adhesion and providing self-renewing and/or proliferative cues. Disclosures: No relevant conflicts of interest to declare.

Circadian Adrenergic Regulation of Bone Marrow Endothelial Adhesion Molecule Expression Impacts Progenitor Recruitment and Engraftment Efficiency

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 398-398
Author(s):  
Yuya Kunisaki ◽  
Christoph Scheiermann ◽  
Daniel Lucas ◽  
Andrew Chow ◽  
Paul S. Frenette
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Peripheral Blood ◽  
Myeloid Cells ◽  
Fold Increase ◽  
Adherent Cells ◽  
Adhesion Molecule Expression ◽  
Vessel Area

Abstract Abstract 398 Previous studies have revealed that hematopoietic stem cells (HSCs) are released into peripheral blood in a circadian manner in a process controlled by the sympathetic nervous system (SNS) through the regulation of CXCL12 levels in the bone marrow (BM) (Mendez-Ferrer et. al. Nature 2008;452:442). Here, we have evaluated the constitutive recruitment of hematopoietic cells back to the BM. We have observed using high-speed multichannel fluorescence intravital microscopy (MFIM) significant circadian oscillations in the number of adherent BM cells in sinusoids with a nadir in the morning (Zeitgeber time, ZT5: 0.97 ± 0.17 adherent cells/ 100 μm2 vessel area) and a peak at night (ZT13: 2.54 ± 0.53 adherent cells/100 μm2, p = 0.007) after adoptive transfer on a 12 hour light-12 hour darkness cycle. Flow cytometric analyses revealed that the majority (∼70 %) of homed BM cells were Gr-1+ Mac-1+ myeloid cells. To investigate the underlying mechanisms, we have examined the expression levels of P- and E-selectins and VCAM-1, essential homing receptors for progenitor cells in the BM, and found that their mRNA and protein expression on BM endothelium oscillated over the course of a day with the peak expression overlapping the time of the highest cell adhesion numbers (ZT13). To examine the role of the SNS in this process, we surgically sympathectomized mice by unilateral section of the superior cervical ganglion (SCGx) whose neurons project into the calvarial vasculature, while performing sham surgery on the contralateral side. Sympathectomy abolished circadian fluctuations in the number of adoptively transferred adherent cells to the denervated calvarial BM compared to the control side in the same animals (nerve-intact side: ZT5 / ZT13: 1.66 ± 0.10 / 2.41 ± 0.08 cells / 100 μm2, p<0.0001; SCGx: ZT5 / ZT13: 1.65 ± 0.09 vs 1.63 ± 0.09 / 100 μm2 vessel area, p=0.90). We then ascertained further the role of adrenergic signals by evaluating mice deficient in b-adrenergic receptors. We found that the oscillations in cell adhesion molecule expression were markedly reduced in β2 (Adrb2-/-) and β3 (Adrb3-/-) adrenergic receptor deficient mice. These results suggest that hematopoietic cell recruitment to the BM is under circadian control, which is dependent on oscillating expression of endothelial selectins and VCAM-1, and regulated by the SNS. To test the relevance of circadian leukocyte recruitment, we investigated whether isoproterenol, a pan-b-adrenergic agonist commonly used in the clinic, could promote hematopoietic progenitor recruitment and thus BM reconstitution after BM transplantation (BMT). Treatment with isoproterenol (5 mg/kg) for 5 days significantly up-regulated expression of P-selectin (1.2-fold increase; p = 0.027), E-selectin (1.5-fold increase; P = 0.003) and VCAM-1 (2.3-fold increase; P=0.006) on BM endothelium in irradiated recipients as determined by flow cytometry of Tie-2+ PECAM-1+ endothelial cells. Consequently, homing of BM cells was dramatically increased (control / isoproterenol: 2.4 ± 0.2 ×104/4.9 ± 0.4 × 104 donor cells/femur; p = 0.0002) as was the number of recruited hematopoietic progenitors (17.0 ± 3.5/74.1 ± 18.8 CFU-C/femur; p = 0.017). In addition, the recovery of mature myeloid cells in peripheral blood was significantly accelerated in 3 weeks after transplantation of 5 × 104 BM cells (0.38 ± 0.21 × 103/1.64 ± 0.50 ×103/μl; p = 0.024). Of importance, using limiting numbers of BM cells (2.5 × 104) for transplantation, isoproterenol treatment markedly improved the survival (median survival time 10 vs 18 days, percent survival at 4 weeks post-BMT 5.8 vs 35.2%; p = 0.0097). These results indicate that the circadian timing of donor cell infusion and/or manipulation of adrenergic signals in the BM microenvironment may improve transplantation outcome through enhanced engraftment efficiency. Disclosures: No relevant conflicts of interest to declare.

