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Heat shock proteins (HSPs) are molecular chaperones that assist proteins in their folding to native structures. HSPs are regarded as possible therapeutic targets in acute myeloid leukaemia (AML). We used bioinformatical approaches to characterize the HSP profile in AML cells from 75 consecutive patients, in addition to the effect of the HSP90 inhibitor 17-DMAG. Patients harbouring a FLT3-internal tandem duplication (FLT3-ITD) were extensively overrepresented in the cluster with high HSP levels, indicating a strong dependence of HSPs in stabilizing FLT3-ITD encoded oncoproteins. FLT3 ligation further increased the levels of HSP90 and its co-chaperone HSP70. HSP90 inhibition had a stronger pro-apoptotic effect for AML cells with FLT3-ITD than for cells with wild-type FLT3, whereas the anti-proliferative effect of HSP90 inhibition was similar for the two patient subsets. HSP90 inhibition altered the constitutive cytokine release profile in an anti-angiogenic direction independent of FLT3 mutational status: (i) pro-angiogenic CXCL8, MMP-2 and MMP-9 showed a stronger decrease than anti-angiogenic CXCL9-11, (ii) the Tie-2 agonist Ang-1 showed a stronger decrease than the potentially antagonistic Ang-2, and (iii) VEGF and HGF levels were decreased. Finally, HSP90 inhibition counteracted the leukaemia-stimulating effect of endothelial cells. Our studies demonstrate that HSP90 inhibition mediates anti-leukaemic effects through both direct and indirect activity.

Although TP53 mutations are rare in acute myeloid leukemia (AML), wild type p53 function is habitually annulled through overexpression of MDM2 or through various mechanisms including epigenetic silencing by histone deacetylases (HDACs). We hypothesized that co-inhibition of MDM2 and HDACs, with nutlin-3 and valproic acid (VPA) would additively inhibit growth in leukemic cells expressing wild type TP53 and induce p53-mediated apoptosis. In vitro studies with the combination demonstrated synergistic induction of apoptosis in AML cell lines and patient cells. Nutlin-3 and VPA co-treatment resulted in massive induction of p53, acetylated p53 and p53 target genes in comparison with either agent alone, followed by p53 dependent cell death with autophagic features. In primary AML cells, inhibition of proliferation by the combination therapy correlated with the CD34 expression level of AML blasts. To evaluate the combination in vivo, we developed an orthotopic, NOD/SCID IL2rγ(null) xenograft model of MOLM-13 (AML FAB M5a; wild type TP53) expressing firefly luciferase. Survival analysis and bioluminescent imaging demonstrated the superior in vivo efficacy of the dual inhibition of MDM2 and HDAC in comparison with controls. Our results suggest the concomitant targeting of MDM2-p53 and HDAC inhibition, may be an effective therapeutic strategy for the treatment of AML.

Endocan is a soluble proteoglycan expressed only by vascular endothelium and is also found circulating in the bloodstream. Inflammatory cytokines as well as proangiogenic growth factors increase its expression, and increased serum levels are found in immunocompetent patients with sepsis. We investigated serum endocan levels in patients with untreated acute myeloid leukemia (AML) and AML patients during chemotherapy-induced bone marrow failure. We observed increased levels in 40 AML patients compared with healthy controls, which was also confirmed in a second cohort. The serum levels decreased after intensive chemotherapy and subsequent severe chemotherapy-induced cytopenia, and increased levels were thereafter observed during bone marrow regeneration. However, even for these severely immunocompromized patients, serum endocan levels increased during complicating bacterial infections before a decrease was seen during antibiotic therapy. To conclude, serum endocan is a disease marker in AML, but serum levels are also affected by complicating infections and bone marrow regeneration.

