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Background: Chronic lymphocytic leukemia (CLL) is the most common hematological malignancy in western countries, characterized by a heterogeneous clinical course. Although genetic studies have identified chromosomal aberrations or specific mutations, epigenetic changes have been poorly characterized in CLL., Methods: We assessed ten-eleven translocations (TET) 1, 2, and 3, isocitrate dehydrogenase (IDH) 1, and 2 messenger RNA (mRNA) expression using real-time PCR on purified leukemic B cells from 214 CLL patients (median follow-up = 75 months, range 1-380), normal peripheral blood B cells ( n  = 20), and umbilical cord blood B cells ( n  = 21). The microenvironment influence was assessed after 24 h co-culture of CLL cells with bone marrow mesenchymal stromal cells (BMSC). Finally, 5-hydroxymethylcytosine level (%5-hmC) was assessed by ELISA in CLL cells alone or with microenvironment stimuli., Results: TET 1 and 3 and IDH2 were decreased in CLL cells compared with healthy B cells ( P  = 0.0221, 0.0013, <0.0001, respectively), while IDH1 was overexpressed ( P  = 0.0037). TET2 and IDH1 were significantly correlated with treatment-free survival (TFS); patients with high TET2/IDH1 expression had a higher median TFS (111 months) than patients with low expression (78 months, P  = 0.0071/0.0123). Moreover, TET1 expression decreased ( P  = 0.0371), while TET3 and IDH2 expression increased ( P  = 0.0273/0.0039) in co-cultures. However, %5-hmC was not correlated with clinical data and was unchanged following microenvironment stimuli., Conclusions: Despite a slight deregulation in CLL cells compared with normal B cells, we identified a significant association between TET/IDH gene expression and prognosis, suggesting that epigenetic changes could potentially be associated with disease progression. Moreover, despite an identical %5-hmC, TET gene expression was influenced by contact with BMSC confirming the crucial role of the microenvironment in CLL pathogenesis.

Richter syndrome (RS) is associated with chemotherapy resistance and a poor historical median overall survival (OS) of 8-10 months. We conducted a phase II trial of standard CHOP-21 (cyclophosphamide, doxorubicin, vincristine, prednisolone every 21 d) with ofatumumab induction (Cycle 1: 300 mg day 1, 1000 mg day 8, 1000 mg day 15; Cycles 2-6: 1000 mg day 1) (CHOP-O) followed by 12 months ofatumumab maintenance (1000 mg given 8-weekly for up to six cycles). Forty-three patients were recruited of whom 37 were evaluable. Seventy-three per cent were aged >60 years. Over half of the patients received a fludarabine and cyclophosphamide-based regimen as prior CLL treatment. The overall response rate was 46% (complete response 27%, partial response 19%) at six cycles. The median progression-free survival was 6·2 months (95% confidence interval [CI] 4·9-14·0 months) and median OS was 11·4 months (95% CI 6·4-25·6 months). Treatment-naïve and TP53-intact patients had improved outcomes. Fifteen episodes of neutropenic fever and 46 non-neutropenic infections were observed. There were no treatment-related deaths. Seven patients received platinum-containing salvage at progression, with only one patient obtaining an adequate response to proceed to allogeneic transplantation. CHOP-O with ofatumumab maintenance provides minimal benefit beyond CHOP plus rutuximab. Standard immunochemotherapy for RS remains wholly inadequate for unselected RS. Multinational trials incorporating novel agents are urgently needed., (© 2016 John Wiley & Sons Ltd.)

