Ruxolitinib in combination with prednisone and nilotinib exhibit synergistic effects in human cells lines and primary cells from myeloproliferative neoplasms.

Ruxolitinib is the front-line non-palliative treatment for myelofibrosis (MF). However, a significant number of patients lose or present suboptimal response, are resistant or have unacceptable toxicity. In an attempt to improve response and avoid the adverse effects of this drug, we evaluated the combination of 17 drugs with ruxolitinib in ex vivo models of peripheral blood mononuclear cells from MF patients and cell lines. We found that the combination ruxolitinib and nilotinib had a synergistic effect against MF cells (ΔEC50 nilotinib, -21.6%). Moreover, the addition of prednisone to combined ruxolitinib/nilotinib improved the synergistic effect in all MF samples studied. We evaluated the molecular mechanisms of combined ruxolitinib/nilotinib/prednisone and observed inhibition of JAK/STAT (STAT5, 69.2+11.8% inhibition) and MAPK (ERK, 29.4+4.5% inhibition) signaling pathways. Furthermore, we found that the triple therapy combination inhibited collagen protein and COL1A1 gene expression in human bone marrow mesenchymal cells. Taken together, we provide evidence that combined ruxolitinib/nilotinib/prednisone is a potential therapy for MF, possibly through the anti-fibrotic effect of nilotinib, the immunomodulatory effect of ruxolitinib and prednisone, and the anti-proliferative effect of ruxolitinib. This combination will be further investigated in a phase Ib/II clinical trial in MF.

Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting.

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G(0) to G(1) phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.

Treatment with bortezomib of human CD4+ T cells preserves natural regulatory T cells and allows the emergence of a distinct suppressor T-cell population.

BACKGROUND: In vitro depletion of alloreactive T cells using the proteasome inhibitor bortezomib is a promising approach to prevent graft-versus-host disease after allogeneic stem cell transplantation. We have previously described the ability of bortezomib to selectively eliminate alloreactive T cells in a mixed leukocyte culture, preserving non-activated T cells. Due to the role of regulatory T cells in the control of graft versus host disease, in the current manuscript we have analyzed the effect of bortezomib in regulatory T cells. DESIGN AND METHODS: Conventional or regulatory CD4(+) T cells were isolated with immunomagnetic microbeads based on the expression of CD4 and CD25. The effect of bortezomib on T-cell viability was analyzed by flow cytometry using 7-amino-actinomycin D staining. To investigate the possibility of obtaining an enriched regulatory T-cell population in vitro with the use of bortezomib, CD4(+) T cells were cultured during four weeks in the presence of anti-CD3 and anti-CD28 antibodies, IL-2 and bortezomib. The phenotype of these long-term cultured cells was studied, analyzing the expression of CD25, CD127 and FOXP3 by flow cytometry, and mRNA levels were determined by RT-PCR. Their suppressive capacity was assessed in co-culture experiments, analyzing proliferation and IFN-gamma and CD40L expression of stimulated responder T cells by flow cytometry. RESULTS: We observed that naturally occurring CD4(+)CD25(+) regulatory T cells are resistant to the pro-apoptotic effect of bortezomib. Furthermore, we found that long-term culture of CD4(+) T cells in the presence of bortezomib promotes the emergence of a regulatory T-cell population that significantly inhibits proliferation, IFN-gamma production and CD40L expression among stimulated effector T cells. CONCLUSIONS: These results reinforce the proposal of using bortezomib in the prevention of graft versus host disease and, moreover, in the generation of regulatory T-cell populations, that could be used in the treatment of multiple T-cell mediated diseases.

Immunophenotypical, morphologic, and functional characterization of maturation-associated plasmacytoid dendritic cell subsets in normal adult human bone marrow.

BACKGROUND: Information about maturation of plasmacytoid dendritic cell precursors (pre-pDCs) in normal bone marrow (BM) remains limited. STUDY DESIGN AND METHODS: Immunophenotypical, morphologic, and functional changes associated with maturation of pre-pDCs were analyzed in adult normal human BM (n = 45). RESULTS: Three pre-pDC maturation stages, with an increasingly higher degree of maturity, were systematically identified: CD34++/HLA-DR++/+++/CD123++/CD45+/++ (Stage I), CD34+/HLA-DR+/++/CD123++/+++/CD45+/++ (Stage II), and CD34-/HLA-DR++/CD123++/+++/CD45++ (Stage III) cells. Lymphoid- and early myeloid-associated molecules, as well as CD86, were coexpressed in Stage I pre-pDCs, being down regulated afterward. CD304 and CD85j appeared in Stage I, progressively increasing their levels thereafter. Interestingly, cutaneous lymphocyte-associated antigen was heterogeneously expressed throughout the maturation, particularly in Stage I pre-pDCs. The morphologic appearance of Stage I pre-pDCs was consistent with their undifferentiated stage, while Stage II/III cells showed morphologic features of more differentiated cells. From the functional point of view, only Stage II and Stage III pre-pDCs were capable of inducing allogeneic T-cell proliferation, the later subset also showing interferon-g secretion; in contrast, Stage I pre-pDCs showed the highest endocytic ability. CONCLUSION: In summary, three maturation stages of pre-pDCs can be identified in adult normal BM, which show unique phenotypical, morphologic, and functional characteristics; these results provide a frame of reference for a better understanding of pDC malignancies.

