Chronic graft-versus-host disease could ameliorate the impact of adverse somatic mutations in patients with myelodysplastic syndromes and hematopoietic stem cell transplantation.

Somatic mutations in patients with myelodysplastic syndromes (MDS) undergoing allogeneic hematopoietic stem cell transplantation (HSTC) are associated with adverse outcome, but the role of chronic graft-versus-host disease (cGVHD) in this subset of patients remains unknown. We analyzed bone marrow samples from 115 patients with MDS collected prior to HSCT using next-generation sequencing. Seventy-one patients (61%) had at least one mutated gene. We found that patients with a higher number of mutated genes (more than 2) had a worse outcome (2 years overall survival [OS] 54.8% vs. 31.1%, p = 0.035). The only two significant variables in the multivariate analysis for OS were TET2 mutations (p = 0.046) and the development of cGVHD, considered as a time-dependent variable (p < 0.001), correlated with a worse and a better outcome, respectively. TP53 mutations also demonstrated impact on the cumulative incidence of relapse (CIR) (1 year CIR 47.1% vs. 9.8%, p = 0.006) and were related with complex karyotype (p = 0.003). cGVHD improved the outcome even among patients with more than 2 mutated genes (1-year OS 88.9% at 1 year vs. 31.3%, p = 0.02) and patients with TP53 mutations (1-year CIR 20% vs. 42.9%, p = 0.553). These results confirm that cGVHD could ameliorate the adverse impact of somatic mutations in patients with MDS with HSCT.

Mesenchymal Stromal Cell Irradiation Interferes with the Adipogenic/Osteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability.

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.

Intra-articular injection of two different doses of autologous bone marrow mesenchymal stem cells versus hyaluronic acid in the treatment of knee osteoarthritis: multicenter randomized controlled clinical trial (phase I/II).

BACKGROUND: Mesenchymal stromal cells are a promising option to treat knee osteoarthritis. Their safety and usefulness must be confirmed and the optimal dose established. We tested increasing doses of bone marrow mesenchymal stromal cells (BM-MSCs) in combination with hyaluronic acid in a randomized clinical trial. MATERIALS: A phase I/II multicenter randomized clinical trial with active control was conducted. Thirty patients diagnosed with knee OA were randomly assigned to intraarticularly administered hyaluronic acid alone (control), or together with 10 × 10(6) or 100 × 10(6) cultured autologous BM-MSCs, and followed up for 12 months. Pain and function were assessed using VAS and WOMAC and by measuring the knee motion range. X-ray and magnetic resonance imaging analyses were performed to analyze joint damage. RESULTS: No adverse effects were reported after BM-MSC administration or during follow-up. BM-MSC-administered patients improved according to VAS during all follow-up evaluations and median value (IQR) for control, low-dose and high-dose groups change from 5 (3, 7), 7 (5, 8) and 6 (4, 8) to 4 (3, 5), 2 (1, 3) and 2 (0,4) respectively at 12 months (low-dose vs control group p = 0.005 and high-dose vs control group p < 0.009). BM-MSC-administered patients were also superior according to WOMAC, although improvement in control and low-dose patients could not be significantly sustained beyond 6 months. On the other hand, the BM-MSC high-dose group exhibited an improvement of 16.5 (12, 19) points at 12 months (p < 0.01). Consistent with WOMAC and VAS values, motion ranges remained unaltered in the control group but improved at 12 months with BM-MSCs. X-ray revealed a reduction of the knee joint space width in the control group that was not seen in BM-MSCs high-dose group. MRI (WORMS protocol) showed that joint damage decreased only in the BM-MSC high-dose group, albeit slightly. CONCLUSIONS: The single intraarticular injection of in vitro expanded autologous BM-MSCs together with HA is a safe and feasible procedure that results in a clinical and functional improvement of knee OA, especially when 100 × 10(6) cells are administered. These results pave the way for a future phase III clinical trial. CLINICAL TRIALS: gov identifier NCT02123368. Nº EudraCT: 2009-017624-72.