Background: Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results., Materials and Methods: We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM)., Results: Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins)., Conclusion: We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by a heterogeneous clinical course. This variability in clinical course has spiked the search for prognostic markers able to predict patient evolution at the moment of diagnosis. Markers demonstrated to be of value are the mutation status of the immunoglobulin heavy chain variable region genes (IGHV) and lipoprotein lipase (LPL) expression. High LPL mRNA expression has been associated with short treatment free (TFS) and decreased overall survival (OS) in CLL. The LPL SNPs rs301 (T

In multiple myeloma (MM), bone marrow mesenchymal stromal cells (BM-MSCs) play an important role in pathogenesis and disease progression by supporting myeloma cell growth and immune escape. Previous studies have suggested that direct and indirect interactions between malignant cells and BM-MSCs result in constitutive abnormal immunomodulatory capacities in MM BM-MSCs. The aim of this study was to investigate the mechanisms that underlie these MM BM-MSCs abnormalities. We demonstrated that MM BM-MSCs exhibit abnormal expression of CD40/40L, VCAM1, ICAM-1, LFA-3, HO-1, HLA-DR and HLA-ABC. Furthermore, an overproduction of IL-6 (1,806 ± 152.5 vs 719.6 ± 18.22 ng/mL; p = 0.035) and a reduced secretion of IL-10 (136 ± 15.02 vs 346.4 ± 35.32 ng/mL; p = 0.015) were quantified in culture medium when MM BM-MSCs were co-cultured with T lymphocytes compared to co-cultures with healthy donor (HD) BM-MSCs. An increased Th17/Treg ratio was observed when T cells were co-cultured with MM BM-MSCs compared to co-cultures with HD BM-MSCs (0.955 vs 0.055). Together, these observations demonstrated that altered immunomodulation capacities of MM BM-MSCs were linked to variations in their immunogenicity and secretion profile. These alterations lead not only to a reduced inhibition of T cell proliferation but also to a shift in the Th17/Treg balance. We identified factors that are potentially responsible for these alterations, such as IL-6, VCAM-1 and CD40, which could also be associated with MM pathogenesis and progression.

MicroRNAs (or miRs) play a crucial role in chronic lymphocytic leukemia (CLL) physiopathology and prognosis. In addition, circulating microRNAs in body fluids have been proposed as new biomarkers. We investigated the expression of matched cellular and serum circulating microRNA-150 by quantitative real-time PCR (qPCR) from purified CD19(+) cells or from CLL serums obtained at diagnosis in a cohort of 273/252 CLL patients with a median follow-up of 78 months (range 7-380) and correlated it to other biological or clinical parameters. We showed that miR-150 was significantly overexpressed in CLL cells/serums compared with healthy subjects (P < 0.0001). Among CLL patients, a low cellular miR-150 expression level was associated with tumor burden, disease aggressiveness and poor prognostic factors. In contrast, a high level of serum miR-150 was associated with tumor burden markers and some markers of poor prognosis. Similarly, cellular and serum miR-150 also predicted treatment-free survival (TFS) and overall survival (OS) in an opposite manner: patients with low cellular/serum miR-150 levels have median TFS of 40/111 months compared with high-level patients who have a median TFS of 122/60 months (P < 0.0001/P = 0.0066). Similar results were observed for OS. We also found that cellular and serum miR-150 levels vary in an opposite manner during disease progression and that cellular miR-150 could be regulated by its release into the extracellular space. Cellular and serum levels of miR-150 are associated with opposite clinical prognoses and could be used to molecularly monitor disease evolution as a new prognostic factor in CLL.

Histone deacetylases (HDAC) play a crucial role in transcriptional regulation and are often deregulated in many cancers. However, global HDAC enzymatic activity has never been investigated in Chronic Lymphocytic Leukemia (CLL). We measured HDAC activity in protein extracts from CD19+ B-cells purified from 114 CLL patients with a median follow-up of 91 months (range: 11-376). HDAC activity was equivalent in CLL and normal B-cells but higher in patients who died during the study than in living patients (152.1 vs. 65.04 pmol; P = 0.0060). Furthermore, HDAC activity correlated with treatment-free survival (TFS; P = 0.0156) and overall survival (OS; P < 0.0001): patients with low HDAC activity (n = 75) had a median TFS and OS of 101 and > 376 months, respectively, whereas patients with high HDAC activity (n = 39) had a median TFS and OS of 47 and 137 months, respectively. Multivariate analyses indicated that HDAC activity is an independent predictor of OS (hazard ratio = 7.68; P = 0.0017). Finally, HDAC activity increased after B-cell receptor stimulation using IgM, suggesting a role for microenvironment stimuli (n = 10; P = 0.0371). In conclusion, high HDAC activity in CLL B-cells is associated with shorter TFS and OS and is an independent marker of OS, refining the use of other prognostic factors. This work provides a biological base for the use of HDAC inhibitors in CLL treatment.