A precision medicine test predicts clinical response after idarubicin and cytarabine induction therapy in AML patients.

Complete remission (CR) after induction therapy is the first treatment goal in acute myeloid leukemia (AML) patients and has prognostic impact. Our purpose is to determine the correlation between the observed CR/CRi rate after idarubicin (IDA) and cytarabine (CYT) 3 + 7 induction and the leukemic chemosensitivity measured by an ex vivo test of drug activity. Bone marrow samples from adult patients with newly diagnosed AML were included in this study. Whole bone marrow samples were incubated for 48 h in well plates containing IDA, CYT, or their combination. Pharmacological response parameters were estimated using population pharmacodynamic models. Patients attaining a CR/CRi with up to two induction cycles of 3 + 7 were classified as responders and the remaining as resistant. A total of 123 patients fulfilled the inclusion criteria and were evaluable for correlation analyses. The strongest clinical predictors were the area under the curve of the concentration response curves of CYT and IDA. The overall accuracy achieved using MaxSpSe criteria to define positivity was 81%, predicting better responder (93%) than non-responder patients (60%). The ex vivo test provides better yet similar information than cytogenetics, but can be provided before treatment representing a valuable in-time addition. After validation in an external cohort, this novel ex vivo test could be useful to select AML patients for 3 + 7 regimen vs. alternative schedules.

[Paroxysmal nocturnal hemoglobinuria]. / Hemoglobinuria paroxística nocturna.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic disorder characterized by the existence of somatic mutations in the PIG-A (phosphatidylinositolglycan complementation class A) gene, which encodes for a protein involved in the biosynthesis of the glycosyl phosphatidylinositol (GPI) molecule that serves as an anchor for many cell surface proteins. This genetic alteration translates into a total or partial deficiency in the PNH clone of surface proteins attached to the cell by a GPI anchor. Evaluation of deficient expression of GPI-associated proteins is currently used for the diagnosis of paroxysmal nocturnal hemoglobinuria. Among other proteins, deficiency of CD55 and CD59 leads to an increased susceptibility of PNH cells to complement-mediated cell lysis. Variable degrees of cytopenia, an increased susceptibility to infections and recurrent thrombotic events are other symptoms of the disease. In this paper we review the recent advances in the knowledge about the pathogenic mechanisms of the disease and the current approaches for the diagnosis and monitoring of PNH patients.

Quantitative analysis of the expression of glycosylphosphatidylinositol-anchored proteins during the maturation of different hematopoietic cell compartments of normal bone marrow.

BACKGROUND: Glycosylphosphatidylinositol-anchored proteins (GPI-AP) are a heterogeneous group of proteins deficiently expressed in patients with paroxysmal nocturnal hemoglobinuria. Up till now, no study has been reported in which the exact patterns of expression of a large number of GPI-AP are quantitatively evaluated in normal bone marrow (BM) cells classified according to their lineage and maturation stage. METHODS: In the present study, we have quantitatively analyzed the expression of eleven different GPI-AP (CD14, CD16, CD24, CD48, CD52, CD58, CD59, CD66b, CD87, CD109 and CD157) during maturation of the neutrophil, monocytic, erythroid, lymphoid, basophil and plasmacytoid dendritic cells (DC) lineages in normal BM as a frame of reference for the understanding of the abnormal patterns of expression of GPI-AP observed in the BM of PNH patients. RESULTS: Our results show that expression of most GPI-AP varies during normal BM maturation, different profiles being frequently observed depending on the cell lineage or the GPI-AP analyzed. CONCLUSION: Overall, these results provide a detailed map GPI-AP expression during normal hematopoietic differentiation, which could serve as a basis for the identification and characterization of changes occurring in PNH.