[Bone marrow microenvironment in chronic myeloid leukemia: implications for disease physiopathology and response to treatment]. / Microambiente medular en la leucemia mieloide crónica: su relación con la enfermedad y la respuesta al tratamiento.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm related to the presence of the BCR-ABL1 fusion gene, linked to t (9;22) (q34;q11). It is originated from an abnormal hematopoietic stem cell, which is characterized as its normal counterparts by long-term self-renewal and multi-lineage differentiation. Both leukemic and quiescent normal hematopoietic stem cells preferentially reside in the osteoblastic niche. Mesenchymal stromal cells (MSC) are located near them, playing a critical role in their regulation. Currently, with tyrosine kinase inhibitor (TKI) therapy, long term clinical responses are achieved in most CML cases. However, late treatment failures may be observed related to the persistence of leukemic stem cells. The interactions between the leukemic stem cell and the microenvironment may be responsible in part for these events. We review the interactions between the leukemic stem cell and BM stroma and its potential clinical and therapeutic implications.

Analysis of incidence, risk factors and clinical outcome of thromboembolic and bleeding events in 431 allogeneic hematopoietic stem cell transplantation recipients.

Allogeneic hematopoietic stem cell transplantation recipients have an increasing risk of both hemorrhagic and thrombotic complications. However, the competing risks of two of these life-threatening complications in these complex patients have still not been well defined. We retrospectively analyzed data from 431 allogeneic transplantation recipients to identify the incidence, risk factors and mortality due to thrombosis and bleeding. Significant clinical bleeding was more frequent than symptomatic thrombosis. The cumulative incidence of a bleeding episode was 30.2% at 14 years. The cumulative incidence of a venous or arterial thrombosis at 14 years was 11.8% and 4.1%, respectively. The analysis of competing factors for venous thrombosis revealed extensive chronic graft-versus-host disease to be the only independent prognostic risk factor. By contrast, six factors were associated with an increased risk of bleeding; advanced disease, ablative conditioning regimen, umbilical cord blood transplantation, anticoagulation, acute III-IV graft-versus-host disease, and transplant-associated microangiopathy. The development of thrombosis did not significantly affect overall survival (P=0.856). However, significant clinical bleeding was associated with inferior survival (P<0.001). In allogeneic hematopoietic stem cell transplantation, significant clinical bleeding is more common than thrombotic complications and affects survival.

Prognostic impact of the number of methylated genes in myelodysplastic syndromes and acute myeloid leukemias treated with azacytidine.

The prognostic impact of the aberrant hypermethylation in response to azacytidine (AZA) remains to be determined. Therefore, we have analyzed the influence of the methylation status prior to AZA treatment on the overall survival and clinical response of myeloid malignancies. DNA methylation status of 24 tumor suppressor genes was analyzed by methylation-specific multiplex ligation-dependent probe amplification in 63 patients with myelodysplastic syndromes and acute myeloid leukemia treated with azacytidine. Most patients (73 %) showed methylation of at least one gene, but only 12 % of patients displayed ≥3 methylated genes. The multivariate analysis demonstrated that the presence of a high number (≥2) of methylated genes (P = 0.022), a high WBC count (P = 0.033), or anemia (P = 0.029) were independent prognostic factors associated with shorter overall survival. The aberrant methylation status did not correlate with the response to AZA, although four of the five patients with ≥3 methylated genes did not respond. By contrast, favorable cytogenetics independently influenced the clinical response to AZA as 64.7 % of patients with good-risk cytogenetic abnormalities responded (P = 0.03). Aberrant methylation status influences the survival of patients treated with AZA, being shorter in those patients with a high number of methylated genes.

Impaired expression of DICER, DROSHA, SBDS and some microRNAs in mesenchymal stromal cells from myelodysplastic syndrome patients.

UNLABELLED: Background Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cell-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze DICER1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS, a DICER1-related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. DESIGN AND METHODS: Mesenchymal stromal cells from 33 bone marrow samples were evaluated. DICER, DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. RESULTS: Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower DICER1 (0.65±0.08 vs. 1.91±0.57; P=0.011) and DROSHA (0.62±0.06 vs. 1.38±0.29; P=0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs. 1.15±0.28; P=0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR-222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. Conclusions This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where DICER1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.

Allogeneic mesenchymal stem cell therapy for refractory cytopenias after hematopoietic stem cell transplantation.

