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1.  Umbilical cord blood expansion with nicotinamide provides long-term multilineage engraftment 
The Journal of Clinical Investigation  2014;124(7):3121-3128.
BACKGROUND. Delayed hematopoietic recovery is a major drawback of umbilical cord blood (UCB) transplantation. Transplantation of ex vivo–expanded UCB shortens time to hematopoietic recovery, but long-term, robust engraftment by the expanded unit has yet to be demonstrated. We tested the hypothesis that a UCB-derived cell product consisting of stem cells expanded for 21 days in the presence of nicotinamide and a noncultured T cell fraction (NiCord) can accelerate hematopoietic recovery and provide long-term engraftment.
METHODS. In a phase I trial, 11 adults with hematologic malignancies received myeloablative bone marrow conditioning followed by transplantation with NiCord and a second unmanipulated UCB unit. Safety, hematopoietic recovery, and donor engraftment were assessed and compared with historical controls.
RESULTS. No adverse events were attributable to the infusion of NiCord. Complete or partial neutrophil and T cell engraftment derived from NiCord was observed in 8 patients, and NiCord engraftment remained stable in all patients, with a median follow-up of 21 months. Two patients achieved long-term engraftment with the unmanipulated unit. Patients transplanted with NiCord achieved earlier median neutrophil recovery (13 vs. 25 days, P < 0.001) compared with that seen in historical controls. The 1-year overall and progression-free survival rates were 82% and 73%, respectively.
CONCLUSION. UCB-derived hematopoietic stem and progenitor cells expanded in the presence of nicotinamide and transplanted with a T cell–containing fraction contain both short-term and long-term repopulating cells. The results justify further study of NiCord transplantation as a single UCB graft. If long-term safety is confirmed, NiCord has the potential to broaden accessibility and reduce the toxicity of UCB transplantation.
TRIAL REGISTRATION. Clinicaltrials.gov NCT01221857.
FUNDING. Gamida Cell Ltd.
doi:10.1172/JCI74556
PMCID: PMC4071379  PMID: 24911148
2.  Inhibition of Nuclear Factor-κB Activation in Pancreatic β-Cells Has a Protective Effect on Allogeneic Pancreatic Islet Graft Survival 
PLoS ONE  2013;8(2):e56924.
Pancreatic islet transplantation, a treatment for type 1 diabetes, has met significant challenges, as a substantial fraction of the islet mass fails to engraft, partly due to death by apoptosis in the peri- and post-transplantation periods. Previous evidence has suggested that NF-κB activation is involved in cytokine-mediated β-cell apoptosis and regulates the expression of pro-inflammatory and chemokine genes. We therefore sought to explore the effects of β-cell-specific inhibition of NF-κB activation as a means of cytoprotection in an allogeneic model of islet transplantation. To this end, we used islets isolated from the ToI-β transgenic mouse, where NF-κB signalling can specifically and conditionally be inhibited in β-cells by expressing an inducible and non-degradable form of IκBα regulated by the tet-on system. Our results show that β-cell-specific blockade of NF-κB led to a prolonged islet graft survival, with a relative higher preservation of the engrafted endocrine tissue and reduced inflammation. Importantly, a longer delay in allograft rejection was achieved when mice were systemically treated with the proteasome inhibitor, Bortezomib. Our findings emphasize the contribution of NF-κB activation in the allograft rejection process, and suggest an involvement of the CXCL10/IP-10 chemokine. Furthermore, we suggest a potential, readily available therapeutic agent that may temper this process.
doi:10.1371/journal.pone.0056924
PMCID: PMC3578930  PMID: 23437272
3.  Improvement of CXCR4 Tracer Specificity For PET Imaging 
Journal of Controlled Release  2011;157(2):216-223.