An Exploratory Study on the Role of Burnout and Devaluation among Staff in Psychiatric Correctional Facilities

2021 ◽  
pp. 0306624X2110102
Author(s):  
Lindamarie Olson ◽  
Robin Gearing ◽  
Berenice Pérez Ramírez ◽  
Luis R. Torres
Keyword(s):  
Mental Illness ◽  
Mexico City ◽  
Exploratory Study ◽  
Large Scale ◽  
Correctional Facility ◽  
Correctional Facilities ◽  
Correctional Staff ◽  
Low Levels

Correctional staff work in demanding job environments that can lead to increased burnout. Research aims were to determine the prevalence of burnout and devaluation of consumers (i.e., individuals in their care) and their families among correctional staff, examine associations between burnout and devaluation of consumers and their families, and determine significant predictors of burnout. This exploratory study investigated 30 correctional staff in a specialized psychiatric correctional facility for individuals with mental illness in Mexico City. Correctional staff experienced low to moderate burnout, reported high levels of devaluation of consumers (DCS), and low levels of devaluation of consumers’ families (DCFS). The implications of burnout on DCS and DCFS warrant large scale research and should compare burnout and devaluation in specialized psychiatric correctional facilities versus general correctional facilities.

The Tetraspanin CD9 Is Involved in Primary Myelofibrosis Dysmegakaryopoiesis Through c-Myb Regulation and Stroma Interactions,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3834-3834
Author(s):  
Christophe Desterke ◽  
Costanza Bogani ◽  
Lisa Pieri ◽  
Alessandro M. Vannucchi ◽  
Bernadette Guerton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Migration ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Actin Polymerization ◽  
Primary Myelofibrosis ◽  
Membrane Expression ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Abstract 3834 Introduction: CD9, a four transmembrane glycoprotein belonging to the tetraspanin family, is suggested to regulate cell motility and adhesion and to play a role in megakaryopoiesis. It has been reported to be a molecular marker of primary myelofibrosis (PMF) being characterized by myeloproliferation, dysmegakaryopoiesis, alterated bone marrow/spleen stroma and extramedullary haematopoiesis. CD9 mRNA has been shown to be overexpressed in CD34+ PMF HPs and its membrane expression level was correlated with platelet counts. Our recent data evidencing an alteration of CD9 expression in PMF megakaryocytes (MK) have encouraged us to investigate whether CD9 participates in the dysmegakaryopoiesis and whether it is involved in the dialogue between MK and stromal cells in PMF patients. Patients and Methods: CD34+ cells were MACS selected from the peripheral blood of PMF patients (n=67) and of unmobilized healthy donors (n=61). Functional studies were performed on MK precursor-derived from CD34+ cells cultured in MK medium with ou without monoclonal antibodies (Syb mAb) or siRNAs targeting CD9. CXCL12-induced MK migration was performed in Boyden chambers. Bone marrow mesenchymal stromal cells (MSC) from healthy donors and PMF patients were cultured in DMEM+10%FCS. Results: Our results showed that CD9 membrane expression was altered on CD34+ cells and on MK precursor-derived from PMF CD34+ cells. Binding of CD9 with Syb mAb restored the in vitro megakaryocyte differentiation process that was altered in patients as shown by an increase in: i) megakaryocytic colony formation in semisolid medium, ii) CD41 and CD62p MK differentiation marker and GATA-1 expression, iii) MK cytoplasmic maturation, iv) apoptotic MK number (reduced AKT phosphorylation and Bcl-XL expression and increased percentage of Annexin+ cells). Activation of CD9 was also associated with regulation of MAPK and AKT-GSK3β pathways whose balance is involved in MK differentiation. Treatment of PMF MK precursors by Syb modulated activation of the MAPK pathway as shown by an increased of p38, JNK and GSK3β phosphorylation and of AP-1 mRNA expression. Taking into account the structure of the tetraspanin molecular network, binding with Syb mAb might also impact the effects associated to the multimolecular complex in which CD9 is involved. This prompted us to study the effects of a molecular silencing of CD9 on the PMF MK differentiation. We showed that, in contrast to the Syb mAb, addition of CD9 siRNA to PMF megakaryocytes reduced their transcriptional program including c-Myb, a transcription factor that is involved in CD9 regulation during megakaryopoiesis. Given the role of CD9 in cell migration, we further investigated whether it could be involved in the megakaryocytic precursor migration observed in patients. We showed that silencing CD9 reduced the CXCL12-dependent megakaryocytic precursor migration as well as the CXCR4 and CXCL12 transcription and that this migration involved actin polymerization. c-Myb siRNA restored CXCR4 and CXCL12 expression and reduced actin polymerization suggesting that CD9 was involved, via c-Myb, in the CXCL12-dependent megakaryocytic precursor migration. Effect of CD9 on cell migration is often interpreted as related to modulation of integrins participating in the integrin/tetraspanin network and of their interaction with mesenchymal stromal cells (MSC). We showed that several genes involving the CD9 partner interactome were over-expressed in MSC from PMF bone marrow as compared to MSC from healthy donors. Preliminary results showing that PMF MK precursors display different behaviour in terms of cell survival and adhesion when co-cultured on bone marrow MSC from PMF patients as compared to healthy donors suggest that interactions between MKs and bone marrow MSC is involved in PMF dysmegakaryopoiesis. Addition of Syb reverses these alterations suggesting the participation of CD9 in the abnormal dialogue between MK and MSC. Conclusion: Our results show a deregulation of CD9 expression in megakaryocytes from PMF patients. They also suggest that CD9 i) participates in PMF dysmegakaryopoieis in terms of MK differentiation and survival and ii) is involved in the increased MK precursor migration through alterations of the CXCL12/CXCR4 axis. Our data further support the role of bone marrow stroma in PMF dysmegakaryopoeisis through CD9 interactions. Disclosures: No relevant conflicts of interest to declare.

