32 results on '"Fierro, P."'
Search Results
2. Development of Chimeric Antigen Receptor-Expressing iPSC-Derived Macrophages with Improved Anti-Tumor Activity
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Pouyanfard, Somayeh, Fierro, Manuel, and Kaufman, Dan S
- Subjects
Stem Cell Research ,Stem Cell Research - Induced Pluripotent Stem Cell ,Biotechnology ,Rare Diseases ,Cancer ,5.2 Cellular and gene therapies ,Immunology ,Cardiorespiratory Medicine and Haematology ,Clinical Sciences ,Paediatrics and Reproductive Medicine - Abstract
Abstract Previous studies by our group demonstrate the ability to routinely derive hematopoietic and immune cells from human pluripotent stem cells. Here, we demonstrate the efficient derivation of macrophages from human induced pluripotent stem cells (iPSCs). These macrophages have phenotypic and genotypic characteristics similar to monocytes/macrophages isolated from human peripheral blood. We also demonstrate the ability to polarize these iPSC-derived macrophages (iPSC-Macs) to M1 and M2 populations. Specifically, M1 iPSC-Macs have pro-inflammatory characteristics including expression of CD40 and CD80 on the cell surface, produce increased amounts of TNF-a and IL-6 detected in the supernatant, as well have increased expression of inflammatory cytokines/chemokines (TNF-a, IL-6, IL-1b, IL-12, CCL2, CCL3 and TRAIL) and increased expression of matrix metalloproteases (MMPs). Function of these iPSC-Macs was initially assessed by phagocytosis of fluorescently-labeled beads. These studies demonstrated both the iPSC-M1 and M2 macrophages efficiently phagocytized these beads, and at similar amounts as their peripheral blood counterparts. Next, we tested the ability of the iPSC-Macs to phagocytize human tumor cells. Using A1847 ovarian tumor cells, we found while the iPSC-Macs alone had limited ability to phagocytize the tumor cells (9%), addition of either an anti-CD47 mAb (41%) or anti-EGFR (41%) lead to markedly increased phagocytosis, with the combination of the 2 antibodies being even better (55% phagocytosis). We then tested iPSC-Macs in vivo against luciferase (luc)-expressing A1847 ovarian cancer cells as a xenograft model in NSG-SGM3 mice that express human IL3, GM-CSF and SCF. Using bioluminescent imaging, we found that the combination of iPSC-Macs with both anti-CD47 and anti-EGFR demonstrated significantly improved anti-tumor activity, with median survival of 75 days, compared to 50-60 days for mice treated with only iPSC-Macs, only mAbs or with iPSC-Macs combined either single mAb. Next, we aimed to use the iPSC platform to produce iPSC-Macs engineered to express chimeric antigen receptors (CARs) to further improve their anti-tumor activity. Here, we developed and tested novel macrophage specific CARs that were stably expressed in undifferentiated iPSCs using transposon-mediated gene transfer, similar to our previous studies to derive iPSC-derived CAR-expressing NK cells that have now been translated into clinical trials. We used an anti-mesothelin (meso) scFv combined with 8 different CAR constructs with distinct intracellular signaling components. We found that the iPSC-Macs could express good levels of the CARs (iPSC-CarMacs). Function was again tested in vitro by phagocytosis of the Meso+ A1847 ovarian cancer cells. The iPSC-CarMacs with a Bai1 stimulatory domain consistently demonstrated the best activity in this assay system. We next tested the anti-meso-iPSC-CarMacs in vivo using the A1847 cells. Again, we demonstrate the iPSC-CarMacs combined with anti-CD47 mAb mediate significantly improved anti-tumor activity using this in vivo model compared to the non-CAR-iPSC-Macs + anti-CD47, p
- Published
- 2021
3. The Decisive Role of Pronounced Hyperferritinemia for Diagnosis and Prognosis in the Spectrum of Hematological Disorders
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Vargas-España, Andres, Fuentes-Martin, Valerie, Arias-Espinosa, Luis, Fierro-Angulo, Oscar Manuel, Russ-Tolmos, Mauricio, de la Puente, Ariel, Toledo, Jose M, Seidman-Sorsby, Alec, Soto Mota, Adrian, and Bourlon De Los Rios, Christianne
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- 2022
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4. Feasibility and Improvement in Survival with a Risk-Adapted Treatment Regimen for Childhood Acute Lymphoblastic Leukemia in a Limited Resource Setting
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Jimenez-Antolinez, Yajaira Valentine, Fierro Sáenz, Samantha, Carrizales Torres, Lilia Edith, Colunga Pedraza, Julia Esther, Ramirez-Duran, Hernan, Gómez-De León, Andrés, Gómez-Almaguer, David, and Gonzalez, Oscar
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- 2022
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5. Rivaroxaban vs warfarin in high-risk patients with antiphospholipid syndrome
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Pengo, Vittorio, Denas, Gentian, Zoppellaro, Giacomo, Jose, Seena Padayattil, Hoxha, Ariela, Ruffatti, Amelia, Andreoli, Laura, Tincani, Angela, Cenci, Caterina, Prisco, Domenico, Fierro, Tiziana, Gresele, Paolo, Cafolla, Arturo, De Micheli, Valeria, Ghirarduzzi, Angelo, Tosetto, Alberto, Falanga, Anna, Martinelli, Ida, Testa, Sophie, Barcellona, Doris, Gerosa, Maria, and Banzato, Alessandra
- Abstract
Rivaroxaban is an effective and safe alternative to warfarin in patients with atrial fibrillation and venous thromboembolism. We tested the efficacy and safety of rivaroxaban compared with warfarin in high-risk patients with thrombotic antiphospholipid syndrome. This is a randomized open-label multicenter noninferiority study with blinded end point adjudication. Rivaroxaban, 20 mg once daily (15 mg once daily based on kidney function) was compared with warfarin (international normalized ratio target 2.5) for the prevention of thromboembolic events, major bleeding, and vascular death in patients with antiphospholipid syndrome. Only high-risk patients triple positive for lupus anticoagulant, anti-cardiolipin, and anti–β2-glycoprotein I antibodies of the same isotype (triple positivity) were included in the study. The trial was terminated prematurely after the enrollment of 120 patients (59 randomized to rivaroxaban and 61 to warfarin) because of an excess of events among patients in the rivaroxaban arm. Mean follow-up was 569 days. There were 11 (19%) events in the rivaroxaban group, and 2 (3%) events in the warfarin group. Thromboembolic events occurred in 7 (12%) patients randomized to rivaroxaban (4 ischemic stroke and 3 myocardial infarction), whereas no event was recorded in those randomized to warfarin. Major bleeding occurred in 6 patients: 4 (7%) in the rivaroxaban group and 2 (3%) in the warfarin group. No death was reported. The use of rivaroxaban in high-risk patients with antiphospholipid syndrome was associated with an increased rate of events compared with warfarin, thus showing no benefit and excess risk. This trial was registered at www.clinicaltrials.govas #NCT02157272.