BACKGROUND: Posttransplant cytopenias are a severe complication after allogeneic stem cell transplantation (allo-SCT) and their origin is often multifactorial or unknown. They are frequently refractory to standard therapy, which may include steroids and/or immunoglobulins. Mesenchymal stem cells (MSCs) are an attractive therapeutic tool in the allo-SCT setting for the ability to enhance engraftment as well as acting as immunosuppressants for graft-versus-host disease. There is no prior experience in the literature of the use of MSCs to treat cytopenias after allo-SCT. CASE REPORTS: In this work we report for the first time four cases of refractory posttransplant cytopenias (three patients with thrombocytopenia and one with neutropenia) that were treated with MSCs from a third-party donor. MSCs were expanded from 100 mL of marrow obtained under standard good manufacturing practice conditions. Most patients received more than one cell dose, and median dose of MSCs administered was 1 × 10(6) /kg. RESULTS: All patients recovered normal blood counts, with a mean follow-up of 12.5 months. There were no adverse events related to MSC administration. CONCLUSION: MSC therapy may contribute to the recovery of refractory posttransplant peripheral cytopenias in patients undergoing allo-SCT.

Bone marrow transplantation extends its scope.

The term hematopoietic stem cell transplantation (HSCT) has completely replaced the most widespread bone marrow transplantation (BMT). This semantic change is based on the fact that not only hematopoietic stem cells with capacity for regenerating haematopoiesis and the immune system of the recipient are located in the BM. It was later observed that is possible to mobilise these cells into the peripheral blood, with the aid of certain cytokines, and then collect them through the process of aphaeresis. Moreover, hematopoietic stem cells from umbilical cord blood have been used successfully, and their use in on the increase. The main objectives of HSCT are, first, to substitute a defective haematopoietic system for a healthy one and, secondly, to allow the use of chemo and/or radiotherapy treatment at what would otherwise be supralethal doses, re-establishing haematopoiesis through the administration of haematopoietic progenitor cells. The complications of HSCT tend to be the result of the various factors including toxicity, release of certain cytokine, immunological processes associated with allo-HSCT (especially GVHD) and the effect of immunosuppressive drugs, as we discussed below.

Titanium and tantalum as mesenchymal stem cell scaffolds for spinal fusion: an in vitro comparative study.

INTRODUCTION: In the last few years, great interest has been focused on tissue engineering as a potential therapeutic approach for musculoskeletal diseases. The role of metallic implants for spinal fusion has been tested in preclinical and clinical settings. Titanium and tantalum have excellent biocompatibility and mechanical properties and are being used in this situation. On the other hand, the therapeutic role of mesenchymal stem cells (MSC) is extensively explored for their multilineage differentiation into osteoblasts. OBJECTIVES: In vitro comparison of titanium and tantalum as MSCs scaffolds. MATERIAL AND METHODS: In the present study, we have compared the in vitro expansion capacity, viability, immunophenotype (both explored by flow cytometry) and multi-differentiation ability of MSC cultured in the presence of either titanium or tantalum fragments. The adherence of MSC to either metal was demonstrated by electron microscopy. RESULTS: Both metals were able to carry MSC when transferred to new culture flasks. In addition, our study shows that culture of MSC with titanium or tantalum improves cell viability and maintains all their biological properties, with no significant differences regarding the metal employed. CONCLUSION: This would support the use of these combinations for clinical purposes, especially in the spinal fusion and reconstruction setting.

Multiparametric comparison of mesenchymal stromal cells obtained from trabecular bone by using a novel isolation method with those obtained by iliac crest aspiration from the same subjects.

Trabecular bone fragments from femoral heads are sometimes used as bone grafts and have been described as a source of mesenchymal progenitor cells. Nevertheless, mesenchymal stromal cells (MSC) from trabecular bone have not been directly compared with MSC obtained under standard conditions from iliac crest aspiration of the same patients. This is the ideal control to avoid inter-individual variation. We have obtained MSC by a novel method (grinding bone fragments with a bone mill without enzymatic digestion) from the femoral heads of 11 patients undergoing hip replacement surgery and compared them with MSC obtained by standard iliac crest aspiration of bone marrow from the same patients. We have shown that trabecular bone MSC obtained by mechanically fragmented femoral heads fulfil the immunophenotypic and multilineage (adipogenic, osteogenic and chondrogenic) differentiation criteria used to define MSC. We have also differentially compared cellular yields, growth kinetics, cell cycle assessment, and colony-forming unit-fibroblast content of MSC from both sources and conclude that these parameters do not significantly differ. Nevertheless, the finding of slight differences, such as a higher expression of the immature marker CD90, a lower expansion time through the different passages, and a higher percentage of cycling cells in the trabecular bone MSC, warrants further studies with the isolation method proposed here in order to gain further knowledge of the status of MSC in this setting.

Effects of imatinib mesylate on normal bone marrow cells from chronic myeloid leukemia patients in complete cytogenetic response.