Autologous mesenchymal stromal cell (MSC)-based therapies offer one of the most promising and safe methods for regeneration or reconstruction of tissues and organs. Routine procedures to obtain adequate amount of autologous stem cells need their expansion through culture, with risks of contamination and cell differentiation, leading to the loss of cell ability for therapies. We suggest the use of human bone marrow (BM) as a physiological bioreactor to produce autologous MSC by injection of autologous platelet-rich plasma activated by recombinant human soluble tissue factor (rhsTF) in iliac crest. A trial on 13 healthy volunteers showed the feasibility and harmlessness of the procedure. The phenotype and cellularity of BM cells were not modified, on day 3 after injection. Endothelial progenitor cells (EPC) were mobilized to the bloodstream, without stimulation of hematopoietic stem cells (HSC). MSC level in BM increased with a specific commitment to preosteoblastic cell population both in vivo and in vitro. This self-stimulation system of BM seems thus to be a promising feasible process 3 days before clinical cell therapy applications.

Background: In multiple myeloma, bone marrow mesenchymal stromal cells support myeloma cell growth. Previous studies have suggested that direct and indirect interactions between malignant cells and bone marrow mesenchymal stromal cells result in constitutive abnormalities in the bone marrow mesenchymal stromal cells., Design and Methods: The aims of this study were to investigate the constitutive abnormalities in myeloma bone marrow mesenchymal stromal cells and to evaluate the impact of new treatments., Results: We demonstrated that myeloma bone marrow mesenchymal stromal cells have an increased expression of senescence-associated β-galactosidase, increased cell size, reduced proliferation capacity and characteristic expression of senescence-associated secretory profile members. We also observed a reduction in osteoblastogenic capacity and immunomodulatory activity and an increase in hematopoietic support capacity. Finally, we determined that current treatments were able to partially reduce some abnormalities in secreted factors, proliferation and osteoblastogenesis., Conclusions: We showed that myeloma bone marrow mesenchymal stromal cells have an early senescent profile with profound alterations in their characteristics. This senescent state most likely participates in disease progression and relapse by altering the tumor microenvironment.

Histone deacetylases (HDACs) play a crucial role in chromatin structure and, consequently, gene expression. Their deregulation has been reported in various cancers. We performed a complete and comprehensive study of the expression of 18 HDACs (including Sirtuin; SIRT) by real-time PCR in a cohort of 200 chronic lymphocytic leukemia (CLL) patients with a median follow-up of 77 mo, and compared it with the results obtained from normal B cells. We also compared HDAC expression at diagnosis and after relapse. We observed significant deregulation (mostly upregulation) of HDACs in CLL. In terms of clinical significance, only HDAC6 was significantly correlated with treatment-free survival (TFS), whereas HDAC3 and SIRT2, 3 and 6 were correlated with overall survival (OS). A multivariate Cox regression stepwise analysis indicated that HDAC6, 7 and 10 and SIRT3 were TFS independent predictors. Interestingly, poor prognosis was associated with an overexpression of HDAC7 and 10 but an underexpression of HDAC6 and SIRT3. Therefore, these factors were combined in a TFS score: patients with a score of 0-1-2, 3 and 4 had a median TFS of 107, 57 and 26 mo, respectively (HR = 4.03, p < 0.0001). For OS, SIRT5 and 6 allowed stratification into 3 groups, with a median OS of > 360, 237 and 94 mo (HR = 6.38, p < 0.0001). However, we could not find statistical differences in HDAC expression after relapse. These results, validated by a 5-fold cross-validation, highlight the complex impact of HDAC expression in CLL clinical course.