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- 2018
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6. Incidence of a first thromboembolic event in asymptomatic carriers of high-risk antiphospholipid antibody profile: a multicenter prospective study
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Pengo, Vittorio, Ruffatti, Amelia, Legnani, Cristina, Testa, Sophie, Fierro, Tiziana, Marongiu, Francesco, De Micheli, Valeria, Gresele, Paolo, Tonello, Marta, Ghirarduzzi, Angelo, Bison, Elisa, Denas, Gentian, Banzato, Alessandra, Padayattil Jose, Seena, and Iliceto, Sabino
- Abstract
Persistent antiphospholipid (aPL) antibodies are occasionally found in subjects without prior history of thromboembolic events (TEs), raising the dilemma of whether to initiate or not a primary thromboprophylaxis. A first TE is considered rare in aPL carriers, but previous studies did not consider the aPL profile nor was the test positivity confirmed in a reference laboratory. In this study, 104 subjects with high-risk aPL profile (positive lupus anticoagulant, anticardiolipin, and anti-β2–glycoprotein I antibodies, triple positivity) confirmed in a reference laboratory, were followed up for a mean of 4.5 years. There were 25 first TEs (5.3% per year): the cumulative incidence after 10 years was 37.1% (95% confidence interval [CI], 19.9%-54.3%). On multivariate analysis, male sex (hazard ratio = 4.4; 95% CI, 1.5-13.1, P = .007) and risk factors for venous thromboembolism (hazard ratio = 3.3; 95% CI, 1.3-8.5, P = .01) were independent predictors for TEs. Aspirin did not significantly affect the incidence of TE. In conclusion, the occurrence of a first TE in carriers of high-risk aPL profile is considerable; it is more frequent among male subjects and in the presence of additional risk factors for venous TE. These data can help in the decision to initiate primary thromboprophylaxis in these subjects.
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- 2011
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7. Eltrombopag for the treatment of the inherited thrombocytopenia deriving from MYH9mutations
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Pecci, Alessandro, Gresele, Paolo, Klersy, Catherine, Savoia, Anna, Noris, Patrizia, Fierro, Tiziana, Bozzi, Valeria, Mezzasoma, Anna Maria, Melazzini, Federica, and Balduini, Carlo L.
- Abstract
Platelet transfusion is currently the primary medical treatment for reducing thrombocytopenia in patients with inherited thrombocytopenias. To evaluate whether stimulating megakaryopoiesis could increase platelet count in these conditions, we treated patients with a severe thrombocytopenia induced by MYH9mutations (MYH9-related disease) with a nonpeptide thrombopoietin receptor agonist, eltrombopag. Twelve adult patients with MYH9-RD and platelet counts of less than 50 × 109/L received 50 mg of eltrombopag orally per day for 3 weeks. Patients who achieved a platelet count higher than 150 × 109/L stopped therapy, those with 100 to 150 platelets × 109/L continued treatment at the same eltrombopag dose for 3 additional weeks, while those with less than 100 platelets × 109/L increased the eltrombopag dose to 75 mg for 3 weeks. Major responses (platelet count of at least 100 × 109/L or 3 times the baseline value) were obtained in 8 patients, minor responses (platelet counts at least twice the baseline value) in 3. One patient did not respond. Bleeding tendency disappeared in 8 of 10 patients with bleeding symptoms at baseline. Mild adverse events were reported in 2 patients. The availability of thrombopoietin mimetics opened new prospects in the treatment of inherited thrombocytopenias. This study is registered at www.clinicaltrials.govas NCT01133860 (European Union Drug Regulating Authorities Clinical Trials number 2008-001903-42).
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- 2010
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8. In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder
- Author
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Palmieri, Camillo, Falcone, Cristina, Iaccino, Enrico, Tuccillo, Franca Maria, Gaspari, Marco, Trimboli, Francesca, De Laurentiis, Annamaria, Luberto, Laura, Pontoriero, Marilena, Pisano, Antonio, Vecchio, Eleonora, Fierro, Olga, Panico, Maria Rosaria, Larobina, Michele, Gargiulo, Sara, Costa, Nicola, Dal Piaz, Fabrizio, Schiavone, Marco, Arra, Claudio, Giudice, Aldo, Palma, Giuseppe, Barbieri, Antonio, Quinto, Ileana, and Scala, Giuseppe
- Abstract
B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-β cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.
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- 2010
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9. iPSC-Derived Natural Killer Cells and Macrophages Synergistically Kill Acute Myeloid Leukemia Blasts
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Goldenson, Benjamin, Fierro, Manuel, Pouyanfard, Somayeh, and Kaufman, Dan S
- Abstract
Despite many advances, the treatment of acute myeloid leukemia (AML) remains challenging, and few patients are cured by therapies other than allogeneic hematopoietic cell transplant. Treatment with natural killer (NK) cells from allogeneic donors is a promising therapy that can achieve remissions in 30-50% of AML patients. To generate an improved cell-based therapy for AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from additional modifications or combination with other therapies to durably cure AML. Based on studies that demonstrate that targeting the CD47 pathway on macrophages and NK cells improves anti-tumor activity and is an effective treatment for patients with AML, we investigated the combination of iPSC-derived NK cells with iPSC-derived macrophages with and without CD47 blockade for the treatment of AML.
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- 2021
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10. MTHFR and SLCO1B1 Gene Polymorphism and Methotrexate Induced Toxicity in Children with Acute Lymphoblastic Leukemia in the Northwest of Mexico
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Fierro Sáenz, Samantha, Portillo Zavala, Claudia Selene, Moreno Brito, Veronica, Dominguez Viveros, Joel, and González Rodríguez, Everardo
- Abstract
Introduction.
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- 2021
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11. E-selectin receptors on human leukocytes
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Nimrichter, Leonardo, Burdick, Monica M., Aoki, Kazuhiro, Laroy, Wouter, Fierro, Mark A., Hudson, Sherry A., Von Seggern, Christopher E., Cotter, Robert J., Bochner, Bruce S., Tiemeyer, Michael, Konstantopoulos, Konstantinos, and Schnaar, Ronald L.
- Abstract
Selectins on activated vascular endothelium mediate inflammation by binding to complementary carbohydrates on circulating neutrophils. The human neutrophil receptor for E-selectin has not been established. We report here that sialylated glycosphingolipids with 5 N-acetyllactosamine (LacNAc, Galß1-4GlcNAcß1-3) repeats and 2 to 3 fucose residues are major functional E-selectin receptors on human neutrophils. Glycolipids were extracted from 1010 normal peripheral blood human neutrophils. Individual glycolipid species were resolved by chromatography, adsorbed as model membrane monolayers and selectin-mediated cell tethering and rolling under fluid shear was quantified as a function of glycolipid density. E-selectin–expressing cells tethered and rolled on selected glycolipids, whereas P-selectin–expressing cells failed to interact. Quantitatively minor terminally sialylated glycosphingolipids with 5 to 6 LacNAc repeats and 2 to 3 fucose residues were highly potent E-selectin receptors, constituting more than 60% of the E-selectin–binding activity in the extract. These glycolipids are expressed on human blood neutrophils at densities exceeding those required to support E-selectin–mediated tethering and rolling. Blocking glycosphingolipid biosynthesis in cultured human neutrophils diminished E-selectin, but not P-selectin, adhesion. The data support the conclusion that on human neutrophils the glycosphingolipid NeuAca2-3Galß1-4GlcNAcß1-3[Galß1-4(Fuca1-3)GlcNAcß1-3]2[Galß1-4GlcNAcß1-3]2Galß1-4GlcßCer (and closely related structures) are functional E-selectin receptors.