Normal patterns of expression of glycosylphosphatidylinositol-anchored proteins on different subsets of peripheral blood cells: a frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Evaluation of the expression of glycosylphosphatidylinositol-anchored membrane proteins (GPI-AP) is currently used for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). In this study, we analyzed the amount of expression of a wide variety of GPI-AP in different subsets of hematopoietic cells present in normal peripheral blood (PB), to establish their normal patterns of expression and provide a frame of reference for the definition of the best combination of GPI-AP and PB cell subsets to be applied in the diagnosis and monitoring of PNH. RESULTS: Our results show variable patterns of expression of different GPI-AP in distinct subsets of normal PB cells. Combined use of CD55 and CD59 represented the most useful dual-marker combination; however, its utility remained suboptimal for several subsets of leukocytes and for platelets. CONCLUSIONS: For some cell subsets such as the neutrophils additional useful markers could be selected from a relatively broad panel (CD16/CD24/CD55/CD59/CD66b/CD157), whereas for other cell subsets the number of useful antigens was either restricted (monocytes: CD14/CD55/CD157; B cells: CD24/CD48/CD52/CD55; CD4+ T cells: CD48/CD52/CD55; eosinophils: CD55/CD59; CD8+ T cells: CD48/CD55) or limited to a single marker (CD48 on CD56low NK cells, CD55 on BDCA3- dendritic cells and CD56high NK cells, and CD59 for red cells), from all antigens analyzed.

Pharmacological profiles of acute myeloid leukemia treatments in patient samples by automated flow cytometry: a bridge to individualized medicine.

BACKGROUND: We have evaluated the ex vivo pharmacology of single drugs and drug combinations in malignant cells of bone marrow samples from 125 patients with acute myeloid leukemia using a novel automated flow cytometry-based platform (ExviTech). We have improved previous ex vivo drug testing with 4 innovations: identifying individual leukemic cells, using intact whole blood during the incubation, using an automated platform that escalates reliably data, and performing analyses pharmacodynamic population models. PATIENTS AND METHODS: Samples were sent from 24 hospitals to a central laboratory and incubated for 48 hours in whole blood, after which drug activity was measured in terms of depletion of leukemic cells. RESULTS: The sensitivity of single drugs is assessed for standard efficacy (EMAX) and potency (EC50) variables, ranked as percentiles within the population. The sensitivity of drug-combination treatments is assessed for the synergism achieved in each patient sample. We found a large variability among patient samples in the dose-response curves to a single drug or combination treatment. CONCLUSION: We hypothesize that the use of the individual patient ex vivo pharmacological profiles may help to guide a personalized treatment selection.

Dasatinib as a bone-modifying agent: anabolic and anti-resorptive effects.

BACKGROUND: Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function. METHODS: For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model. RESULTS: Dasatinib inhibited the platelet derived growth factor receptor-ß (PDGFR-ß), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2-5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1-2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVß3 integrin and CCR1 expression. CONCLUSIONS: Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease.

Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus: comparison with bone marrow mesenchymal stromal cells from the same subjects.

STUDY DESIGN: To identify mesenchymal stromal cells (MSC) from degenerate human nucleus pulposus (NP) and compare them with bone marrow (BM) MSC. OBJECTIVE: To test whether MSC obtained from NP and BM from the same subjects share similar biologic characteristics. SUMMARY OF BACKGROUND DATA: Recent studies have proposed biologic strategies for the treatment of intervertebral disc degeneration, including cell therapy. Bone marrow (BM) MSC could be an attractive approach to restore disc function, and there is evidence that NP may contain MSC-like cells. METHODS: Tissue samples were obtained from degenerate lumbar NP and from iliac crest of the same 16 patients with degenerative disc diseases, undergoing discectomy and fusion procedures. MSC isolated from both sources were compared regarding their expansion time, immunophenotype, differentiation ability, and molecular analysis. RESULTS: In all cases, MSC from NP were isolated and expanded. They fulfil nearly all morphological, inmunophenotypical, and differentiation criteria described by the International Society of Cell Therapy for MSC, with the exception that NP-MSC are not able to differentiate into adipocytes. Slight differences were observed with BM-MSC from the same subjects. CONCLUSION: The NP contains mesenchymal stem cells. These cells were quite similar to mesenchymal stem cells from BM, with the exception of their adipogenic differentiation ability. These findings suggest that we may treat intervertebral disc degeneration by cell therapy (MSC from BM) and by stimulating endogenous MSC from NP.

Both CD133(+) cells and monocytes provide significant improvement for hindlimb ischemia, although they do not transdifferentiate into endothelial cells.