Information on the effects of imatinib mesylate (IM) on the non-clonal bone marrow (BM) cell compartment is scanty. We have analyzed the gene expression profile of BM hematopoietic cells after IM therapy in 20 patients with chronic myeloid leukaemia (CML) in complete cytogenetic response (CCyR) and compared it with that of normal volunteer donors by oligonucleotide microarrays. In CCyR CML samples, IM induces a decrease in proliferation as well as increase in apoptosis and ubiquitination in residual non-clonal BM cells. In addition, IM diminishes cell-to-cell adhesion and downregulates the expression of the erythropoietin (EPO) receptor gene. The latter was confirmed by RT-PCR.

Prospective comparative analysis of the angiogenic capacity of monocytes and CD133+ cells in a murine model of hind limb ischemia.

BACKGROUND AIMS: The aim of this study was to compare prospectively the vasculogenic capacity of two cell sources, monocytes and CD133+ cells. METHODS: Cells were obtained from healthy donors by adherence or magnetic selection. Animals studies were performed in a model of hind limb ischemia and different groups were established according to type and number of cells infused. Revascularization was measured by sequential blood flow analysis using a laser Doppler device and by assessing capillary density in the ischemic muscles. In order to locate the infused cells, immunofluorescence and immunocytochemistry techniques were performed and analyzed by light and confocal microscopy. RESULTS: During the study period there was a significant improvement in both limb perfusion and capillary density in mice treated with either human monocytes or CD133+ cells (P<0.05) compared with non-treated mice. No cells were detected as incorporated into the vessels when 1 x 10(5) cells were used but with higher doses (1 x 10(6)) a few human cells were observed integrated into the vessels in both groups of treated mice. Supernatants of both cell types showed vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and platelet-derived growth factor- AB (PDGF-AB) expression. CONCLUSIONS: Treatment with human monocytes or CD133+ cells improves blood perfusion and capillary density in a murine model and both cell types seem to stimulate vasculogenesis in a fairly similar way.

Biocompatibility of two model elastin-like recombinamer-based hydrogels formed through physical or chemical cross-linking for various applications in tissue engineering and regenerative medicine.

Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein-based ones, such as elastin-like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix-like hydrogels through either physical or chemical cross-linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase-expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR-based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL-1ß, IL-4, IL-6, and IL-10 concentrations were measured by enzyme-linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine-related applications.

Posttransplant hematopoiesis in patients undergoing sibling allogeneic stem cell transplantation reflects that of their respective donors although with a lower functional capability.

We tested the principle of whether patient long-term hematopoiesis following allogeneic stem cell transplantation (allo-SCT) reflects the characteristics of the hematopoiesis of their respective donor. For this purpose, we analyzed bone marrow (BM) hematopoiesis using long-term cultures (LTC), delta assays, and clonogeneic assays as well as CD34+ cells and their subsets by flow cytometry in a series of 37 patients undergoing allo-SCT, and we compared it to that of their respective human leukocyte antigen-matched sibling donors in a paired study performed more than 1 year after the transplant procedure. Interestingly, the main factor that influenced post-allo-SCT BM hematopoiesis in the long term was donor hematopoiesis. Nevertheless, compared to their respective donors, patients exhibited a significantly lower number of colony-forming units granulomonocytic, burst-forming units erythroid, and immature progenitors (CD34++/CD38dim/CD90+/CD133+ cells, LTC-initiating cells, and colonies generated in the delta assay). Moreover, BM stromal function was diminished in patients undergoing allo-SCT compared to their donors. In addition, the presence of chronic graft-versus-host disease under immunosuppressive treatment also conditioned an impaired hematopoietic function. In summary, our study shows that BM hematopoiesis evaluated more than 1 year after an allo-SCT mainly reproduces that of their respective donors, although with a significantly decreased in vitro activity.

Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients.

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

Effect of pre-transplant cumulative doses of chemotherapeutic drugs on early and long-term hematological recovery after autologous bone-marrow transplantation for lymphoma.