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- 2008
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12. Interleukin 2 (IL 2) and Interferon-γ Production by T Lymphocytes From Patients With B-Chronic Lymphocytic Leukemia: Evidence That Normally Released IL 2 Is Absorbed by the Neoplastic B Cell Population
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Foa, Robert, Giovarelli, Mirella, Jemma, Cristina, Fierro, Maria Teresa, Lusso, Paolo, Ferrando, Maria Luisa, Lauria, Francesco, and Forni, Guido
- Abstract
The capacity of T lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) to release interleukin 2 (IL 2) and interferon (IFN)-γ was assessed following various stimuli. The spontaneous release of IL 2 and IFN-γ was practically absent both with B-CLL and normal T lymphocytes. By contrast, after stimulation with phytohemagglutinin (PHA) or with PHA plus 12-O-tetradecanoylphorbol-13-acetate, the production of IL 2 and IFN-γ by B-CLL T lymphocytes was similar to that of normal T lymphocytes, irrespective of the reversed T lymphocyte subset distribution (OKT4/OKT8 ratio) observed in B-CLL. However, the titer of IL 2 was greatly reduced when autologous leukemic B cells were added to the culture system. Unlike IL 2, the presence of leukemic B cells did not affect the titer of IFN-γ in the culture supernatants. The indication that IL 2 may be adsorbed in vivo by the neoplastic B cells was further confirmed by the demonstration of the IL 2 receptor (revealed by anti-Tac monoclonal antibody) on the leukemic B cells, particularly following mitogenic stimulation, and by the evidence that exogenous IL 2 can be directly absorbed by untreated B-CLL cells. These findings suggest that the ability of B-CLL T lymphocytes to release IFN-γ and IL 2 is preserved, but that IL 2 may be rapidly removed by the neoplastic B-CLL cells, thus contributing to the well-documented T lymphocyte abnormalities present in this disease.
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- 1985
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13. Lymphokine-activated killer (LAK) cell activity in B and T chronic lymphoid leukemia: defective LAK generation and reduced susceptibility of the leukemic cells to allogeneic and autologous LAK effectors
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Foa, R, Fierro, MT, Raspadori, D, Bonferroni, M, Cardona, S, Guarini, A, Tos, AG, di Celle, PF, Cesano, A, and Matera, L
- Abstract
The capacity to generate lymphokine-activated killer (LAK) cells and the susceptibility of the neoplastic cells to both allogeneic and autologous LAK effectors were studied in B and T chronic lymphoproliferative disorders. While in B-cell chronic lymphocytic leukemia (B-CLL) the depressed natural killer function could be restored after a 7-day incubation with recombinant interleukin (IL-2), B-CLL mononuclear cells showed a reduced LAK activity compared with normal LAK cells. Furthermore, in all but 1 of the 20 B-CLL samples tested the leukemic cells were totally resistant to autologous LAK effectors. In most cases the leukemic cells were also resistant to normal allogeneic LAK cells. Competition experiments demonstrated that the patients' LAK cells, as well as normal LAK effectors, were capable of recognizing B-CLL cells, pointing, therefore, to a postbinding cytolytic defect. In hairy cell leukemia (HCL) an overall reduced LAK activity against allogeneic targets was documented, but, at variance from B-CLL, hairy cells were often susceptible to the lytic effect of normal LAK cells, and in half of the cases tested the neoplastic population was also sensitive in an autologous system. Similarly to B- CLL, in the great majority of T chronic lymphoproliferative disorders studied, the pathologic cells were resistant to normal and autologous LAK effectors and a defective LAK generation was found. These results demonstrate that in most B and T chronic leukemias the LAK function is defective and, when inducible, does not appear directed against the leukemic population. The possibility of exploiting an immunotherapeutic approach with IL-2/LAK cells in the management of chronic lymphoproliferative disorders does not gain support by these findings.
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- 1990
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14. Production of tumor necrosis factor-alpha by B-cell chronic lymphocytic leukemia cells: a possible regulatory role of TNF in the progression of the disease
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Foa, R, Massaia, M, Cardona, S, Tos, AG, Bianchi, A, Attisano, C, Guarini, A, di Celle, PF, and Fierro, MT
- Abstract
Tumor necrosis factor-alpha (TNF) is a cytokine that displays a pleomorphic array of effects on different cell populations. Evidence is presented that TNF may be constitutively produced by B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) cells and that it may play a relevant role in these diseases. These conclusions are based on the presence of circulating levels of TNF in the serum of 20 of the 24 patients tested (83.3%), while undetectable values were found in normal sera. The suggestion that the increased serum levels were due to the leukemic cell population is strengthened by the evidence that purified B-CLL and HCL cells may constitutively release variable degrees of TNF. These levels markedly increase after incubation with interferon gamma or phytohemagglutinin (PHA) plus phorbol myristate acetate (PMA). The cellular release of TNF by primary B-CLL cells was significantly (P less than .001) higher in B-CLL stage O-I patients compared with stage II-III patients. The demonstration that, in B-cell chronic lymphoproliferative disorders, the pathologic cells may release TNF was further confirmed by the presence of the mRNA for this cytokine in primary and/or in pre-activated cells. Recombinant TNF was capable of inducing a proliferative signal only in a minority of cases (4/24); in most cases it was ineffective, and, in a few, it reduced the degree of proliferation. Furthermore, in costimulatory experiments with interleukin-2 and PHA plus PMA, TNF was ineffective. On the other hand, when primary B-CLL cells were incubated in the presence of an anti-TNF antibody, in 8 of 12 independent experiments a 2- to 15-fold increase in thymidine uptake was documented. Taken together, these results suggest that TNF may play a regulatory role in the progression of the neoplastic clone in B-cell chronic lymphoproliferative disorders and may be implicated in some of the side effects associated with these diseases.
