Servicio de Hematología, Hospital Clínico Universitario and Centro de Investigación del Cáncer (CIC); Servicio de Citometría, Universidad de Salamanca, Centro en Red de Medicina Regenerativa y ...Terapia Celular de Castilla y León, Spain
Correspondence: José A. Pérez Simón, Servicio de Hematología, Hospital Clínico Universitario y CIC, Paseo de San Vicente s/n, 37007, Salamanca, Spain. E-mail: pesimo{at}usal.es
Background: Mesenchymal stem cells are multilineage non-hematopoietic progenitor cells that play a key role in supporting the lymphohematopoietic system. Their distribution in bone marrow and secondary lymphoid organs allows an intimate interaction with T- and B-lymphocytes. While their effect on T-lymphocytes has been extensively analyzed, data on the effect of mesenchymal stem cells on B cells are more limited. We analyzed the effects of mesenchymal stem cells on B-lymphocytes and the pathways involved in these effects.
Design and Methods: The effect of MSC on the proliferation and viability of B cells was evaluated using MTT assays, annexin/7-amino-actinomycin D and propidium iodide staining. The B-cell maturation pattern was established using flow cytometry based on the expression of different markers related to the differentiation of B cells, such as CD38, CD138, CD19 and CCR7, and to the expression of surface and intracellular immunoglobulins. Finally, western blot assays were used to identify the pathways involved in the effects of mesenchymal stem cells on B-lymphocytes.
Results: Mesenchymal stem cells increased viability and blocked the cell cycle of B-lymphocytes in the G 0 /G 1 phase. In vitro exposure of B cells to plasmacytoid dendritic cells induced B-cell differentiation as shown by an increased number of CD38 ++ /CD138 ++ cells, which also displayed higher levels of cytoplasmic immunoglobulin and lower levels of CD19, CCR7 and surface immunoglobulin. Interestingly, this maturation pattern was inhibited by adding mesenchymal stem cells to the culture. Finally, mesenchymal stem cells modified the phosphorylation pattern of the extracellular response kinase 1/2 and p38 pathways which are both involved in B-cell viability, proliferation and activation.
Conclusions: Mesenchymal stem cells increase B-cell viability while inhibiting proliferation, arresting B-lymphocytes in the G 0 /G 1 phase of the cell cycle. The presence of mesenchymal stem cells blocked B-cell differentiation as assessed by flow cytometry. Finally, mesenchymal stem cells modified the activation pattern of the extracellular response kinase and the p38 mitogen-activated protein kinase pathways in B-lymphocytes.
Key words: mesenchymal stem cells, B lymphocytes, cell survival, B-cell differentiation.
To identify mesenchymal stromal cells (MSC) from degenerate human nucleus pulposus (NP) and compare them with bone marrow (BM) MSC.
To test whether MSC obtained from NP and BM from the same subjects ...share similar biologic characteristics.
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.
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.
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.
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.
MSCs products as well as their derived extracellular vesicles, are currently being explored as advanced biologics in cell-based therapies with high expectations for their clinical use in the next few ...years. In recent years, various strategies designed for improving the therapeutic potential of mesenchymal stromal cells (MSCs), including pre-conditioning for enhanced cytokine production, improved cell homing and strengthening of immunomodulatory properties, have been developed but the manufacture and handling of these cells for their use as advanced therapy medicinal products (ATMPs) remains insufficiently studied, and available data are mainly related to non-industrial processes. In the present article, we will review this topic, analyzing current information on the specific regulations, the selection of living donors as well as MSCs from different sources (bone marrow, adipose tissue, umbilical cord, etc.), in-process quality controls for ensuring cell efficiency and safety during all stages of the manual and automatic (bioreactors) manufacturing process, including cryopreservation, the use of cell banks, handling medicines, transport systems of ATMPs, among other related aspects, according to European and US legislation. Our aim is to provide a guide for a better, homogeneous manufacturing of therapeutic cellular products with special reference to MSCs.
Aims
Imatinib is considered the standard first‐line treatment in newly diagnosed patients with chronic‐phase myeloid leukaemia (CML). Several imatinib population pharmacokinetic (popPK) models have ...been developed. However, their predictive performance has not been well established when extrapolated to different populations. Therefore, this study aimed to perform an external evaluation of available imatinib popPK models developed mainly in adult patients, and to evaluate the improvement in individual model‐based predictions through Bayesian forecasting computed by each model at different treatment occasions.
Methods
A literature review was conducted through PubMed and Scopus to identify popPK models. Therapeutic drug monitoring data collected in adult CML patients treated with imatinib was used for external evaluation, including prediction‐ and simulated‐based diagnostics together with Bayesian forecasting analysis.
Results
Fourteen imatinib popPK studies were included for model‐performance evaluation. A total of 99 imatinib samples were collected from 48 adult CML patients undergoing imatinib treatment with a minimum of one plasma concentration measured at steady‐state between January 2016 and December 2020. The model proposed by Petain et al showed the best performance concerning prediction‐based diagnostics in the studied population. Bayesian forecasting demonstrated a significant improvement in predictive performance at the second visit. Inter‐occasion variability contributed to reducing bias and improving individual model‐based predictions.
Conclusions
Imatinib popPK studies developed in Caucasian subjects including α1‐acid glycoprotein showed the best model performance in terms of overall bias and precision. Moreover, two imatinib samples from different visits appear sufficient to reach an adequate model‐based individual prediction performance trough Bayesian forecasting.