CD99 Is Highly Expressed in Acute Myeloid Leukemia (AML) and Presents a Viable Therapeutic Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1540-1540 ◽  
Author(s):  
Vijaya Pooja Vaikari ◽  
Miran Jang ◽  
Mojtaba Akhtari ◽  
Houda Alachkar
Keyword(s):  
Bone Marrow ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Normal Bone Marrow ◽  
Potential Therapeutic Target ◽  
Normal Bone ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal proliferation and block of differentiation of myeloid precursors. Overall survival for patients with AML remains dismal (<50% for younger patients and <10% for older patients) due to high relapse rate. In search for novel therapeutic targets in AML, we compared gene expression data of normal hematopoietic vs AML cells from 7 datasets (GSE13159, GSE13164, GSE7186, GSE1159, GSE995, GSE31174 and TCGA Leukemia) available on Oncomine. We identified CD99 to be significantly upregulated in AML cells compared with normal cells in all data sets with available measurements of CD99 expression (median ranking 155, p = 0.013); other genes among the top 10 genes identified in this analysis were FLT3 (median ranking 102, p<0.001) and WT1 (median ranking 120, p<0.001); both are known to play a role in AML. CD99 was significantly over-expressed (p<0.001) in 542 AML patients as compared with PBMCs from 74 healthy donors from the GSE13159 dataset. In the GSE13164 dataset, CD99 was significantly over-expressed in 257 AML patients as compared with PBMCs from 58 healthy donors. Consistently, in the GSE7186 dataset, CD99 was significantly over-expressed (p<0.001) in 23 AML patients as compared with 6 normal bone marrow samples and in the GSE1159 dataset, CD99 was significantly over-expressed (p=0.001) in 285 AML patients as compared with 5 normal bone marrow, and 3 normal blood samples. We also analyzed CD99 expression in cells obtained from 23 patients with AML and sorted according to their CD34 and CD38 expression levels (GSE3077 dataset). We found that CD99 expression was significantly higher (p<0.001) in the CD34+CD38+ and CD34+CD38- subpopulation compared with CD34-CD38- and CD34-CD38+; suggesting a possible role of CD99 in AML stem cells. Interestingly, analysis of three datasets (GSE22848, GSE6891, GSE15434) via R2: Genomics Analysis and Visualization Platform showed a correlation between CD99 expression and the presence of FLT3-ITD mutation. In the GSE22848 dataset, CD99 was significantly over-expressed (p=0.007) in 48 patients with FLT3-ITD as compared with 189 patients with FLT3 wildtype. In the GSE6891 dataset, 126 patients with FLT3-ITD had a significant over-expression of CD99 (p=0.006) as compared with 334 patients with FLT3 wildtype and in the GSE15434 dataset 90 FLT3-ITD positive patients had significantly higher levels of CD99 (p<0.001) as compared with 161 patients with the wildtype gene. CD99 (E2, MIC2), a 32-kD cell surface glycoprotein, is known to be involved in the transendothelium migration of neutrophils, T-cell adhesion, and T-cell death by a caspase-independent pathway. In cancer cells, CD99 was found to be highly expressed on the cell surface of Ewing's sarcoma tumors and in gliomas. Importantly, CD99 expression levels were found to be correlated with tumor invasiveness and with lower survival rates. In order to examine the role of CD99 in AML, we assessed CD99 expression by flow cytometry in nine AML cell lines (KG-1, KG-1A, MOLM13, MV4-11, Kasumi-1, THP-1, NB4, U937, UOC-M1); we found CD99 to be expressed in all cell lines. To determine whether CD99 is a potential therapeutic target in AML, we treated leukemia cells with anti-CD99 mAb (mAb 0662) at 5 µg/mL and analyzed cell viability 48 hours post-treatment. We found significant decrease in cell viability; 15% in MV4-11 cells (p=0.02), 32% in MOLM13 cells (p=0.002) and 18% in THP-1 cells (p<0.001) as compared with untreated controls measured by Alamar blue assay. Furthermore, inhibiting CD99 led to a decrease in migration of MV4-11, MOLM13 and THP-1 cells when analyzed using a trans-well migration assay. In conclusion, CD99 is highly expressed in AML, and this expression is significantly higher in less differentiated leukemia cells and in patients with FLT3-ITD mutation. Functional studies using CD99 antibodies revealed a possible role of this gene in cell survival and cell migration. Further studies are needed to establish CD99 as a potential therapeutic target and further investigations are ongoing to determine the mechanism by which CD99 regulates cell survival in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Changes in the Main Bone Marrow Cell Subpopulations May Potentially Contribute to the Initiation of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2494-2494
Author(s):  
Shlomit Yehudai-Reshef ◽  
Rawan Sabah ◽  
Tal Gabay ◽  
Tsila Zuckerman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Viability ◽  
Myeloid Leukemia ◽  
Healthy Controls ◽  
Healthy Donors ◽  
Cluster 2 ◽  
Acute Myeloid