To address a number of questions regarding the experimental use of bone marrow (BM) stem cells in hindlimb ischemia, including which is the best cell type (e.g., purified hematopoietic stem cell or monocytes), the best route of delivery [intramuscular (IM) or intravenous (IV)], and the mechanism of action (transdifferentiation or paracrine effects), we have compared the neovascularization capacities of CD133(+) stem cells and monocytes (CD11b(+)) from the BM of Tie2-GFP mice either via IV or IM in a murine severe hindlimb ischemia model. To test the effect of cytokine administration, an extra group received BM conditioned medium. Peripheral blood flow as well as capillary density and GPF-positivity detection in ischemic muscles was evaluated 7, 14, and 21 days postinjection. In addition, CD133(+) and CD11b(+) cells from transgenic animals were cultured in vitro with angiogenic media for 7, 14, and 21 days to assess GFP expression. In all four cell-treated groups, blood flow and capillary density significantly recovered compared with the mice that received no cells or conditioned medium. There were no differences with respect to cell types or administration routes, with the exception of a faster flow recovery in the CD133(+)-treated cell group. We did not find GFP(+) cells in the ischemic muscles and there was no GFP expression after in vitro proangiogenic culture. Our study shows that both purified CD133(+) stem cells and myeloid mononuclear cells, either IM or IV administered, have similar neoangiogenic ability. Nevertheless, transdifferentiation into endothelial cells is not the mechanism responsible for their beneficial effect.

Detailed immunophenotypic characterization of different major and minor subsets of peripheral blood cells in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a deficient expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs), due to somatic mutations of the phosphatidylinositolglycan complementation Class A (PIG-A) gene. STUDY DESIGN AND METHODS: In this study, the expression of a high number of GPI-APs on different subsets of peripheral blood (PB) cells from 14 PNH patients and their potential association with underlying genetic abnormalities has been analyzed. RESULTS: This study confirms the existence of variable patterns of expression of different GPI-APs on both major and minor PB-cell subsets from PNH patients. The size of the PNH clone within PB neutrophils and monocytes was systematically higher than that of other cell populations. Genetic changes were detected in the PIG-A gene in 5 of 13 cases analyzed. Interestingly, the reactivity for many GPI-APs was significantly higher on different subsets of normal PB cells from PNH patients than those observed on healthy volunteers. CONCLUSION: The best combination of markers for the diagnostic screening of PNH would include evaluation of CD14 on monocytes and of CD16 on neutrophils, although further analysis of CD55 and CD59 expression may contain additional clinically useful information. Clear association between the genetic changes detected in the PIG-A gene in 5 of 13 cases analyzed, and the phenotypic profile of PNH cells has not been found. Additionally, an abnormally higher expression of several GPI-APs among normal residual cells from PNH patients in comparison to healthy donors was observed, suggesting that factors other than the PIG-A mutation could determine the phenotypic profile of PB cells in PNH.

Comparative analysis of different flow cytometry-based immunophenotypic methods for the analysis of CD59 and CD55 expression on major peripheral blood cell subsets.

BACKGROUND: Flow cytometry-based immunophenotypic techniques for the analysis of CD55 and CD59 expression on the major cell populations present in blood are the preferred method for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH). METHODS: In the present study, we comparatively analyze the effects of stain-lyse-and-then-wash techniques and lyse-wash-and-then-stain procedures on the detection of both CD55 and CD59 expression on the major peripheral blood (PB) leucocyte subsets, as analyzed by flow cytometry. Our major goal was to establish the minimum amounts of anti-CD55 and anti-CD59 reagents required to be added to a minimum volume of blood, which would allow an optimal staining for both antigens on red cells, platelets, and leucocytes present in a single tube. RESULTS: Our results show that upon comparing stain-lyse-and-then-wash techniques with lyse-wash-and-then-stain protocols, the presence of important amounts of red cells at the time peripheral blood leucocytes are stained for CD55 and CD59 is associated with a significantly (P < 0.01) lower and more heterogeneous pattern of antigen expression on almost all major PB leucocyte subsets, supporting the need to use red cell lysing procedures prior to the staining of leucocytes. Identical, optimal patterns of antigen staining for CD55 and CD59 were obtained upon incubating 3 microL of blood with 10 microL of each of these monoclonal antibody (mAb) reagents (protein concentration of 0.05 microg/microL and 0.2 microg/microL respectively) for 30 min (room temperature [RT]) using a non-lyse-non-wash sample preparation procedure. This latter procedure allowed for the simultaneous analysis of CD55 and CD59 expression on red cells, platelets, neutrophils, monocytes, and lymphocytes present in the sample through the combined staining of CD55 and CD59 with CD64-fluorescein isothiocyante (FITC) plus CD61-peridinin chlorophyll protein (PerCP) and CD45-PerCP. CONCLUSIONS: In summary, our results show that the sample preparation protocol has a significant impact on the quality of the staining obtained for the CD55 and CD59 antigens on the major PB leucocyte subsets; additionally, we propose a simple and reliable stain-non-lyse-non-wash method for the simultaneous analysis of CD55 and CD59 expression on PB red cells, platelets, neutrophils, monocytes, and lymphocytes, which could be reached through the use of two triple stainings.