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- 1990
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15. Interleukin 2 (IL 2) and interferon-gamma production by T lymphocytes from patients with B-chronic lymphocytic leukemia: evidence that normally released IL 2 is absorbed by the neoplastic B cell population
- Author
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Foa, R, Giovarelli, M, Jemma, C, Fierro, MT, Lusso, P, Ferrando, ML, Lauria, F, and Forni, G
- Abstract
The capacity of T lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) to release interleukin 2 (IL 2) and interferon (IFN)-gamma was assessed following various stimuli. The spontaneous release of IL 2 and IFN-gamma was practically absent both with B-CLL and normal T lymphocytes. By contrast, after stimulation with phytohemagglutinin (PHA) or with PHA plus 12-O- tetradecanoylphorbol-13-acetate, the production of IL 2 and IFN-gamma by B-CLL T lymphocytes was similar to that of normal T lymphocytes, irrespective of the reversed T lymphocyte subset distribution (OKT4/OKT8 ratio) observed in B-CLL. However, the titer of IL 2 was greatly reduced when autologous leukemic B cells were added to the culture system. Unlike IL 2, the presence of leukemic B cells did not affect the titer of IFN-gamma in the culture supernatants. The indication that IL 2 may be adsorbed in vivo by the neoplastic B cells was further confirmed by the demonstration of the IL 2 receptor (revealed by anti-Tac monoclonal antibody) on the leukemic B cells, particularly following mitogenic stimulation, and by the evidence that exogenous IL 2 can be directly absorbed by untreated B-CLL T lymphocytes to release IFN-gamma and IL 2 is preserved, but that IL 2 may be rapidly removed by the neoplastic B-CLL cells, thus contributing to the well-documented T lymphocyte abnormalities present in this disease.
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- 1985
- Full Text
- View/download PDF
16. Defective lymphokine-activated killer cell generation and activity in acute leukemia patients with active disease
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Foa, R, Fierro, MT, Cesano, A, Guarini, A, Bonferroni, M, Raspadori, D, Miniero, R, Lauria, F, and Gavosto, F
- Abstract
In 26 myeloid and lymphoid acute leukemia patients at presentation the capacity to generate interleukin-2 (IL-2)-induced lymphokine-activated killer (LAK) cells effective against the natural killer (NK)-resistant Raji cell line, as well as the susceptibility of the blasts to normal peripheral blood (PB) LAK cells and to autologous LAK effectors was analyzed. The overall PB LAK activity against Raji cells was significantly lower in acute leukemia patients compared with normal controls (mean, 1,473 +/- 971 SD LU/10(8) LAK effectors v 3,340 +/- 1,862; P less than .001). The sensitivity of the blasts to autologous LAK cells was also significantly lower than to normal LAK effectors (517 +/- 593 LU/10(8) LAK effectors v 1,304 +/- 1,066; P less than .01). When the data were analyzed independently, four patterns of behavior could be recognized. The relatively largest group (9 of 26) included patients in whom effective LAK cells could be generated against the Raji line, but in whom the blasts were resistant to autologous PB-LAK effectors while being susceptible to normal LAK cells (defective specific LAK activity). In 5 of 26 cases, an incapacity to generate LAK activity against both allogeneic and autologous target cells was observed (defective LAK generation). In six further cases, the blasts were resistant to both allogeneic and autologous LAK populations, though the latter were effective against the Raji line (resistant blasts). The same defects could also be shown with bone marrow-derived LAK cells. Only in six cases did the leukemic blasts appear susceptible to autologous and allogeneic LAK cells. In four patients the analysis could be repeated at remission, and in three a restoration of the LAK function against the primary blasts was recorded. In the 10 cases studied at relapse, the blasts were resistant to autologous LAK effectors in nine and to normal LAK in seven. These data demonstrate that in most acute leukemia patients with active disease, a defect of the LAK machinery, either a deficient generation of LAK cells or the resistance of the blasts to LAK effectors, may be documented, pointing therefore to a possible contributory role of the LAK system in the control of leukemic cell growth. In view of the frequent normalization of the autologous LAK activity at the time of remission, immunotherapy with IL-2/LAK cells should be primarily aimed to patients with minimal residual disease.
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- 1991
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17. Latent Tuberculosis in Hematopoietic Stem Cell Transplantation: Diagnostic and Therapeutic Strategies to Prevent Disease Activation in an Endemic Population
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Bourlon, Christianne, Fierro-Angulo, Oscar Manuel, Garcia-Ramos, Jesus A, Camacho-Hernandez, Rocio, Demichelis, Roberta, and Acosta-Medina, Aldo A
- Abstract
Demichelis: AMGEN: Research Funding, Speakers Bureau; Abbvie: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Shire: Speakers Bureau.
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- 2019
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18. Risk Factors for Induction Related Mortality in Adults Patients with Acute Lymphoblastic Leukemia: Report from a Hispanic Population
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Inclan-Alarcon, Sergio I, Bourlon, Christianne, Fierro-Angulo, Oscar Manuel, Garcia-Ramos, Jesus A, Riviello-Goya, Santiago, and Demichelis-Gómez, Roberta
- Abstract
No relevant conflicts of interest to declare.
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- 2018
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19. Risk Factors of Thromboembolic Disease in Men with Prostate Cancer: Report from a Hispanic Population
- Author
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Luna-Santiago, Rodrigo, Bourlon, Maria T, Jasqui-Bucay, Ariel, Jasqui-Bucay, Alan, Fierro-Angulo, Oscar Manuel, Garcia-Ramos, Jesus A, Rosales-Sotomayor, Guillermo, Martiñon-Gutierrez, Silvia, Velazquez, Hugo E, Castro, Carlos A, and Bourlon, Christianne
- Abstract
No relevant conflicts of interest to declare.