Tumors expressing the chemokine receptor CXCR4 have been reported to be more aggressive and to produce more metastatic seeding in specific organs, such as the bone marrow. However, evaluation of tumors for CXCR4 expression requires testing of ex vivo biopsy samples, and is not routinely done in cancer management. In prior work to address this issue, we and others have developed tracers for positron emission tomography (PET) that targeted CXCR4, but in addition to binding to CXCR4 these tracers also bound to red blood cells (and to other unrelated targets) in vivo. Here we report two new tracers based on the CXCR4 peptide antagonist 4F-benzoyl-TN14003 (T140) that bind to CXCR4, but not to undesired targets. These tracers, NOTA-NFB and DOTA-NFB, show slight reductions in both 1) binding affinities for CXCR4 and 2) inhibition of CXCL12 induced migration, compared to T140, in vitro. Both NOTA-NFB and DOTA-NFB specifically accumulate in CXCR4-positive, but not CXCR4-negative, tumor xenografts in mice and allow clear visualization of CXCR4 expression by PET. Evaluation of NOTA-NFB and DOTA-NFB for their potential to mobilize immune cells and progenitor cells from the bone marrow to the peripheral blood revealed slightly reduced, but still comparable, results to the parent molecule T140. The tracers reported here may allow the evaluation of CXCR4 expression in primary tumors and metastatic nodules, and enable better informed, more personalized treatment for patients with cancer.
doi:10.1016/j.jconrel.2011.09.076
PMCID: PMC3259211  PMID: 21964282
T140; CXCR4 imaging; PET; copper-64
4.  Role of CXCR4 in the Pathogenesis of Acute Myeloid Leukemia 
Theranostics  2013;3(1):34-39.
The Chemokine receptor CXCR4 and its ligand stromal derived factor-1 (SDF-1/CXCL12) are important players involved in cross-talk between leukemia cells and the bone marrow (BM) microenvironment. CXCR4 expression is associated with poor prognosis in AML patients with and without the mutated FLT3 gene.
CXCL12 which is constrictively secreted from the BM stroma and AML cells is critical for the survival and retention of AML cells within the BM. In vitro, CXCR4 antagonists were shown to inhibit the migration of AML cells in response to CXCL12. In addition, such antagonists were shown to inhibit the survival and colony forming potential of AML cells and abrogate the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. In vivo, using immune deficient mouse models, CXCR4 antagonists were found to induce the mobilization of AML cells and progenitor cells into the circulation and enhance anti leukemic effects of chemotherapy. The hypothesis that CXCL12/CXCR4 interactions contribute to the resistance of AML cells to signal transduction inhibitor- and chemotherapy-induced apoptosis is currently being tested in a series of Phase I/II studies in humans.
doi:10.7150/thno.5150
PMCID: PMC3563079  PMID: 23382784
CXCR4; CXCL12; AML; Bone marrow; Microenvironment.
5.  Characterization of Cyclin E Expression in Multiple Myeloma and Its Functional Role in Seliciclib-Induced Apoptotic Cell Death 
PLoS ONE  2012;7(4):e33856.
Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator – cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator - cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion–mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.
doi:10.1371/journal.pone.0033856
PMCID: PMC3338814  PMID: 22558078
6.  Involvement of CCR6/CCL20/IL-17 Axis in NSCLC Disease Progression 
PLoS ONE  2011;6(9):e24856.
Objectives
Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.
Methods
A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.
Results
CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.
Conclusion
Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.
doi:10.1371/journal.pone.0024856
PMCID: PMC3174223  PMID: 21949768
7.  The Chemokine CXCL16 and Its Receptor, CXCR6, as Markers and Promoters of Inflammation-Associated Cancers 
PLoS ONE  2009;4(8):e6695.
Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes.
doi:10.1371/journal.pone.0006695
PMCID: PMC2723911  PMID: 19690611
8.  Interaction between CXCR4 and CCL20 Pathways Regulates Tumor Growth 
PLoS ONE  2009;4(4):e5125.