Background: Interactions with the microenvironment, such as bone marrow mesenchymal stromal cells and nurse-like cells, protect chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis. This protection is partially mediated by the chemokine SDF-1α (CXCL12) and its receptor CXCR4 (CD184) present on the chronic lymphocytic leukemia cell surface., Design and Methods: Here, we investigated the ability of AMD3100, a CXCR4 antagonist, to sensitize chronic lymphocytic leukemia cells to chemotherapy in a chronic lymphocytic leukemia/mesenchymal stromal cell based or nurse-like cell based microenvironment co-culture model., Results: AMD3100 decreased CXCR4 expression signal (n=15, P=0.0078) and inhibited actin polymerization/migration in response to SDF-1α (n=8, P<0.01) and pseudoemperipolesis (n=10, P=0.0010), suggesting that AMD3100 interferes with chronic lymphocytic leukemia cell trafficking. AMD3100 did not have a direct effect on apoptosis when chronic lymphocytic leukemia cells were cultured alone (n=10, P=0.8812). However, when they were cultured with SDF-1α, mesenchymal stromal cells or nurse-like cells (protecting them from apoptosis, P<0.001), chronic lymphocytic leukemia cell pre-treatment with AMD3100 significantly inhibited these protective effects (n=8, P<0.01) and decreased the expression of the anti-apoptotic proteins MCL-1 and FLIP. Furthermore, combining AMD3100 with various drugs (fludarabine, cladribine, valproïc acid, bortezomib, flavopiridol, methylprednisolone) in our mesenchymal stromal cell co-culture model enhanced drug-induced apoptosis (n=8, P<0.05) indicating that AMD3100 could mobilize chronic lymphocytic leukemia cells away from their protective microenvironment, making them more accessible to conventional therapies., Conclusions: Taken together, these data demonstrate that interfering with the SDF-1α/CXCR4 axis by using AMD3100 inhibited chronic lymphocytic leukemia cell trafficking and microenvironment-mediated protective effects. Combining AMD3100 with other drugs may, therefore, represent a promising therapeutic approach to kill chronic lymphocytic leukemia cells.

The co-infusion of mesenchymal stromal cells (MSCs) with hematopoietic stem cells could improve the hematopoietic engraftment after cord blood transplant. Adult bone marrow is the major source of MSCs for cell therapy. However, bone marrow aspiration involves an invasive procedure and, in the case of a cord blood transplant, requires the use of a third party. The umbilical cord matrix, called Wharton's jelly (WJ), was previously shown to be a valuable source of MSCs. However, the process of cell separation is not standardized and needs to be optimized. In this study, we focused on the efficiency of the isolation procedure and expansion of cells from WJ MSCs isolated from human full-term umbilical cords. MSCs were isolated from the WJ without enzyme digestion or dissection. The procedure was based only on the plastic adhesion capacities of MSCs. Briefly, umbilical cord segments of 5-10 cm were cut longitudinally and plated with the WJ onto a plastic surface for 5 days in an appropriate culture medium. After removing the cord segment, the culture was pursued until subconfluency. The number of cells and their phenotypes, clonogenic capacities, differentiation capacities, immunomodulation, and hematopoietic supportive functions were evaluated. Using this method, we were able to isolate MSCs from all human umbilical cords analyzed (n = 50). We obtained a mean of 1.4 × 10(8) cells at the second passage and >7 × 10(9) cells at the third. The expanded cells expressed characteristic markers and presented typical functional properties of MSCs such as differentiation capacities, immunologic properties, and hematopoietic supportive functions. In conclusion, we have established a simple, rapid, and reproducible protocol to isolate abundant MSCs from short segments of umbilical cords.