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- 2018
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20. Anti-CCR4 Monoclonal Antibody, Mogamulizumab, Demonstrates Significant Improvement in PFS Compared to Vorinostat in Patients with Previously Treated Cutaneous T-Cell Lymphoma (CTCL): Results from the Phase III MAVORIC Study
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Kim, Youn H., Bagot, Martine, Pinter-Brown, Lauren, Rook, Alain H., Porcu, Pierluigi, Horwitz, Steven M., Whittaker, Sean, Tokura, Yoshiki, Vermeer, Maarten, Zinzani, Pier Luigi, Sokol, Lubomir, Morris, Stephen, Kim, Ellen, Ortiz-Romero, Pablo L., Eradat, Herbert, Scarisbrick, Julia, Tsianakas, Athanasios, Elmets, Craig, Dalle, Stephane, Fisher, David C., Halwani, Ahmad S., Poligone, Brian, Greer, John P., Fierro, Maria Teresa, Khot, Amit, Moskowitz, Alison J., Dwyer, Karen, Moriya, Junji, Humphrey, Jeffrey, Hudgens, Stacie, Grebennik, Dmitri O., Tobinai, Kensei, and Duvic, Madeline
- Abstract
Kim: Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; Soligenix: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Portola: Consultancy, Research Funding; Neumedicine: Research Funding; miRagen: Research Funding; Merck: Research Funding; Medivir: Membership on an entity's Board of Directors or advisory committees; Kyowa-Kirin-Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Consultancy, Research Funding; Horizon Pharma: Consultancy, Research Funding; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tetralogic: Research Funding; Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Bagot: Innate Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Actelion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Kyowa: Membership on an entity's Board of Directors or advisory committees. Horwitz: Kyowa-Hakka-Kirin: Consultancy, Research Funding; Mundipharma: Consultancy; ADCT Therapeutics: Research Funding; Forty-Seven: Consultancy, Research Funding; Infinity/Verastem: Consultancy, Research Funding; HUYA: Consultancy; Millenium/Takeda: Consultancy, Research Funding; BMS: Consultancy; Seattle Genetics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Aileron Therapeutics: Research Funding. Whittaker: Celgene: Honoraria; Galderma: Research Funding. Vermeer: Innate Pharma safety board for IPH4102-101: Membership on an entity's Board of Directors or advisory committees. Zinzani: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; J&J: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sokol: Spectrum Pharmaceuticals: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy. Kim: Kyowa Kirin Pharmaceutical Development, Inc.: Other: Clinical trials investigator; Solgenix: Other: Clinical trials investigator; Actelion: Consultancy; Cutaneous Lymphoma Foundation: Membership on an entity's Board of Directors or advisory committees; US Cutaneous Lymphoma Consortium: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy. Ortiz-Romero: ACTELION: Consultancy; 4SC: Consultancy; Innate Pharma: Consultancy; Takeda: Consultancy; MEDA: Research Funding. Eradat: Abbvie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding; Novartis: Research Funding; Celgene: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding. Scarisbrick: 4SC: Consultancy; Takeda: Consultancy; Mallinckrodt: Consultancy; Innate Pharma: Consultancy; Actelion: Consultancy. Elmets: NCI: Research Funding; Veterans Administration: Research Funding; California Wine Grape Assn: Research Funding; Solegenix: Research Funding; Idera: Research Funding; Elorac: Research Funding; Ferndale Labs: Consultancy, Research Funding; Astellas Pharma: Research Funding. Dalle: Kyowa Hakko Kirin Pharmaceutical: Research Funding. Fisher: Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Halwani: Amgen: Research Funding; Pharmacyclics: Research Funding; Takeda: Research Funding; Genetech Inc.: Research Funding; Roche/Genentech Inc.: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squib: Research Funding; Kyowa Hikko Kirin: Research Funding; AbbVie: Research Funding; Immune Design: Research Funding; Miragen: Research Funding. Poligone: Actelion Pharmaceutical: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; Kyowa Hakko Kirin: Research Funding; Soligenix: Research Funding. Khot: Celgene: Consultancy; Janssen: Consultancy; Amgen: Other: Travel Grant. Moskowitz: Incyte: Research Funding; Takeda: Honoraria; Bristol Myers-Squibb: Consultancy, Research Funding; ADC Therapeutics: Research Funding; Seattle Genetics: Honoraria, Research Funding. Dwyer: Kyowa Kirin Pharmaceutical Development, Inc.: Employment. Moriya: Kyowa Kirin Pharmaceutical Development, Inc.: Employment. Humphrey: KYOWA KYRIN PHARMACEUTICAL DEVELOPMENT: Employment. Hudgens: Clinical Outcomes Solutions: Consultancy, Research Funding. Grebennik: Kyowa Kirin Pharmaceutical Development, Inc.: Employment. Tobinai: AbbVie: Research Funding; Chugai: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Zenyaku Kogyo: Honoraria; GlaxoSmithKline: Research Funding; Takeda: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Servier: Research Funding; Janssen: Honoraria, Research Funding; HUYA Bioscience: Honoraria; Daiichi Sankyo Co., Ltd: Consultancy, Honoraria; Mundipharma: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding. Duvic: MDACC: Other: Safety Oversight Committee, Research Funding.
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- 2017
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21. Impact Of The Number Of Prior Treatment Lines Received On The Distribution Of Peripheral Blood Leukocyte Subsets In Advanced-Stage B-Cell Chronic Lymphocytic Leukemia (CLL)
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Grigore, Georgiana, Barrena, Susana, Perez-Andres, Martin, Fierro, Miriam, González, Marcos, Rabasa, Pilar, Medina, Angeles, Tomás, José Francisco, Solano, Fernando, De la Serna, Javier, Marco, Jose García, Allegue, Mª José, Loscertales, Javier, PÉrez, Inmaculada, Olave, Teresa, Almeida, Julia, and Orfao, Alberto
- Abstract
It is currently well-known that B-cell chronic lymphocytic leukemia (CLL) patients have an impaired immune function -particularly in advanced disease-, which significantly contributes to a higher risk of infections. The introduction of new effective therapeutic agents, such as the purine analogues (or alkylating agents with concomitant properties of purine analogues) plusanti-CD20 monoclonal antibodies, have significantly increased the rate of complete responses in CLL, but so far their impact on the overall immune function and the spectrum of infections occurring in CLL patients after therapy, remains to be fully understood.To evaluate the effect of the number of treatment lines received on the different normal circulating leucocyte cell populations, including normal B-cell subsets, in advanced-stage treated CLL.The distribution of peripheral blood (PB) leukocytes was analyzed in 85 untreated CLL patients, and compared to that of 63 patients who had previously been treated with 1 line of treatment (n=39) or >1 line of treatment (n=24), and who failed to respond. Analysis was performed by 8-color flow cytometry with monoclonal antibodies against CD3, CD4, CD5, CD8, TCRgd, CD19, CD20, CD27, CD38, CD45, CD56, sIgM, sIgA, sIgG, sIgLambda and sIgKappa.The absolute count of circulating malignant B cells was not significantly different (p>0.05) in the untreated vs. previously treated patients who received 1 or >1 line of treatment (77,627±84,211 vs 67,994±72,087 vs 59,282±74,206 cells/uL; respectively). In contrast, as compared to untreated patients, PB normal B cells were found to be reduced in patients who had received either 1 line or >1 line of treatment (89±142 vs 36±57 and 23±32 cells/uL, p=0.004 and p<0.001, respectively), but at similar levels between the two groups of previously treated patients (p>0.05). When dissecting the normal B-cell subsets, therapy-related decreased B-cell numbers were mostly due to a reduced number of circulating memory B cells (67±98 vs 21±45 and 15±26 cells/uL; p=0.001 and p<0.001, respectively), including all memory isotypes: IgM (24±31 vs 5±13 and 5±6 cells/uL, p<0.001), IgG (25±59 vs 10±32 and 6±13 cells/uL; p=0.008) and IgA (18±30 vs 5±10 and 6±12 cells/uL; p=0.001 and p=0.003). No significant differences were found as regards the absolute count of immature (5±11 vs 8±26 and 3±11 cells/uL; p>0.05) and naïve (16±55 vs 6±18 and 4±8 cells/uL; p>0.05) B cells, nor for circulating plasma cells (3±18 vs 5±21 and 2±6 cells/uL; p>0.05), regardless of the therapy status.As compared to untreated patients, the absolute count of CD4+ T cells and CD4/CD8 double negative TCRαβ cells were significantly lower in patients with >1 line of treatment (1,836±1,340 vs 1,256±1,027 and 131±165 vs 56±97 cells/uL, p=0.03 and p=0.007 respectively) but not in those who had received only 1 line (1,477±1,349 and 201±747 cells/uL; p>0.05). In contrast, therapy did not show a significant impact on the absolute count of PB T CD8+ and TCRgd cells. No statistically significant differences were observed in the number of PB innate immune subpopulations including, neutrophils, eosinophils, basophils, monocytes, NK cells and dendritic cells.While there are no differences regarding the number of leukemic cells, previously treated patients have significantly reduced counts of total and memory (all isotypes) normal B-cell subsets when compared to untreated patients. Together with this, CD4+ helper T cells could also be compromised after more than 1 line of treatment. Monitoring of these therapy-related immune defects could contribute to a better management of infectious complications in advanced-stage CLL patients.No relevant conflicts of interest to declare.