Introduction: In acute myeloid leukemia (AML), differentiation and proliferation of early progenitors is inhibited, leading to blast accumulation in the bone marrow (BM) and in the peripheral blood (PB). Hematopoietic stem cells (HSCs) reside in specialized BM microenvironment (ME), mainly containing osteocytes and adipocytes, derived from BM mesenchymal stem cells (MSCs). Recent studies suggest a possible role of the ME in various hematological malignancies, that either serves as a potential source of an altered milieu or mediates abnormal interaction with HSCs. BM ME contributes to disease pathogenesis, chemotherapy resistance and recurrence in certain types of hematological cancers, but there is limited evidence that BM cells themselves may initiate AML. BM derived MSCs are characterized by a potential to mainly differentiate to adipocytes, osteocytes or chondrocytes. Our experiments have revealed an imbalance between osteocytes and adipocytes in the BM of AML patients that is not observed in the BM of healthy donors. We hypothesize that this imbalance could result in the development of leukemic stem cells/pre-leukemic cells, leading to leukemia initiation. The current study aimed to characterize the composition of AML BM derived adipocytes and osteocytes and evaluate the ability of these subpopulations to support AML cell viability. Methods: Secreted levels of adipogenic and osteogenic biomarkers in the BM plasma were measured using ELISA. The potential of AML MSCs to differentiate to adipocytes and osteocytes was assessed using FACS analysis. Exome sequencing was applied for genetic alteration screening in AML MSCs. Obtained adipocytes and osteocytes were separately co-cultured with AML cells and colony forming unit (CFU) assays were used to evaluate AML cell viability. ProcartaPlex immunoassay was used to evaluate levels of cytokines and other potential mediators secreted to the conditioned media. Results: The study included BM samples of 10 AML patients and 13 healthy donors. Levels of secreted FABP4 and osteocalcin biomarkers were reduced in AML plasma (279.7±164 pg/ml and 0.00057±0.017 pg/ml, respectively) compared to that of healthy controls (1230±1502 pg/ml and 0.0016±0.00052 pg/ml, respectively), with P=0.02 for FABP4 and P=0.017 for osteocalcin. According to adipocyte cell differentiation, patients were divided into two clusters. Cluster-1 demonstrated a higher expression level of FABP4 (mean 65.2±11.4%) compared to healthy controls (mean: 47.5±19.2 %; P=0.05), while cluster-2 exhibited a lower level of FABP4 expression (mean 30.78±6.1%) than healthy controls (mean 47.6±19.2%; P=0.01). A nonsynonymous somatic mutation in the AHNAK2 gene (V3209L) was found in patients' mesenchymal stromal cells. A statistically significant reduction in AML cell proliferation (P<0.01) was revealed following co-culture with adipocyte cells of cluster-1 patients. Of the 10 cytokines tested, only ANG1 levels were significantly elevated in the co-culture of adipocytes from cluster-1 (189.5±17.3pg/ml), cluster-2 (258.08±34.33pg/ml) or osteocytes (392.04±58.3pg/ml) with AML cells of the same patients compared to the levels observed in controls (0.002±0.052pg/ml for adipocytes and 168.663pg/ml for osteocytes; P<0.01). Conclusions: The current study has demonstrated that changes in the secretion of stromal specific biomarkers and in the MSC ability to differentiate to adipocytes and osteocytes indicate impaired functional properties of BM MSCs in AML. Within the adipocyte subpopulation in the AML BM, clusters with either increased or inhibited adipogenesis have been revealed. These variations in adipocyte and osteocyte cells reflect interpatient heterogeneity in AML BM subpopulations. The AHNAK2 gene mutation found in AML MSCs in our study and the previously suggested role of this gene in adipocyte differentiation regulation, may explain in part the phenotype diversity observed in AML patients. Adipocyte cells, derived from an AML patient, were found to inhibit normal hematopoiesis in HSCs of the same patient. This specific crosstalk may be related to the elevated levels of ANG1 mediating abnormal signals from adipocytes to HSCs. Overall, the findings of this study may support the contribution of abnormal composition of the main BM subpopulations to leukemogenesis and could ultimately pave the way to novel therapeutic strategies targeting the BM niche. Disclosures No relevant conflicts of interest to declare.

scholarly journals Large-scale seroepidemiology identifies a nephro-vascular syndrome associated with autoimmune reactivity to tau

2021 ◽  
Author(s):  
Andreia D. Magalhães ◽  
Marc Emmenegger ◽  
Elena De Cecco ◽  
Manfredi Carta ◽  
Karl Frontzek ◽  
...  
Keyword(s):  
Large Scale ◽  
Potential Role ◽  
University Hospital ◽  
Arterial Embolism ◽  
Urinary Disorders ◽  
Healthy Donors ◽  
Embolism And Thrombosis ◽  
Antibody Titers ◽  
Autoimmune Reactivity

