Myeloperoxidase oxidized LDL interferes with endothelial cell motility through miR-22 and heme oxygenase 1 induction: possible involvement in reendothelialization of vascular injuries.

Cardiovascular disease linked to atherosclerosis is the leading cause of death worldwide. Atherosclerosis is mainly linked to dysfunction in vascular endothelial cells and subendothelial accumulation of oxidized forms of LDL. In the present study, we investigated the role of myeloperoxidase oxidized LDL (Mox-LDL) in endothelial cell dysfunction. We studied the effect of proinflammatory Mox-LDL treatment on endothelial cell motility, a parameter essential for normal vascular processes such as angiogenesis and blood vessel repair. This is particularly important in the context of an atheroma plaque, where vascular wall integrity is affected and interference with its repair could contribute to progression of the disease. We investigated in vitro the effect of Mox-LDL on endothelial cells angiogenic properties and we also studied the signalling pathways that could be affected by analysing Mox-LDL effect on the expression of angiogenesis-related genes. We report that Mox-LDL inhibits endothelial cell motility and tubulogenesis through an increase in miR-22 and heme oxygenase 1 expression. Our in vitro data indicate that Mox-LDL interferes with parameters associated with angiogenesis. They suggest that high LDL levels in patients would impair their endothelial cell capacity to cope with a damaged endothelium contributing negatively to the progression of the atheroma plaque.

A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

BACKGROUND: Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation. METHODS: We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes. RESULTS: The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo. CONCLUSIONS: We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

Downregulation of microRNA-24 and -181 parallels the upregulation of IFN-γ secreted by activated human CD4 lymphocytes.

IFN-γ is a cytokine with important roles in the innate and adaptive immune responses. This cytokine is secreted by activated T cells, NK cells and macrophages. Studies on the regulation of human IFN-γ expression had been previously focused on the promoter region. Consequently, the role of microRNAs (miRs) in this regulation has not been investigated yet. As miR-24 and miR-181 were found to have potential target sites in IFN-γ mRNA 3'UTR, we assessed their impact on IFN-γ expression by co-stimulating PB CD4+ T cells with anti-CD3, anti-CD28, IL-12, and IL-18. This co-stimulation cocktail induced an abundant secretion of IFN-γ together with a down-regulation of miR-24, and miR-181. Existence of a link between these two phenomena was further substantiated by transfection and transduction assays that showed that these two miRs negatively regulate IFN-γ expression by directly binding to their target sites in the mRNA. Thus, identifying target sites for miR-24 and miR-181 in IFN-γ-3'UTR points to a novel regulatory mechanism of this crucial gene.

The impact of molecular biology techniques on the management of newly diagnosed chronic myeloid leukemia patients in chronic phase. A review.

Since the introduction of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), the management of this disease has completely changed. The aim has been first to bring to the patient a maximal response and to identify at different time-points what could be considered an optimal response (which is to render the progression free survival as important as possible). To achieve this, new molecular tools were needed, the most important being the real time quantitative PCR (RT-qPCR), to measure the number of remaining transcripts after several period of treatment. The second important tool was the sequencing of the BCR-ABL kinase domain to identify potential mutations giving rise to resistance to imatinib first and next to second generation TKIs. This technique, much more sensitive than cytogenetics, has allowed the definition of important levels of transcripts (the major molecular response i.e. a three log reduction and the complete molecular response i.e. a 4.5 log reduction) the first ensuring a long term PFS on treatment, the second allowing the birth of studies looking at whether it would be possible to discontinue the treatment in this group of patients.

Improved tolerability by a modified intermittent treatment schedule of dasatinib for patients with chronic myeloid leukemia resistant or intolerant to imatinib.

Intermittent dosing of dasatinib with a once daily regimen has been shown to reduce side effects while preserving clinical efficacy in early and advanced phase chronic myeloid leukemia (CML). Yet, hematologic toxicity and fluid retention demand a dose modification or treatment discontinuation in selected patients. Patients resistant or intolerant to imatinib were retrospectively evaluated based on the toxicity-guided administration of a dose-reduced dasatinib regimen. Patients were treated with an on/off regimen (3 to 5 days on, 2 to 4 days off) to allow regression of dasatinib-dependent off-target toxicity. Patients were followed up by routine hematologic and cytogenetic assessment and molecular monitoring to safeguard clinical response to the altered drug schedule. Thirty-three CML patients primarily in chronic phase with imatinib intolerance (n = 11) or resistance (n = 22) were investigated. Nonexclusive reasons for dose reduction were hematologic toxicity (17/33, 51%) and pleural effusions (18/33, 55%). On/off treatment with a weekend drug holiday significantly reduced pleural effusions and hematologic toxicity. Eighteen of 31 (58%) patients showed effective disease control despite reduced total weekly dasatinib doses, either demonstrated by achieving an improved response level (12/31) or keeping the response level achieved by conventional continuous dosing (6/31). Of note, 10/12 patients with subsequently improved response have been treated for a minimum of 6 months with continuous dosing dasatinib regimens without having achieved the response level achieved after allowing drug holiday. Weekend treatment interruption of dasatinib allows continuation of dasatinib treatment for patients suffering from side effects. These data mandate prospective investigation of alternative intermittent targeting regimens.

Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia.

BACKGROUND: MicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules whose deregulation of expression can contribute to human disease including the multistep processes of carcinogenesis in human. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as type 2 diabetes, pulmonary disease, colorectal cancer, and gastric cancer among others; however, defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that could serve as a biomarker for diagnosis or in the follow-up has not been done yet. METHODS: TaqMan miRNA microarray was performed to identify deregulated miRNAs in the plasma of AML patients. Quantitative real-time RT-PCR was used to validate the results. Receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s). RESULTS: The plasma expression level of let-7d, miR-150, miR-339, and miR-342 was down-regulated whilst that of let-7b, and miR-523 was up-regulated in the AML group at diagnosis compared to healthy controls. ROC curve analyses revealed an AUC (the areas under the ROC curve) of 0.835 (95% CI: 0.7119- 0.9581; P<0.0001) and 0.8125 (95% CI: 0.6796-0.9454; P=0.0005) for miR-150, and miR-342 respectively. Combined ROC analyses using these 2 miRNAs revealed an elevated AUC of 0.86 (95% CI: 0.7819-0.94; P<0.0001) indicating the additive effect in the diagnostic value of these 2 miRNAs. QRT-PCR results showed that the expression level of these two miRs in complete remission AML patients resembled that of healthy controls. CONCLUSIONS: Our findings indicated that plasma miR-150 and miR-342 are novel important promising biomarkers in the diagnosis of AML. These novel and promising markers warrant validation in larger prospective studies.

MicroRNA profile of circulating CD4-positive regulatory T cells in human adults and impact of differentially expressed microRNAs on expression of two genes essential to their function.

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

Differentiation associated regulation of microRNA expression in vivo in human CD8+ T cell subsets.

BACKGROUND: The differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown. METHODS: In order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway. RESULTS: We found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease. CONCLUSIONS: This study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions.

Cytotoxic responses to BLV tax oncoprotein do not prevent leukemogenesis in sheep.

Delta retrovirus-mediated leukemogenesis is dependent on the oncogenic potential of Tax. It is not clear, however, whether Tax-specific immune responses play a role in leukemia onset and progression. Using the BLV-associated leukemia model in sheep, we found that Tax-specific cytotoxic responses induced by DNA immunization or viral infection of naïve animals were not predictive of disease outcome and did not prevent tumor development. Furthermore, provirus and tax may be absent from blood for extended periods, emphasizing the relevance of surveying other compartments during chronic lymphoproliferative disorders. Our results support the conclusion that Tax-specific cytotoxic responses, even during the initial phase of infection, are not sufficient to prevent leukemogenesis.

Dendritic cells generated in clinical grade bags strongly differ in immune functionality when compared with classical DCs generated in plates.

Mature dendritic cells (DCs) represent, by far, the most potent antigen-presenting cells. The development of clinical grade techniques to produce them in large numbers has rendered possible their use in clinical trials. It is therefore crucial to assess the DCs characteristics according to the methodology used to generate them, to improve the comparison and standardization of these trials. We thus compared DCs generated and matured in culture plates (pla-DCs) or in clinical grade bags (bag-DCs) by analyzing, their secretion of bioactive interleukin (IL)-12 and their capacity to induce in-vitro primary responses. We also used several molecular techniques to better characterize the functional differences between the 2 type of DCs. Mature bag-DCs displayed a mature phenotype, but did not secrete significant amounts of IL-12 and failed to initiate primary immune responses. Molecular analyses performed on immature bag-DCs showed them already engaged in a particular maturation process (early activation of nuclear factor kappa B and beta-catenin). Using microarrays, we found underexpression of receptors for the maturation cocktail in bag-DCs. In mature bag-DCs, we found crucial genes (IL-12, chemokines, and costimulatory and adhesion molecules) down-regulated. Electrophoertic mobility shift assay and Western blots showed a normal activation profile in mature pla-DCs, but not in bag-DCs where the Mek/Erk pathway was still activated. Our results strongly suggest that differentiation of monocytes into DCs in bags generates immature DCs already engaged in an inefficient type of activation, with down-regulation of genes involved in response to the maturation cocktail. This results in mature DCs unable to induce T(H)1-type responses.