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- 2013
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22. Functional Influence of CXCL12 Regulating miRNAs in Mesenchymal Stem Cells (MSCs).
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Arabanian, Laleh S., Fierro, Fernando, Poitz, David M., Strasser, Ruth H., Bornhaeuser, Martin, Ehninger, Gerhard, Stölzel, Friedrich, and Illmer, Thomas
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No relevant conflicts of interest to declare.
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- 2012
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23. Regulation of β1-Integrin by Mir-134 in Mesenchymal Stromal Cells – Implications for Mesenchymal Stromal Cell Adherence and Hematopoietic Stem Cell Interaction
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Stölzel, Friedrich, Poitz, David M., Arabanian, Laleh S., Friedrichs, Jens, Docheva, Denitsa, Schieker, Matthias, Fierro, Fernando A., Platzbecker, Uwe, Strasser, Ruth H., Bornhäuser, Martin, Werner, Carsten, Ehninger, Gerhard, and Illmer, Thomas
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Platzbecker: Amgen: Consultancy; GlaxoSmithKline: Consultancy; Celgene: Consultancy; Novartis: Consultancy.
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- 2012
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24. A Randomized Clinical Trial of Lenalidomide and Dexamethasone with and without Autologous Stem Cell Transplant in Patients with Newly Diagnosed Multiple Myeloma: Interim Study Results,
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Dai, Lijun, O'Sullivan, Amy, Kennedy, Ryan, Abbas, Mohammad, Shuai, Yongli, Passero, Vida Almario, Andreas, Carrie, Gardner, Diane, Redner, Robert L, Roodman, G. David, Marks, Stanley M., Raptis, Anastasios, Hou, Jing-Zhou, Petro, Daniel, Sun, Min, Evans, Terry, Pietragallo, Louis V., Waas, John K., Viverette, J. Franklin, Osborn, Jennifer, Reyes, Vincent, Pinkerton, Richard, Crandall, Theodore, Fierro, Ronald, Volkin, Robert, Normolle, Daniel, Mapara, Markus Y., and Lentzsch, Suzanne
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High dose chemotherapy combined with autologous stem cell transplantation (ASCT) as opposed to conventional chemotherapy improved progression free survival (PFS) and overall survival (OS) in multiple myeloma (MM) and is currently the standard of care for newly diagnosed MM patients less than 65 years old. Over the last decade, novel agents such as lenalidomide or bortezomib have dramatically improved MM outcomes with similar response rates as ASCT and the role of upfront ASCT has become more controversial. Therefore the goal of this randomized clinical trial is to determine the role of upfront ASCT in newly diagnosed myeloma patients receiving novel agent lenalidomide and low-dose dexamethasone induction.Patients aged ≥18 years with newly confirmed, measurable MM in stage 2 and 3 (Salmon Durie) and meeting CRAB criteria were enrolled. Patients were randomized to transplant (Arm A) or to non-transplant (Arm B). Patients in Arm A received 4 cycles of lenalidomide (25mg days 1 – 21) plus low-dose dexamethasone (40mg days 1,8,15,22) followed by ASCT conditioned with 200 mg/m2 melphalan (LD+ASCT); Arm B patients received 8 cycles of lenalidomide plus low-dose dexamethasone (LD alone). Both arms received stem cell collection after 4 cycles of therapy if patients achieved at least a partial remission (PR). Patients with stable disease (SD) or progressive disease (PD) went off study. The primary objective was to compare best response. The secondary endpoints included duration of response (DOR), progression free survival (PFS), overall survival (OS) and evaluation of secondary malignancies in both arms.From February 2008 to May 2011, 44 patients with newly diagnosed MM were randomized. The patient characteristics were as follow: median age of the patients was 61.7 years (range 48∼75), 45.5% female and 55.5% male patients, ISS stage I 31%, II 51% and III 18%. 40 patients were eligible for evaluation and 20 patients were randomized to Arm A or Arm B, respectively. The data were analyzed according to latest IMWG response criteria (Blood. 2011 May 5;117(18):4691–5). In an intention to treat analysis, patients in Arm A (LD + ASCT), achieved a 100% Overall Response Rate (ORR) with 40% PR (n=8) and 60% Very Good Partial Response (VGPR) (n=12). In Arm B (LD only) the ORR was 75% (n=15), including 15% CR (n=3), 35% VGPR (n=7), 25% PR (n=5), 20% SD (n=4) and 5% PD (n=1). The ORR was significantly superior in the LD+ASCT group compared to LD alone (p=0.047). After a median follow-up of 25.3 months, 17 patients have PD (8 in LD+ASCT and 9 in LD alone), 6 have died (1 in LD+ASCT and 5 in LD alone). DOR, PFS and OS were not significantly different in both groups. OS showed a trend to be superior in patients treated with LD+ASCT (p=0.08). (Table 1). One patient in the LD+ASCT arm developed MDS 13 months after start of therapy.Our interim analysis of an ongoing clinical study suggests that treatment of newly diagnosed MM patients with lenalidomide plus low-dose dexamethasone induction followed by upfront ASCT resulted in significantly improved ORR. There was no difference in terms of DOR or PFS with a trend of superior OS in the LD+ASCT group. The study requires careful interpretation based on the low patient number and relatively short follow up, but supports the continued role of upfront consolidative ASCT in newly diagnosed MM patients. The incidence of secondary malignancy was low with the development of 1 MDS. Updated data on response and overall survival will be available at the time of presentation.Roodman: Amgen: Consultancy; Millennium Pharmaceuticals: Consultancy. Raptis:Millennium: Speakers Bureau; Celgene Corp: Speakers Bureau; Eisai: Speakers Bureau. Lentzsch:Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.
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- 2011
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25. Regulatory Potential of miRNAs on CXCL12 Expression in Human Mesenchymal Stem Cells (MSC)
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Illmer, Thomas, Heder, Carolin, Stölzel, Friedrich, Fierro, Fernando, and Ehninger, Gerhard
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No relevant conflicts of interest to declare.
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- 2011
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26. Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived From Human Embryonic Stem Cells.
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Gruenloh, William, Kambal, Amal, Sondergaard, Claus, McGee, Jeannine, Fierro, Fernando Antonio, Olson, Scott, and Nolta, Jan A.
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No relevant conflicts of interest to declare.
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- 2009
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27. Hydralazine + Magnesium Valproate as Epigenetic Treatment for Myelodysplastic Syndrome (MDS). Preliminary Results of a Phase II Trial.