The microtubule-associated protein tau is involved in several neurodegenerative diseases and is currently being investigated as a plasma biomarker for the detection and monitoring of Alzheimer's disease and as an immunotherapeutical target in clinical trials. We assessed plasma anti-tau IgG reactivity in 40'098 unselected patients visiting a university hospital and healthy blood donors. We found that 4.97% patients and 1.58% healthy donors had natural anti-tau antibody titers >1.8 log10(EC50). In a multivariate model, female sex (P<0.001), age (P<0.001), cystitis (RR 1.59, 95%CI 1.14-2.16, P=0.004), other urinary disorders (RR 1.23, 95%CI 1.03-1.45, P=0.018), chronic kidney disease (RR 1.20, 95%CI 1.01-1.41, P=0.033), arterial embolism and thrombosis (RR 1.56, 95%CI 1.02-2.25, P=0.026) and atherosclerosis (RR 1.35, 95%CI 1.09-1.1.66, P=0.004) were independent predictors of anti-tau autoantibodies. We therefore conclude that anti-tau autoimmunity is associated with a systemic syndrome that includes vascular, kidney and urinary disorders. The expression of tau in these extraneural tissues suggests a potential role of autoimmunity in this syndrome.

Myeloid Derived Suppressor Cell Expansion Correlates With Increased regulatory T Cells In Myelodysplastic Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2766-2766
Author(s):  
Astrid Olsnes Kittang ◽  
Shahram Kordasti ◽  
Kristoffer Evebø Sand ◽  
Pilar Perezabellan ◽  
Thomas Seidl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Immune Surveillance ◽  
Immune Dysregulation ◽  
Suppressive Effect ◽  
Hla Dr ◽  
Healthy Donors