Mesenchymal stromal cells (MSC) can be expanded from different sources. We compared the influence of inflammation and TLR ligation on the phenotype and function of MSC derived from bone marrow (BM), adipose tissue (AT), and Wharton's jelly (WJ). WJ-MSC were featured by a lack of TLR4 expression. While inflammation upregulated TLR3 in all three MSC types, TLR4 upregulation was observed only on BM-MSC. TLR ligation increased the production of inflammatory cytokines in BM- and AT-MSC but not in WJ-MSC and augmented anti-inflammatory cytokines in AT-MSC. Although inflammation increased in all MSC types the secretion of inflammatory cytokines, additional TLR triggering did not have further effect on WJ-MSC. The immunosuppressive potential of WJ-MSC on MLR was affected neither by inflammation nor by TLR triggering. This resistance was related to an overproduction of HGF. These data indicate that MSC source could be of importance while designing immunomodulating cell therapy in transplantation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: As mesenchymal stromal cells (MSCs) have been proposed as a tool for management or prevention of graft-vs-host disease, we investigated their immunoregulatory properties, their expression of adhesion molecules and galectin-1, and the impact of environment context on these functions., Materials and Methods: The effects of MSCs on T-cell proliferation were analyzed using carboxyfluorescein diacetate N-succinimidyl ester labeling. We evaluated the expression of adhesion molecules and galectin-1 by MSCs and the impact of an inflammatory or infectious environment on these expressions. Using neutralizing antibodies against adhesion molecules and a galectin-1 inhibitor, we assessed the role of these molecules in MSC functions., Results: MSCs inhibition of T-cell proliferation depended on MSC concentrations, cell contact, and culture environment. Expression of adhesion molecules and secretion of galectin-1 by MSCs are tightly regulated. Coculture with activated T cells upregulated expression of CD54 (intercellular adhesion molecule 1) and CD58 (lymphocyte function-associated antigen 3) and secretion of galectin-1 by MSCs. Interestingly, in an inflammatory or infectious environment, expression of adhesion molecules and galectin-1 by MSCs was differentially modulated. Furthermore, blocking galectin-1 activity prevented the suppressive potential of MSCs. Neutralization of adhesion molecule activity had no effect on MSC inhibition., Conclusion: Galectin-1 plays an important role in MSC immunoregulatory functions, which are depending on cell environment. The present study provides new insights concerning MSC physiology and will increase the safety and efficiency of MSCs in clinical settings., (Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Several markers have been proposed to predict the outcome of chronic lymphocytic leukemia (CLL) patients. However, discordances exist between the current prognostic factors, indicating that none of these factors are totally perfect., Methodology/principal Findings: Here, we compared the prognostic power of new RNA-based markers in order to construct a quantitative PCR (qPCR) score composed of the most powerful factors. ZAP70, LPL, CLLU1, microRNA-29c and microRNA-223 were measured by real time PCR in a cohort of 170 patients with a median follow-up of 64 months (range3-330). For each patient, cells were obtained at diagnosis and RNA was extracted from purified CD19 cells. The best markers were included in a qPCR score, which was thereafter compared to each individual factor. Statistical analysis showed that all five RNA-based markers can predict treatment-free survival (TFS), but only ZAP70, LPL and microRNA-29c could significantly predict overall survival (OS). These three markers were thus included in a simple qPCR score that was able to significantly predict TFS and OS by dividing patients into three groups (0/3, 1-2/3 and 3/3). Median TFS were >210, 61 and 24 months (P<0.0001) and median OS were >330, 242 and 137 months (P<0.0001), respectively. Interestingly, TFS results were also confirmed in Binet stage A patients (P<0.0001). When compared to other classical factors, this score displays the highest univariate Cox hazard ratio (TFS: HR=9.45 and OS: HR=13.88) but also provides additional prognostic information., Conclusions: In our hands, this score is the most powerful tool for CLL risk stratification at the time of diagnosis.

Aberrant expression of microRNAs has been recently associated with chronic lymphocytic leukemia (CLL) outcome. Although disease evolution can be predicted by several prognostic factors, a better outcome individualization in a given patient is still of utmost interest. Here, we showed that miR-29c and miR-223 expression levels decreased significantly with progression from Binet stage A to C were significantly lower in poor prognostic subgroups (defined by several prognostic factors) and could significantly predict treatment-free survival (TFS) and overall survival (OS). Furthermore, we developed a quantitative real-time polymerase chain reaction (qPCR) score combining miR-29c, miR-223, ZAP70, and LPL (from 0 to 4 poor prognostic markers) to stratify treatment and death risk in a cohort of 110 patients with a median follow-up of 72 months (range, 2-312). Patients with a score of 0/4, 1/4, 2/4, 3/4, and 4/4 had a median TFS of greater than 312, of 129, 80, 36, and 19 months, respectively (hazard ratio, HR(0/4 < 1/4 < 2/4 < 3/4 < 4/4) = 17.00, P < .001). Patients with a score of 0-1/4, 2-3/4, and 4/4 had a median OS of greater than 312, of 183 and 106 months, respectively (HR(0/4 < 1/4 < 2/4 < 3/4 < 4/4) = 13.69, P = .001). This score will help to identify, among the good and poor prognosis subgroups, patients who will need early therapy and thus will require a closer follow-up.