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Candelaria, Myrna, Herrera, Aquileo, Labardini, Juan, Gonzalez-Fierro, Aurora, Trejo-Becerril, Catalina, de la Cruz-Hernandez, Erick, la Torre-Talavera, Brenda A, and Duenas-Gonzalez, Alfonso
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DNMT inhibitors decitabine and azacitidine are the current standard of MDS treatment. On the other hand, HDACs inhibitors are also being tested against this condition. These two drug classes synergize on their gene reactivating and anticancer activities. The combination of hydralazine and valproate (TRANSKRIP R/L), a DNMTs and HDACs inhibitors respectively is being developed as epigenetic therapy under the “Drug Repositioning” concept against common solid tumors in combination with chemo or radiation, as single agent against cutaneous lymphoma and against MDS.To evaluate the clinical efficacy and safety of TRANSKRIP R/L against MDS.Patients with previously treated MDS and having with normal hepatic and renal function were included. After signing informed consent the acetylator phenotype was determined to prescribe hydralazine dose (slow acetylators 82 mg, daily; fast acetylators 183 mg, daily). Valproate was dosed at 30 mg/kg/day. Both drugs were given daily until progression. Clinical and laboratory evaluations were done weekly. Response was graded with the International Working Group criteria. Toxicity was evaluated by OMS criteria. Bone marrow aspiration was performed every six weeks to evaluate global and gene specific DNA demethylation and histone acetylation.From November 2007 to November 2008, twelve patients were included (5 female, 7 male). Median age ± SD was 59.5 ± 19.78 (range 23-79) years; median time from diagnosis to inclusion was 11.57 months (range 3- 23). Median of previous treatment was 3 (range 1-5). RCMD was diagnosed in 10 cases, and RAEB in two. Many (33 %) had a normal initial caryotype, followed by multiple, complex changes (25 %), two cases had -7 and isolated cases with -5q syndrome, +8, and 11q 23 were documented. According with IPSS, they were graded as intermedial-1 (58 %) or intermedial- two (42 %). Response was evaluated in nine cases: Global response was documented in 55.5 %, including one CR [lasting 10 w], one PR [lasting 6 w], four with an hematological improvement [with a mean time of duration of 25 w]; and one SD. Only two patients (22%) progressed to acute leukemia after 75 and 105 weeks of treatment. The mean increase of blood parameters is summarized in the following table:Transfusional requirements decreased from 3 units monthly to 0.3 for RBC and from 1 to 0.16 monthly for platelets. The main toxicity was grade 1 & 2 somnolence in 70 % of pts, followed by grade 1 nausea in 57 % of them. None severe toxicity was documented. Mean ± SD valproate and hydralazine serum levels were: 68.2 ± 24.45 ug/mL and 211.59 ± 66.45 ng/mL, respectively. The results of pharmacokinetics, DNA methylation and histone acetylation will be presented at the meeting.An hematological improvement was documented in most of the patients. These preliminary results are encouraging, since actually approved demethylating agents for MDS have shown a clearly decrease of hematological derivates transfusion, but not CR o PR. TRANSKRIP R/L administered to previously treated MDS patients is safe and shows promising clinical activityNo relevant conflicts of interest to declare.
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- 2009
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28. Assessment of the Impact of Enforced Expression of Platelet Derived Growth Factor Beta in Human Mesenchymal Stem Cells and Contribution to Blood Flow Restoration.
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Sondergaard, Claus, Fierro, Fernando Antonio, McGee, Jeannine, and Nolta, Jan A.
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No relevant conflicts of interest to declare.
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- 2009
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29. MicroRNA Mir-23a Regulates SDF-1alpha Expression in Human Bone Marrow-Derived Mesenchymal Stem Cells
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Fierro, Fernando, Poitz, David, Nolta, Jan A., Bornhaeuser, Martin, Ehninger, Gerhard, and Illmer, Thomas
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MicroRNAs (miRNAs) can regulate hematopoietic stem/progenitor cells (HSPC) by modulation of intrinsic cell components such as transcription factors and receptors. In addition, miRNAs could play a role in the microenvironment were HSPC host and consequently affect HSPC via extrinsic factors, which to our knowledge is an hypothesis that has not been tested. Since the chemokine stromal derived factor 1alpha (SDF-1alpha) is essential for both homing and retention of HSPC in the bone marrow, we tested if miRNAs expressed by human bone marrow-derived mesenchymal stem cells (MSC; a key source of SDF-1alpha), could potentially inhibit SDF-1alpha expression. In deed, using luciferase reporter-systems we show specific binding of miR-23a to the 3′UTR of SDF-1alpha. Consequently, transfection of MSCs with a precursor miR-23a (pre-miR-23a) leads to a 30% reduction of SDF-1alpha at both mRNA and protein levels. In contrast, inhibition of endogenous miR-23a with anti-sense oligonucleotides (anti-miR-23a), leads to a significant increase of SDF-1alpha also at both mRNA (30%) and protein (10%) levels as compared to controls (scramble pre-/anti-miR). As a result, migration of CD34+ HSPC in transwell assays is strongly affected upon overexpression or inhibition of miR-23a in MSCs (with pre-miR-23a 35% less migration; with anti-miR-23a 20% more migration, as compared to respective controls). Interestingly, transforming growth factor beta 1 (TGF-beta1) inhibits SDF-1alpha expression in MSCs in a concentration dependent manner, while miR-23a is increased under same experimental settings. Even more, silencing endogenous miR-23a significantly reduces the effect of TGF-beta1 on SDF-1alpha mRNA and protein levels, suggesting that at least in part TGF-beta1 inhibits SDF-1alpha expression via increasing miR-23a levels. This is to our knowledge the first established connection between miRNA biology and HSPC-niche related factors.