BACKGROUND AIMS. Pro-angiogenic cytokines can affect myeloma cell proliferation directly and indirectly through stimulation of cancer-associated angiogenesis. METHODS. We investigated how peripheral blood stem cell (PBSC) collection affected plasma angioregulatory cytokine levels in 15 consecutive myeloma patients. RESULTS. Plasma levels of hepatocyte growth factor (HGF) were significantly increased prior to apheresis in patients compared with donors, and a further increase was detected immediately after PBSC apheresis. HGF levels decreased within 24 h, but were still higher than the levels in healthy donors, whose HGF levels were not altered by platelet apheresis. Pre-apheresis levels of other angioregulatory cytokines, angiopoietin-2 and vascular endothelial growth factor (VEGF), were also increased in patients, whereas angiopoietin-1, angiogenin and basic fibroblast growth factor levels did not differ from healthy controls. PBSC harvesting decreased angiopoietin-1 and VEGF levels, increased the microvascular endothelial cell marker endocan levels but did not affect the other mediators. CONCLUSIONS. Our results show that PBSC apheresis alters systemic angioregulatory profiles in myeloma patients. This cytokine modulation is not a general characteristic of all apheresis procedures and was not seen in healthy platelet donors.

Clinical and epidemiological studies have suggested the development of acquired immunity in individuals previously infected with Giardia lamblia. However, there are no data on the long-term cellular immunity and genotype cross-reactivity. An outbreak of assemblage B giardiasis in a nonendemic area made it possible to evaluate the long-term cellular mediated immunity and its specificity toward the 2 Giardia assemblages known to infect humans.Peripheral blood mononuclear cells from 19 individuals infected with Giardia assemblage B 5 years previously and from 10 uninfected controls were cultured with antigens from assemblage A and B Giardia trophozoites for 6 days. Cell-mediated immunity was measured by a (3)H-thymidine proliferation assay and flow cytometric analysis of activation markers HLA-DR, CD45RO, CD25, and CD26 in T-cell subsets.Proliferation responses were significantly elevated in the group previously exposed to Giardia for nearly all Giardia antigens tested. Individual responses toward Giardia trophozoite whole cell, cytosolic, and excretory-secretory antigens from both assemblages correlated well. Activation marker responses were mainly seen in CD4 T cells.G. lamblia infection induces long-term, albeit variable, cellular immune responses that are not assemblage specific and that are largely driven by CD4 T-cell activation.

Further improvements in allogeneic stem-cell transplantation will probably depend on a better balance between immunosuppression to control graft-versus-host disease and immunological reconstitution sufficient to ensure engraftment, reduction of infection-related mortality and maintenance of post-transplant antileukemic immune reactivity. The chemokine network is an important part of the immune system, and, in addition, CXCL12/CXCR4 seem to be essential for granulocyte colony-stimulating factor-induced stem-cell mobilization. Partial ex vivo graft T-cell depletion based on the expression of specific chemokine receptors involved in T-cell recruitment to graft-versus-host disease target organs may also become a future therapeutic strategy; an alternative approach could be pharmacological inhibition (single-receptor inhibitors or dual-receptor inhibitors) in vivo of specific chemokine receptors involved in this T-cell recruitment. Future clinical studies should therefore be based on a better characterization of various immunocompetent cells, including their chemokine receptor profile, both in the allografts and during post-transplant reconstitution.

Platelet transfusions are mainly used for patients with thrombocytopenia due to bone marrow failure, especially cancer patients developing severe chemotherapy-induced thrombocytopenia (e.g. patients with acute leukemia or other hematologic malignancies). A prophylactic transfusion strategy is now generally accepted in developed countries. Some clinical data, however, support the use of a therapeutic transfusion strategy at least for certain subsets of these patients. Several methodological approaches can then be used to evaluate the outcome of platelet transfusions, including peripheral blood platelet increments and bleeding assessments. Several factors will influence the efficiency of platelet transfusions; fever and ongoing hemorrhage are among the most important patient-dependent factors, but the number and quality of the transfused platelets are also important. The quality of transfused platelets can be evaluated by analyzing platelet activation, metabolism or senescence/apoptosis. Only evaluation of metabolism is included in international guidelines, but high-throughput methods for evaluation of activation and senescence/apoptosis are available and should be incorporated into routine clinical practice if future studies demonstrate that they reflect clinically relevant platelet characteristics. Finally, platelet transfusions have additional biological effects that may cause immunomodulation or altered angioregulation; at present it is not known whether these effects will influence the long-time prognosis of cancer patients. Thus, several questions with regard to the optimal use of platelet transfusions in cancer patients still need to be answered.