BACKGROUND AND OBJECTIVES: It has recently been demonstrated that autologous bone-marrow transplantation (ABMT) is feasible in heavily pretreated patients who do not mobilize peripheral blood progenitor cells (PBPC), suggesting that bone marrow (BM) progenitor-cells are not as sensitive to chemotherapy as are PBPC. However, information regarding the impact of previous chemotherapy on the performance of BM grafts is scanty. DESIGN AND METHODS: We have retrospectively analyzed 40 consecutive lymphoma patients treated with the BEAM regimen and ABMT at our institution. The impact of the chemotherapeutic drugs (individual cumulative doses) received before transplant on stem-cell yield and hematologic recovery was investigated. Univariate analysis failed to identify any variable that significantly affected progenitor-cell content. RESULTS: Regarding the impact of pre-transplant chemotherapy on early engraftment, only cumulative doses of cytarabine (r=0.28, p=0.04) and cisplatin (r=0.32,p=0.02) had a negative influence on neutrophil recovery (to >0.5x10(9)/L), but the significance of this was not maintained in multivariate analysis. We did not find any chemotherapeutic drug that negatively affected platelet recovery (to >20x10(9)/L). By contrast, administration of several drugs, including doxorubicin, procarbazine, nitrogen mustard, cytarabine and cisplatin, significantly delayed complete trilineage reconstitution. In multivariate analysis, only previous doxorubicin retained statistical significance (p=0.014). INTERPRETATION AND CONCLUSIONS: Our results show that pre-transplant chemotherapy has little or no influence on progenitor-cell yield and short-term engraftment after ABMT. In contrast, we found that cumulative doxorubicin doses have an independent influence on long-term engraftment. In heavily pretreated lymphoma patients in whom poor PBPC mobilization is expected, BMT may represent an attractive option.

Long-term bone marrow culture data are the most powerful predictor of peripheral blood progenitor cell mobilization in healthy donors.

BACKGROUND AND OBJECTIVES: There is wide interindividual variation in progenitor cell mobilization. The present study was aimed to analyze steady state hematopoiesis in healthy donors and its influence on hematopoietic progenitor cell (HPC) mobilization. DESIGN AND METHODS: Bone marrow (BM) was aspirated from 72 healthy donors prior to administration of recombinant human granulocyte colony-stimulating factor (G-CSF). Analyses of CD34+ cells and semisolid cultures as well as long-term cultures were performed from BM or leukapheresis products. RESULTS: Male donors showed a higher number of BFU-E (p=0.007) and committed progenitors (p=0.05), a better stromal layer (p=0.02), and higher long-term bone marrow culture (LT-BMC) counts (p<0.05) when compared to those in female donors. When correlating the culture pattern of the BM with the data from the leukapheresis products, we observed that the number of the immature progenitors in BM correlated significantly with both the number of CD34 + cells and CFU-GM in the first leukapheresis. Univariate analysis revealed that the following variables had a beneficial impact on the number of CD34+ cells: male sex, body weight >73 Kg, G-CSF schedule and results of LT-BMC, although in the multivariate analysis only the number of CFU-GM obtained after LT-BMC showed a significant influence (p<0.001). INTERPRETATION AND CONCLUSIONS: These results confirm the interindividual variation in HPC mobilization among healthy subjects, with LT-BMC counts being the most reliable predictor, expressing the behavior of the immature progenitors and their relationship with the microenvironment.

Comparative effect of two pan-class I PI3K inhibitors used as anticancer drugs on human T cell function.

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer and, thus, PI3K has been recognized as an attractive molecular target for novel anti-cancer therapies. However, the effect of PI3K inhibitors on T-cell function, a key component of antitumor immunity, has been scantly explored. The objective of this study was to investigate the effect on human T-cell activation of two PI3K inhibitors currently being tested in clinical trials: PX-866 and BKM120. Their activity against a leukemic T cell line was also assessed. For that purpose, Jurkat cells or anti-CD3/anti-CD28 stimulated human peripheral blood mononuclear cells were cultured in the presence of different concentrations of PX-866 or BKM120 and their effect on T-cell proliferation, apoptosis, expression of activation markers and cytokine secretion was analyzed by flow cytometry. In addition, Akt and Erk phosphorylation was analyzed by Western blotting. Both PX-866 and BKM120 decreased viability of Jurkat cells and blocked cell cycle progression. Regarding primary T cells, both compounds similarly inhibited expression of activation markers and cytokine secretion, although they did not induce apoptosis of stimulated T cells. Interestingly, we found differences in their ability to block T-cell proliferation and IL-2 secretion, exerting BKM120 a more potent inhibition. These disparate effects could be related to differences observed in PI3K/Akt and RAS/MEK/ERK signaling between PX-866 and BKM120 treated cells. Our results suggest that, when selecting a PI3K inhibitor for cancer therapy, immunosuppressive characteristics should be taken into account in order to minimize detrimental effects on immune function.