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- 2008
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30. Localization of Hematopoietic Stem Cells in Coculture with Mesenchymal Stromal Cells Impacts on Phenotype and Cell Cycle Status
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Jing, Duohui, Alakel, Nael, Fierro, Fernando, Mueller, Katrin, Bornhaeuser, Martin, Ehninger, Gerhard, and Ordemann, Rainer
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Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became clear that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD34 purified HSC expansion and differentiation demonstrating a promoting impact of MSC on adherent HSC in comparison to non adherent HSC in terms of phenotype, migration capacity and clonogenicity. Performing phase contrast microscopy and confocal microscopy we are able to distinguish HSC which are located on the surface of a MSC monolayer (phase-bright cells) and HSC which are covered by MSC monolayer (phase-dim cells). Both HSC fractions and the non-adherent cells were isolated separately by performing serial washing steps. All three fractions were analyzed at fixed time points during the first week of co-culture in term of cell cycle progression, proliferation, maturation and cell division accompanied differentiation. First we performed propidium iodide (PI) staining for cell cycle analysis revealing that the phase-bright cells contained the highest percentage of G2 cells in comparison to the non adherent cells and the phase-dim cells; 13.9 ±1.0% vs 1.3 ±1.2% vs 2.7 ±2.0%, p<0.001. The data indicate the facilitating impact of MSC on HSC in performing mitosis which is however depending on the location of interaction. When HSC are released into supernatant (non adherent cells) or covered by MSC, G2 phase was significantly down-regulated. Next we studied the proliferation capacity of the separate cell fractions. Consistent with the data of cell cycle, cell number of phase-bright faction increased much faster than the other two fractions during the first 4 days suggesting that the MSC surface in vitro is the predominant location of HSC proliferation. Next we investigated the phenotype of HSC. According to FACS analysis results (CD34+CD38-) phase-dim cells revealed a more immature phenotype in comparison to the non adherent cells and the phase-bright cells. During the first four days 80% of phase-dim cells remained CD34+CD38-, while cells of the phase-bright- and the non adherent fraction exhibited a significant more mature phenotype. Performing cell division tracking using CFSE we were able to show that over time number of divisions of phase-dim cells were significantly diminished in comparison to the other two cell fractions in co-cultures. In addition, phase-dim cells started to lose CD34 at the 7thgeneration, while non-adherent and phase-bright cells already lost CD34 at the 4thgeneration. These data suggest that “stemness” of HSC was rather preserved in the cell fraction which was covered by MSC monolayer than in the cell fraction on the surface of MSC. In conclusion we demonstrate HSC in distinct locations in vitro showing different behaviors in terms of phenotype and proliferation. It becomes evident that not only the cell to cell contact matters but also the localization of contact. Further experiments are needed to investigate NOD/SCID repopulation potential of the different cell fractions.
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- 2008
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31. Hematopoietic Progenitor Cells Harbouring BCR-ABL Exhibit Increased Adhesiveness but an Impaired Migration and Homing Potential.
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Fierro, Fernando A., Taubenberger, Anja, Ehninger, Gerhard, Mueller, Daniel, Bornhaeuser, Martin, and Illmer, Thomas
- Abstract
Recent studies suggest that one of the most important treatment resistance mechanisms of cells with the BCR-ABL translocation could be the interaction with the hematopoietic niche. During the contact with the respective microenvironment, leukemic stem and progenitor cells acquire both a state of cell adhesion mediated drug resistance (CAM-DR) and may change their proliferation and differentiation potential. Studies on the effect of the BCR-ABL oncogene on cell adhesion and migration have yielded conflicting results due to different cell type models (primary cells vs. transduced cell lines), non-standardized quantification methods and diverse substrates (fibronectin vs. bone marrow stromal cells) studied. In the present work, we investigated the adhesion properties of 32D cells which were stably transformed with the BCR-ABL fusion transcript in comparison to an empty vector transformed control. The commonly used panning-style adhesion assay determining the percentage of hematopoietic cells adhering to a stroma cell line (M2-10B4) clearly demonstrated higher adhesion capacity of BCR-ABL transformed cells. Accordingly, an atomic force microscopy (AFM)-based method to measure adhesion forces of a single leukemic cell over stroma cells, showed an increased ‘stickyness’ of BCR-ABL transformed cells. AFM experiments demonstrated highly different forces that were needed to retract attached 32D-vector cells as compared to 32D/BCR-ABL cells (1000 pN vs. 3500 pN; p<0.01). This higher superficial adhesion of BCR-ABL transformed cells was associated with a decreased migration and homing potential of adhering cells into the stromal layer (quantified by cell tracking movies analysis) and cobblestones formation which was analysed by confocal laser scanning microscopy. The differences found between control and BCR-ABL-expressing cells were reversible by pre-incubation with 0.5 μM Imatinib Mesylate (IM), suggesting that altered adhesion is a tyrosine kinase-activity dependent process. To further gain insight in the mechanisms of adhesion we used a cDNA microarray (Affymetrix-Gene chip 430A 2.0) for the identification of BCR-ABL related gene expression with special focus on adhesion associated genes. P-Selectin upregulation (40 folds) in cells harbouring BCR-ABL was seen as the most dramatic change in adhesion related molecules. Consequently, a monoclonal antibody against P-selectin inhibited adhesion capacity of BCR-ABL cells on M2-10B4. In conclusion, we describe an increased adhesion capacity of 32/BCR-ABL transformed cells which is associated with an up-regulation of P-selectin expression. Moreover, whilst 32D/BCR-ABL cells show a high potential to primarily attach to the stroma layer, physiological functions like cobblestone formation is impaired by a reduced migration and consecutively impaired homing potential of the cells into the supporting niches formed by the feeder cells.
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- 2006
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32. Inhibition of PDGFRβ by Imatinib Mesylate Suppresses Proliferation and Alters Differentiation of Human Mesenchymal Stem Cells In Vitro.
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Fierro, Fernando, Illmer, Thomas, Jing, Duhoui, Le Coutre, Philip, Ehninger, Gerhard, Boxberger, Sabine, and Bornhaeuser, Martin
- Abstract
Recent data show that the tyrosine kinase inhibitor Imatinib mesylate (IM) also affects normal hematopoietic stem cells (HSC), T lymphocyte activation and dendritic cell function not relying on the specific inhibition of bcr-abl activity. Mesenchymal stem cells (MSC) have been identified in the bone marrow (BM) as multipotent non-hematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of visceral mesoderm. MSC interact with HSC, influencing their homing and differentiation through cell-cell contact and the production of factors including chemokines We evaluated possible effects of IM in vitro on human bone marrow-derived MSC. Screening the activity of fourty-two receptor tyrosine kinases by a phospho-receptor tyrosine kinase (RTK)-array revealed an exclusive inhibition of platelet-derived growth factor receptor (PDGFRβ) by IM which consequently affects downstream targets of PDGFRβ as Akt and Erk1/2 signalling pathways in a concentration and time dependent manner. Furthermore, perinuclear multivesicular bodies harbouring PDGFRβ were found within 18–20 hours culture of MSC in the presence of 5 μM IM. Cell proliferation and clonogenicity (evaluated as the capability to form colony forming units - fibroblasts (CFU-F)) of MSC were significantly inhibited by IM in a concentration dependent fashion. IM inhibits significantly the differentiation process of MSC into osteoblasts as evaluated by decreased alkaline phosphatase activity and reduced calcium phosphate precipitates. In contrary, differentiation of MSC into adipocytes was strongly favoured in presence of IM. All these functional deficits described, probably contribute to an observed 50% reduction in the support of clonogenic hematopoietic stem cells, as evaluated by a long term culture-initiating cells (LTC-IC)-based assay. In summary our experiments show that IM inhibits the capacity of human MSC to proliferate and to differentiate into the osteogenic lineage, favouring adipogenesis. This effect is mainly mediated by an inhibition of PDGFRβ autophosphorylation leading to a more pronounced inhibition of PI3K/Akt compared to Erk1/2 signalling. This work confirms the role of PDGFRβ recently described for the proliferation and differentiation potential of MSC and provides a first possible explanation for the altered bone metabolism found in certain patients treated with IM.
- Published
- 2006
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