Platelet concentrates contain soluble mediators derived from both platelets and contaminating leukocytes. During platelet transfusion these mediators are transferred, and transfusion-induced modulation of the cytokine network may then occur, possibly contributing to transfusion reactions, immunomodulation, or affecting residual leukemic cells. In this prospective observational study, we investigate the effect of platelet transfusion on the systemic levels of platelet-derived cytokines, chemokines and interleukins in an unselected group of acute leukaemia patients with severe chemotherapy-induced cytopenia. We investigated 31 platelet transfusions involving pre-storage, white blood cell-reduced, gamma-irradiated or pathogen-inactivated, photochemically-treated platelet concentrates received by 10 unselected patients. Peripheral blood plasma samples were collected before, immediately after, one hour, and 24 hours after the transfusions. Sampling from platelet concentrates was performed immediately before transfusion. A total of 31 soluble mediators were examined. Ten healthy controls matched for age and gender were included. Despite heterogeneity in patients and platelet concentrates, significantly increased plasma concentrations were detected for the platelet-derived mediators, platelet-derived growth factor, β-thromboglobulin, transforming growth factor-β (TGF-β), CCL5, and CXCL4, 1 hour and/or immediately after platelet transfusions. The plasma levels of vascular endothelial growth factor and soluble CD40 ligand were not altered by platelet transfusion. Certain interleukins (IL-1β, IL-2, IL-4, IL-6, IL-9, and IL-12), as well as interferon-γ showed a minor, transient decrease in systemic plasma levels during the first hour following transfusion. Platelet transfusions modulate the systemic cytokine network in acute leukaemia patients with severe, chemotherapy-induced cytopenia.

Protein and gene networks centred on the regulatory tumour suppressor proteins may be of crucial importance both in carcinogenesis and in the response to chemotherapy. Tumour suppressor protein p53 integrates intracellular data in stress responses, receiving signals and translating these into differential gene expression. Interpretation of the data integrated on p53 may therefore reveal the response to therapy in cancer. Proteomics offers more specific data - closer to "the real action" - than the hitherto more frequently used gene expression profiling. Integrated data analysis may reveal pathways disrupted at several regulatory levels. Ultimately, integrated data analysis may also contribute to finding key underlying cancer genes. We here proposes a Partial Least Squares Regression (PLSR)-based data integration strategy, which allows simultaneous analysis of proteomic data, gene expression data and classical clinical parameters. PLSR collapses multidimensional data into fewer relevant dimensions for data interpretation. PLSR can also aid identification of functionally important modules by also performing comparison to databases on known biological interactions. Further, PLSR allows meaningful visualization of complex datasets, aiding interpretation of the underlying biology. Extracting the true biological causal mechanisms from heterogeneous patient populations is the key to discovery of new therapeutic options in cancer.

Matrix metalloproteinases (MMPs) comprise a large family of zinc-dependent endopeptidases, which are best known for their ability to degrade essentially all components of the extracellular matrix (ECM). By breaking down ECM, MMPs may remove physical barriers, thus allowing cells to migrate and potentially invade other tissues. Recent evidence, however, shows that the proteolytic activities of MMPs also affect several fundamental physiological processes. Primary human acute myeloid leukemia (AML) cells often show constitutive release of several MMPs and chemokines, and there seems to be a crosstalk between the MMP system and the chemokine network. Firstly, the nuclear factor-κB (NF-κB) system represents a common regulator at the transcriptional level both for MMPs (e.g. MMP-1 and MMP-9) and for the constitutive release of several chemokines (CCL2-4/CXCL1/8) by primary human AML cells. Secondly, the crosstalk at the molecular level probably includes MMP-mediated structural alteration and activation of constitutively released chemokines involved in AML cell migration (e.g. CXCL12) and stimulation of bone marrow angiogenesis (e.g. CXCL8). Thirdly, at a functional level the two systems interact because the chemokine network plays a role in similar physiological processes as the MMPs, including AML cell proliferation and migration and local regulation of angiogenesis. Both the chemokine system and MMPs are currently being evaluated as targets in anti-angiogenesis/cancer therapy and may also have potential therapeutic implications in AML. This review introduces the different members of the MMP family and describes their interactions with the chemokine network and the possible involvement of MMPs together with chemokines in leukemogenesis and chemosensitivity in AML.