Valproate treatment of human cord blood CD4-positive effector T cells confers on them the molecular profile (microRNA signature and FOXP3 expression) of natural regulatory CD4-positive cells through inhibition of histone deacetylase.

Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.

Inflammation modifies the pattern and the function of Toll-like receptors expressed by human mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are involved in tissue repair and in the regulation of immune responses. MSC express Toll-like receptors (TLR) known to link innate and adaptive immunity. We hypothesized that TLR signaling could influence human MSC (hMSC) function. Here, we show that hMSC express TLR1, TLR2, TLR3, TLR4, TLR5, and TLR6 but not TLR7, TLR8, TLR9, and TLR10. In inflammatory conditions mimicked by culturing hMSC in an inflammatory environment, TLR2, TLR3, and TLR4 are upregulated, whereas TLR6 is downregulated. Interleukin (IL)-1 beta, IL-6, IL-12p35 and transforming growth factor-beta mRNAs are constitutively expressed by hMSC. Inflammation leads to an increase in IL-1 beta, IL-6, IL-12p35, and transforming growth factor-beta transcription and is characterized by IL-23p19 and IL-27p28 transcription. In this setting, poly(I:C) further augments IL-6, IL-12p35, IL-23p19, and IL-27p28 transcription, whereas lipopolysaccharide (LPS) increases IL-23p19 and IL-27p28 transcription. By upregulating TLR3 and TLR4 transcription, inflammation increases the hMSC responsiveness to LPS and poly(I:C), leading to a proinflammatory shift in their cytokine profile. The hMSC osteogenic potential does not change after TLR triggering but stimulation with LPS and poly(I:C) results in a decrease in their immunosuppressive capabilities. In conclusion, TLR activation in hMSC may affect their function and could modify their in vivo fate, especially in an inflammatory context.

Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have been shown to possess immunomodulatory functions and proposed as a tool for managing or preventing graft-versus-host disease (GvHD) as well as promoting clinical transplantation tolerance. We investigated the capacity of human bone marrow (BM) MSC to modulate the proliferation of T cells obtained from peripheral blood (PB) and umbilical cord blood (CB). We addressed the importance of the MSC:T-cell ratio, requirement for cell contact and impact of soluble factors on the MSC-mediated effects. We also analyzed whether regulatory T cells could be modulated by MSC in co-cultures. METHODS: The effect of different MSC concentrations on T-cell proliferation induced by allogeneic, mitogenic or CD3/CD28 stimulation was analyzed using bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) labeling. The level of regulatory T cells was assessed using quantitative real-time polymerase chain reaction (PCR) and flow cytometry analysis. RESULTS: MSC induced a dose- and contact-dependent inhibition of T-cell proliferation but lymphocytes from CB and PB were differentially affected. At low concentrations, MSC supported both CB and PB T-cell proliferation, rather than inhibiting their proliferation. This supportive effect was contact independent and soluble factors such interleukin-6 (IL-6) appeared to be involved. Interestingly, among the expanded T-cell population in both CB and PB, regulatory T cells were increased and were a part of the new cells promoted by MSC at low doses. CONCLUSIONS: MSC represent an attractive tool for reducing the lymphocyte response by inhibiting T-cell activation and proliferation as well as promoting tolerance by maintaining and promoting the expansion of regulatory cells. Nevertheless, the dual ability of MSC to either sustain or suppress T-cell proliferation according to conditions should be considered in the context of clinical applications.

Epigenetics and leukemia: unraveling oncogenic processes in the BLV ovine model.

Bovine Leukemia Virus (BLV)-induced B-cell leukemia in sheep is a valuable large animal model for investigating oncogenic mechanisms, particularly those associated with human T-cell leukemia virus 1 (HTLV-1). Multiple factors including viral genes, genetic and epigenetic alterations, and the host immune system are likely to contribute and cooperate in the leukemogenesis of adult T-cell leukemia (ATL) in human and B-cell leukemia in sheep. While considerable effort has been made to explore the role of viral determinants in the transformation process, the participation of host-related mechanisms has been poorly addressed. We discuss recent evidence from sheep studies in the context of the growing knowledge that has accumulated in the field of epigenetics in human cancer. These results support the hypothesis that epigenetic events, which were initially identified as a causative mechanism of virus silencing, are also major players in host gene regulation. Future studies in sheep will increase the number of genes identified that are aberrantly regulated by epigenetic processes and identify potential biomarkers which may be used as therapeutic targets in leukemia.