Background Aims: The transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production., Methods: The migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel in the presence and absence of stromal cell-derived (SDF)-1alpha, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels., Results: We have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli., Conclusions: Our observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.

Background: Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC) could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes., Methods: BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin). By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3., Results: Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin). Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin). Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3), NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate receptor channel (AMPA3) in the presence of two agonist glutamate, AMPA or CNQX antagonist., Conclusion: Our results demonstrate that BM-MSC have the potential to differentiate in neuronal cells with specific gene expression and functional properties. BM-MSC are thus promising candidates for cell-based therapy of neurodegenerative diseases.

Objective: Chronic lymphocytic leukemia (CLL) cells develop chemoresistance over time associated with defects in apoptosis pathway. Novel treatment strategies are required to overcome resistance of cells to commonly used agents. The effects of valproic acid (VPA), an antiepileptic drug with histone deacetylase inhibitory activity, on mononuclear cells isolated from 40 CLL patients were evaluated., Methods: CLL cells were treated with increasing doses of VPA (0.5, 1, 2, and 5 mM). The mode of cytotoxic drug action was determined by annexin binding, DNA fragmentation, and caspase activation., Results: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in the 40 CLL patients. VPA treatment induced apoptotic changes in CLL cells including phosphatidylserine externalization and DNA fragmentation. The mean apoptotic rates were similar between IgV(H) mutated and unmutated patients, the latter presenting a more aggressive clinical course. VPA induced apoptosis via the extrinsic pathway involving engagement of the caspase-8-dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA significantly increased sensitivity of leukemic cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and led to downregulation of c-FLIP (L) expression. VPA caused no potentialization of TRAIL-induced apoptosis on normal B cells. In addition, VPA overcame the prosurvival effects of bone marrow stromal cells., Conclusion: These findings point out that the combination of TRAIL and VPA, at clinically relevant concentration, may be valuable in the treatment of CLL.

Background: Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. The mutational status of the immunoglobulin variable heavy chain region (IGHV) has been used to classify patients into 2 groups in terms of overall survival (OS) and clinical characteristics, but the labor-intensive nature and the cost of this time-consuming analysis has prompted investigations of surrogate markers., Methods: We developed a standardized quantitative real-time reverse transcription-PCR (qPCR) method to measure zeta-chain (TCR)-associated protein kinase (ZAP70) mRNA in purified CD19(+) cells. We evaluated this and other methods (flow cytometry analyses of ZAP70 and CD38 proteins and qPCR analysis of lipoprotein lipase mRNA) in a cohort of 108 patients (median follow-up, 82 months) to evaluate any associations with IGHV mutational status, OS, and treatment-free survival (TFS)., Results: The association between qPCR-measured ZAP70 and IGHV mutational status was statistically significant [chi(2) (1) = 50.95; P <0.0001], and the value of Cramer's V statistic (0.72) indicated a very strong relation. This method also demonstrated sensitivity, specificity, and positive and negative predictive values of 87.8%, 85.7%, 87.5%, and 86%, respectively. ZAP70 expression was significantly associated with OS (P = 0.0021) and TFS (P <0.0001). ZAP70(+) patients had significantly shorter median TFS (24 months) than ZAP70(-) patients (157 months) (P <0.0001). Moreover, qPCR-measured ZAP70 expression has greater prognostic power than IGHV mutational status and the other prognostic markers tested., Conclusions: ZAP70 mRNA quantification via qPCR is a strong surrogate marker of IGHV mutational status and a powerful prognostic factor.