NF-κB usually has antiapotptic effects and is involved in regulation of cell proliferation and intercellular communication. This is also true for the malignant cells in acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS), including the malignant stem cells. However, both AML and MDS patients are heterogeneous with regard to the effect of pharmacological NF-κB inhibition, and the final effect will probably also depend on the pharmacological agent used for the inhibition, e.g. proteasomal inhibitiors versus specific inhibitors. Even though initial studies suggest that NF-κB inhibitors have antileukemic effects, their future clinical use will also depend on their toxicity profile.

Disease-stabilizing therapy with the histone deacetylase inhibitor valproic acid and all-trans retinoic acid (ATRA) has been investigated in acute myelogenous leukemia (AML) in a number of trials. Experimental studies suggest that valproic acid induces a broad chemoresistant phenotype in human AML cells; however, clinical observations combining valproic acid with conventional therapy in a disease-stabilizing setting have not been reported that would confirm this as a clinical issue. We describe five patients receiving oral treatment with low-dose oral 6-mercaptopurin and/or hydroxyurea together with ATRA+valproric acid+theophylline. Hyperleukocytosis was controlled by low doses of the cytotoxic drugs, no unexpected toxicity appeared and the increases in normal peripheral blood cell counts induced by ATRA+valproic acid+theophylline were maintained during therapy. In two patients increasing blast counts later occurred during chemotherapy; a change to the alternative cytotoxic drug was then effective in controlling hyperleukocytosis. We conclude that valproic acid+ATRA+theophylline combined with 6-mercaptopurin or hydroxyurea can be safe and effective in palliative treatment of human AML.

Clonal heterogeneity detected by karyotyping is a biomarker associated with adverse prognosis in acute myeloid leukemia (AML). Constitutive activation of the phosphatidylinositol-3-kinase-Akt-mechanistic target of rapamycin (PI3K-Akt-mTOR) pathway is present in AML cells, and this pathway integrates signaling from several upstream receptors/mediators. We suggest that this pathway reflects biologically important clonal heterogeneity. We investigated constitutive PI3K-Akt-mTOR pathway activation in primary human AML cells derived from 114 patients, together with 18 pathway mediators. The cohort included patients with normal karyotype or single karyotype abnormalities and with an expected heterogeneity of molecular genetic abnormalities. Clonal heterogeneity reflected as pathway mediator heterogeneity was detected for 49 patients. Global gene expression profiles of AML cell populations with and without clonal heterogeneity differed with regard to expression of ectopic olfactory receptors (a subset of G-protein coupled receptors) and proteins involved in G-protein coupled receptor signaling. Finally, the presence of clonal heterogeneity was associated with adverse prognosis for patients receiving intensive antileukemic treatment. The clonal heterogeneity as reflected in the activation status of selected mediators in the PI3K-Akt-mTOR pathway was associated with a different gene expression profile and had an independent prognostic impact. Biological heterogeneity reflected in the intracellular signaling status should be further investigated as a prognostic biomarker in human AML. publishedVersion