Chimeric antigen receptor (CAR) T cells are a novel class of anti-cancer therapy in which autologous or allogeneic T cells are engineered to express a CAR targeting a membrane antigen. In Europe, ...tisagenlecleucel (Kymriah™) is approved for the treatment of refractory/relapsed acute lymphoblastic leukemia in children and young adults as well as relapsed/refractory diffuse large B-cell lymphoma, while axicabtagene ciloleucel (Yescarta™) is approved for the treatment of relapsed/refractory high-grade B-cell lymphoma and primary mediastinal B-cell lymphoma. Both agents are genetically engineered autologous T cells targeting CD19. These practical recommendations, prepared under the auspices of the European Society of Blood and Marrow Transplantation, relate to patient care and supply chain management under the following headings: patient eligibility, screening laboratory tests and imaging and work-up prior to leukapheresis, how to perform leukapheresis, bridging therapy, lymphodepleting conditioning, product receipt and thawing, infusion of CAR T cells, short-term complications including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, antibiotic prophylaxis, medium-term complications including cytopenias and B-cell aplasia, nursing and psychological support for patients, long-term follow-up, post-authorization safety surveillance, and regulatory issues. These recommendations are not prescriptive and are intended as guidance in the use of this novel therapeutic class.
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.
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.
Dasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), 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, αVβ3 integrin and CCR1 expression.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Bleeding is a frequent complication after allogeneic haematopoietic stem cell transplantation (HSCT) and may affect survival. The purpose of this study was to determine the incidence and risk ...factors for life‐threatening bleeding after HSCT by retrospective evaluation of 491 allogeneic HSCT recipients. With a median follow‐up of 33 months, 126 out of 491 allogeneic HSCT recipients experienced a haemorrhagic event (25·7%) and 46 patients developed a life‐threatening bleeding episode (9·4%). Pulmonary and gastrointestinal bleeding were the most common sites for life‐threatening bleeding, followed by central nervous system. In multivariate analyses, the presence of severe thrombocytopenia after day +28 and the development of grade III–IV acute graft‐versus‐host disease (GVHD) or thrombotic microangiopathy (TMA) retained their association with life‐threatening bleeding events. The overall survival at 3 years among patients without bleeding was 67·1% for only 17·1% for patients with life‐threatening bleeding (P < 0·001). In conclusion, life‐threatening bleeding is a common complication after allogeneic HSCT. Prolonged severe thrombocytopenia, acute grade III–IV GVHD and TMA were associated with its development.
Introduction
The ability of mesenchymal stromal cells (MSC) to suppress T‐cell function has prompted their therapeutic use for graft‐versus‐host disease (GVHD) control. However, as MSC also modulate ...the activity of NK cells, which play an important role in graft‐versus‐leukemia (GVL) reaction, their administration could hamper this beneficial effect of allogeneic hematopoietic stem cell transplantation. MSC can be expanded from several sources, especially bone marrow and fat, but it is not well established if the cell source makes a difference in their immunoregulatory capacity.
Objective
The aim of this study was to compare the immunomodulatory effect of MSC derived from bone marrow (BM‐CSM) or adipose tissue (AT‐MSC) on NK cells, to determine whether the use of MSC from one or the other origin could be more favorable to preserve NK cell activity and, therefore, GVL.
Methods
Human NK cells were stimulated with IL‐15 in the presence of BM‐MSC or AT‐MSC. The effect of both MSC populations on NK cell proliferation, cell cycle progression, and CD56 expression was analyzed by flow cytometry. Cytokine secretion was measured by ELISA, and cytotoxic activity was assessed by calcein release assays.
Results
Although both BM‐MSC and AT‐MSC induced a similar inhibition of NK cell proliferation, only BM‐MSC decreased significantly NK cell cytotoxic activity and showed a trend for a higher reduction of IFN‐γ secretion.
Conclusion
These results suggest that, in the context of GVHD inhibition, the use of AT‐MSC rather than BM‐MSC could further preserve NK cell activity and, thus, favor GVL.
Early hematopoiesis is a continuous process in which hematopoietic stem and progenitor cells (HSPCs) gradually differentiate toward specific lineages. Aging and myeloid malignant transformation are ...characterized by changes in the composition and regulation of HSPCs. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize an enriched population of human HSPCs obtained from young and elderly healthy individuals.
Based on their transcriptional profile, we identified changes in the proportions of progenitor compartments during aging, and differences in their functionality, as evidenced by gene set enrichment analysis. Trajectory inference revealed that altered gene expression dynamics accompanied cell differentiation, which could explain aging-associated changes in hematopoiesis. Next, we focused on key regulators of transcription by constructing gene regulatory networks (GRNs) and detected regulons that were specifically active in elderly individuals. Using previous findings in healthy cells as a reference, we analyzed scRNA-seq data obtained from patients with myelodysplastic syndrome (MDS) and detected specific alterations of the expression dynamics of genes involved in erythroid differentiation in all patients with MDS such as TRIB2. In addition, the comparison between transcriptional programs and GRNs regulating normal HSPCs and MDS HSPCs allowed identification of regulons that were specifically active in MDS cases such as SMAD1, HOXA6, POU2F2, and RUNX1 suggesting a role of these transcription factors (TFs) in the pathogenesis of the disease.
In summary, we demonstrate that the combination of single-cell technologies with computational analysis tools enable the study of a variety of cellular mechanisms involved in complex biological systems such as early hematopoiesis and can be used to dissect perturbed differentiation trajectories associated with perturbations such as aging and malignant transformation. Furthermore, the identification of abnormal regulatory mechanisms associated with myeloid malignancies could be exploited for personalized therapeutic approaches in individual patients.
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.
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