The chemokine receptor CXCR4 and its ligand CXCL12 is overexpressed in the majority of tumors and is critically involved in the development and metastasis of these tumors. CXCR4 is expressed in malignant tumor cells whereas its ligand SDF-1 (CXCL12) is expressed mainly by cancer associated fibroblasts (CAF). Similarly to CXCR4, the chemokine CCL20 is overexpressed in variety of tumors; however its role and regulation in tumors is not fully clear. Here, we show that the chemokine receptor CXCR4 stimulates the production of the chemokine CCL20 and that CCL20 stimulates the proliferation and adhesion to collagen of various tumor cells. Furthermore, overexpression of CCL20 in tumor cells promotes growth and adhesion in vitro and increased tumor growth and invasiveness in vivo. Moreover, neutralizing antibodies to CCL20 inhibit the in vivo growth of tumors that either overexpress CXCR4 or CCL20 or naturally express CCL20. These results reveal a role for CCL20 in CXCR4-dependent and -independent tumor growth and suggest a therapeutic potential for CCL20 and CCR6 antagonists in the treatment of CXCR4- and CCL20-dependent malignancies.
doi:10.1371/journal.pone.0005125
PMCID: PMC2659745  PMID: 19340288
9.  Atypical PKC-ζ regulates SDF-1–mediated migration and development of human CD34+ progenitor cells 
Journal of Clinical Investigation  2005;115(1):168-176.
The chemokine stromal cell–derived factor–1 (SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. We report the direct involvement of atypical PKC-ζ in SDF-1 signaling in immature human CD34+-enriched cells and in leukemic pre-B acute lymphocytic leukemia (ALL) G2 cells. Chemotaxis, cell polarization, and adhesion of CD34+ cells to bone marrow stromal cells were found to be PKC-ζ dependent. Overexpression of PKC-ζ in G2 and U937 cells led to increased directional motility to SDF-1. Interestingly, impaired SDF-1–induced migration of the pre-B ALL cell line B1 correlated with reduced PKC-ζ expression. SDF-1 triggered PKC-ζ phosphorylation, translocation to the plasma membrane, and kinase activity. Furthermore we identified PI3K as an activator of PKC-ζ, and Pyk-2 and ERK1/2 as downstream targets of PKC-ζ. SDF-1–induced proliferation and MMP-9 secretion also required PKC-ζ activation. Finally, we showed that in vivo engraftment, but not homing, of human CD34+-enriched cells to the bone marrow of NOD/SCID mice was PKC-ζ dependent and that injection of mice with inhibitory PKC-ζ pseudosubstrate peptides resulted in mobilization of murine progenitors. Our results demonstrate a central role for PKC-ζ in SDF-1–dependent regulation of hematopoietic stem and progenitor cell motility and development.
doi:10.1172/JCI200521773
PMCID: PMC539190  PMID: 15630457
10.  Subsecond Induction of α4 Integrin Clustering by Immobilized Chemokines Stimulates Leukocyte Tethering and Rolling on Endothelial Vascular Cell Adhesion Molecule 1 under Flow Conditions 
Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, α4β1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.
PMCID: PMC2193239  PMID: 10952719
adhesion; integrin; endothelium; chemokine; shear flow
11.  Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function 
Journal of Clinical Investigation  2000;106(11):1331-1339.
The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNA-damaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.
PMCID: PMC381461  PMID: 11104786
12.  The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow 
Journal of Clinical Investigation  1999;104(9):1199-1211.
The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34+ progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34+ cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34+ cells under flow, but, in the presence of surface-bound SDF-1, CD34+ progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction between ICAM-1 and its integrin ligand, which coimmobilized with SDF-1. Human CD34+ cells accumulated efficiently on TNF-activated human umbilical cord endothelial cells in the absence of SDF-1, but they required immobilized SDF-1 to develop firm integrin-mediated adhesion and spreading. In the absence of selectins, SDF-1 also promoted VLA-4–mediated, Gi protein–dependent tethering and firm adhesion to VCAM-1 under shear flow. To our knowledge, this is the first demonstration that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34+ progenitors into firm adhesion by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1.
PMCID: PMC409822  PMID: 10545519

Results 1-12 (12)