In vitro study of matrix metalloproteinase/tissue inhibitor of metalloproteinase production by mesenchymal stromal cells in response to inflammatory cytokines: the role of their migration in injured tissues.

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.

Human natural Treg microRNA signature: role of microRNA-31 and microRNA-21 in FOXP3 expression.

Treg are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate currently. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Treg and identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3' untranslated region (3' UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had an effect on FOXP3 expression levels. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3' UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Transduction of the remaining three miR had no direct effect on FOXP3 expression or on the phenotype and will remain the subject of future investigations. In conclusion, not only have we identified and validated a miR signature for human natural Treg, but also unveiled some of the mechanisms by which this signature was related to the control of FOXP3 expression in these cells.

Prevalence, determinants, and outcomes of nonadherence to imatinib therapy in patients with chronic myeloid leukemia: the ADAGIO study.

Imatinib mesylate (imatinib) has been shown to be highly efficacious in the treatment of chronic myeloid leukemia (CML). Continuous and adequate dosing is essential for optimal outcomes and with imatinib treatment possibly being lifelong, patient adherence is critical. The ADAGIO (Adherence Assessment with Glivec: Indicators and Outcomes) study aimed to assess prospectively over a 90-day period the prevalence of imatinib nonadherence in patients with CML; to develop a multivariate canonical correlation model of how various determinants may be associated with various measures of nonadherence; and to examine whether treatment response is associated with adherence levels. A total of 202 patients were recruited from 34 centers in Belgium, of whom 169 were evaluable. One-third of patients were considered to be nonadherent. Only 14.2% of patients were perfectly adherent with 100% of prescribed imatinib taken. On average, patients with suboptimal response had significantly higher mean percentages of imatinib not taken (23.2%, standard deviation [SD] = 23.8) than did those with optimal response (7.3%, SD = 19.3, P = .005; percentages calculated as proportions x 100). Nonadherence is more prevalent than patients, physicians, and family members believe it is, and therefore should be assessed routinely. It is associated with poorer response to imatinib. Several determinants may serve as alert signals, many of which are clinically modifiable.

Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA.

BACKGROUND: Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. DESIGN AND METHODS: In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. RESULTS: Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70(+) patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70(+) patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70(+) cells had better adhesion/migration capacities and only ZAP70(+) patient cells responded to microenvironment contact by CXCR4 downregulation. CONCLUSIONS: We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70(+) cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease.

Preemptive management of Epstein-Barr virus reactivation after hematopoietic stem-cell transplantation.

BACKGROUND: Epstein-Barr virus (EBV) reactivation after hematopoietic stem-cell transplantation can lead to posttransplant lymphoproliferative disease (PTLD), which carries a high mortality rate. Among therapeutic and prophylactic options being developed, B-cell depletion with monoclonal antibodies is encouraging. Because viral load after transplantation is correlated with PTLD occurrence, we developed a preemptive attitude based on polymerase chain reaction (PCR)-guided rituximab administration. METHODS: We monitored 115 transplant patients with a quantitative PCR for EBV DNA performed on whole-blood samples. Criteria for treatment initiation were a single PCR above 40,000 DNA genome copies per milliliter (gCop/mL) or two rising values above 10,000 gCop/mL. Weekly rituximab infusion at the dose of 375 mg/m was administered until negative PCR results were available. We evaluated the incidence of EBV reactivation and PTLD development. RESULTS: Nineteen patients (16.5%) met the criteria for treatment. Incidence of reactivation was the same in high-risk and standard-risk patients (12 vs. 7, P=0.38). One patient developed PTLD after discontinuation of therapy due to a serious adverse event. No other serious adverse events were noticed. Viral load disappeared after a median of three cycles of therapy, and weekly monitoring allowed prompt intervention. No PTLD-related death was observed, all-cause mortality in the treated population was 68%. CONCLUSIONS: Our PCR-guided and rituximab-based preemptive approach to avoid PTLD after allogeneic hematopoietic stem-cell transplantation is feasible but probably overtreated patients. Prospective trials are strongly needed, they should use uniform PCR techniques and consider higher threshold values for treatment initiation.