Abstract Background T cell mediated immune dysregulation is an established feature of Myelodysplastic syndrome (MDS). We have shown previously that the number of T regulatory cells (Tregs) are increased in high risk MDS and associate with disease progression. In low risk disease however, T helper-17 cells (Th17) are increased which correlates with augmented bone marrow apoptosis. Nevertheless, the role of other components of immune system in MDS pathogenesis is still emerging. Myeloid derived suppressor cells (MDSCs) are a group of immature myeloid cells with a potent immunosuppressive effect which are expanded in an inflammatory environment. MDSCs suppress T cells by depriving them of arginine and cysteine, as well as the secretion of Interleukin (IL)-10, Transforming Growth Factor (TGF)-β and induction of Tregs. While, the role of MDSCs in negatively regulating the anti-tumour immune responses and in determining the T cell polarity have been demonstrated in solid tumours, this information is lacking in MDS. The aim of this study was to investigate the number and function of MDSCs in MDS and potential contribution to immune dysregulation in MDS. Material and Methods MDSCs and Treg numbers were assessed in peripheral blood (PB) and bone marrow (BM). In total we have analysed PB from 24 MDS patients of whom 12 patients had concurrent BM samples. PB samples from 11 age-matched healthy donors (HD) were also included. The WHO subtypes were: RC, RARS or RCMD (n=16, 67%) and 8 patients (33%) had RAEB. Cell surface staining and flow-cytometry was performed following red cell-lysis using the following markers: Live/dead dye, (eBioscience), Lineage markers (CD3, CD16, CD19, CD20, CD56,), CD33, CD34, HLA-DR, CD11b, CD15, CD66b, CD14, CX3CR1, CXCR3 and CXCR4. MDSCs were stained for intracellular TGF-β and IL-10 following fixation and permeabilisation, and they also stained positive for Arginase-1. To evaluate MDSCs function, CFSE stained CD3+CD4+CD25- (T-effectors) from MDS patients were stimulated by anti-CD3/CD28 antibodies. Cells were cultured with: T-effectors alone, T-effectors + CD3+/CD4+/CD25high (Tregs), (ratio 2:1); T-effectors + Tregs + HLA-DR-/CD14+ (MDSC) (ratio 2:1:1). Results MDSCs in the PB were higher in patients with RAEB compared to both healthy donors (HD) and non-RAEB MDS (2.6% v 1.03% p=0.0004) & (2.6% v 2% p=0.02). The absolute numbers of MDSCs were also higher in RAEB compared to non-RAEB subtypes (0.17 x 109/L v 0.08 x 109/L, p=0.02) and in Intermediate (INTR)/High (HR)/Very high (VHR) risk groups in comparison with Very low (VLR)/ Low (LR) risk group (0.31 x 109/L v 0.08 x 109/L, p=0.01). MDSCs frequency in MDS patients was higher in BM than in PB (45% v 2.6%, p=0.002). The PB MDSCs were mainly Monocytic (M)-MDSCs (0.6 % in PB v 0.3% in BM, p=0.02). M-MDSCs were also expressing higher CX3CR1, (MFI 350 v 8849, p=0.008) and higher CXCR4 (MFI 1846 v 2498, p=0.0156) compared to G-MDSCs. Granulocytic (G)-MDSC percentages were increased in INT/HR/VHR compared to VLR/LR (0.7% v 0.2%, p=0.0089) and HD (0.2% v 0.7%, p=0.0385). G-MDSCs were also more frequent in the peripheral blood of RAEB compared to non-RAEB subtypes (0.5% v 0.1% p=0.0299). G-MDSCs from MDS patients produce higher amount of IL-10/TGF-β compared to Monocytic (M)- MDSCs, evaluated by intracellular staining (IL-10 R-MFI, 7.9 v 3.8, p=0.003 and TGF-β R-MFI 15 v 4.8, p=0.024). To evaluate the suppressive effect of MDSCs, autologous Tregs and T-effectors from MDS patients (one INTR and one LR) were co-cultured in the presence and absence of MDSCs. Presence of MDSCs, not only increased the T-effector suppression (figure 1) but also induced Tregs proliferation. In patients with RAEB we also found a positive correlation between absolute counts of MDSC and CD27+FOXP3+CD4+Tregs (Spearman R=0.9, p=0.0374). Discussion Our data show that MDSCs are increased in MDS patients, particularly in high-risk disease, compared to healthy donors. The expanded MDSCs express chemokine receptors and secrete IL-10 and TGF-β, and potentiate the suppressive effect of Tregs. In RAEB patients, numbers of MDSCs are positively correlated with Treg numbers. Data suggests that increased MDSCs play an important role in immunopathogenesis of MDS by suppressing the immune-surveillance against the dysplastic clone. Inhibition of MDSCs (i.e. by arginase inhibitor) could reverse the immunosuppressive environment and re-establish immune-surveillance in MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inferring the selection window in antimicrobial resistance using deep mutational scanning data and biophysics-based fitness models

2017 ◽  
Author(s):  
Pouria Dasmeh ◽  
Anh-Tien Ton ◽  
Caroline Quach ◽  
Adrian W.R. Serohijos
Keyword(s):  
Antimicrobial Resistance ◽  
Genetic Parameters ◽  
Catalytic Efficiency ◽  
Single Step ◽  
Mutant Selection ◽  
Selection Window ◽  
Low Levels ◽  
Lower Limits ◽  
Selection Of

AbstractMutant-selection window (MSW) hypothesis in antimicrobial resistance implies a range for antimicrobial concentration that promotes selection of single-step resistant mutants. Since the inception and experimental verification, MSW has been at the forefront of strategies to minimize development of antimicrobial resistance (AR). Setting the upper and lower limits of MSW requires an understanding of the dependence of selection coefficient of arising mutations to antimicrobial concentration. In this work, we employed a biophysics-based and experimentally calibrated fitness model to estimate MSW in the case of Ampicillin and Cefotaxime resistance in E.coli TEM-1 beta lactamase. In line with experimental observations, we show that selection is active at very low levels of antimicrobials. Furthermore, we elucidate the dependence of MSW to catalytic efficiency of mutants, fraction of mutants in the population and discuss the role of population genetic parameters such as population size and mutation rate. Altogether, our analysis and formalism provide a predictive model of MSW with direct implications in the design of dosage strategies.

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