Previous studies of T cell reconstitution following allogeneic stem cell transplantation have described long-lasting T cell defects, including decreased levels of autocrine proliferating CD4+ T cells. However, T cell functions during the early phase of conditioning-induced, pre-engraftment pancytopenia have not been characterized previously. We used a whole blood assay to investigate T cell proliferation and cytokine release during the period of pre-engraftment cytopenia. The study included 13 acute leukemia patients receiving myeloablative conditioning followed by transplantation of G-CSF-mobilised peripheral blood stem cells derived from HLA-matched family donors. Maximal proliferation and cytokine release could not be reached by anti-CD3 stimulation alone, but was dependent on the presence of additional costimulation with anti-CD28. Circulating T cells showed a broad cytokine release profile after activation, and the highest levels were detected for IFNgamma, GM-CSF and IL-6. Correlation analyses showed that TNFalpha/IL-4/IL-5/IL-13 in particular were released as a separate cluster, IFNgamma and GM-CSF correlated strongly, whereas IL-17 showed a weak correlation to IL-6 only. The capacity of circulating T cells derived during pre-engraftment cytopenia to release high levels of IFNgamma, IL-6 and IL-17 in response to in vitro activation with anti-CD3+anti-CD28 showed statistically significant correlations with later acute GVHD. We conclude that allotransplanted patients have a functional T cell system even during the pre-engraftment period of severe pancytopenia.

The diterpene ester ingenol-3-angelate (referred to as PEP005) is derived from the plant Euphorbia peplus. Crude euphorbia extract causes local toxicity and transient inflammation when applied topically and has been used in the treatment of warts, skin keratoses and skin cancer. PEP005 is a broad range activator of the classical (α, β, γ) and novel (δ, ε, η, θ) protein kinase C isoenzymes. Direct pro-apoptotic effects of this drug have been demonstrated in several malignant cells, including melanoma cell lines and primary human acute myelogenous leukemia cells. At micromolar concentrations required to kill melanoma cells this agent causes PKC-independent secondary necrosis. In contrast, the killing of leukemic cells occurs in the nanomolar range, requires activation of protein kinase C δ (PKCδ) and is specifically associated with translocation of PKCδ from the cytoplasm to the nuclear membrane. However, in addition to this pro-apoptotic effect the agent seems to have immunostimulatory effects, including: (i) increased chemokine release by malignant cells; (ii) a general increase in proliferation and cytokine release by activated T cells, including T cells derived from patients with chemotherapy-induced lymphopenia; (iii) local infiltration of neutrophils after topical application with increased antibody-dependent cytotoxicity; and (iv) development of specific anti-cancer immune responses by CD8(+) T cells in animal models. Published studies mainly describe effects from in vitro investigations or after topical application of the agent, and careful evaluation of the toxicity after systemic administration is required before the possible use of this agent in the treatment of malignancies other than skin cancers.

The Tie-2 receptor can bind its agonistic ligand Angiopoietin-1 (Ang-1) and the potential antagonist Ang-2. Tie-2 can be expressed both by primary human acute myeloid leukaemia (AML) cells and endothelial cells, and Tie-2-blocking antibodies are now being evaluated in clinical trials for cancer treatment.We investigated the effects of Tie-2-blocking antibodies, exogenous Ang-2 and pharmacological agents on AML cell proliferation and the release of angioregulatory mediators.Tie-2-blocking antibodies had a growth inhibitory effect on human AML cells co-cultured with microvascular endothelial cells, but this inhibition was not observed when leukaemic cells were co-cultured with fibroblasts or osteoblasts. AML cell viability in co-cultures was not altered by anti-Tie-2. Furthermore, anti-Tie-2 decreased hepatocyte growth factor (HGF) levels and increased CXCL8 levels in co-cultures, whereas the levels of endocan (a proteoglycan released by endothelial cells) were not altered. The only significant effects of exogenous Ang-2 were decreased levels of HGF and endocan. Constitutive AML cell release of agonistic Ang-1 was decreased by the proteasomal inhibitor bortezomib and the specific IkappaB-kinase/NFkappaB inhibitor BMS-345541.We conclude that various strategies for inhibition of Tie-2-mediated signalling should be considered in AML therapy, possibly in combination with other antiangiogenic strategies.

BACKGROUND: Autologous stem cell transplantation (ASCT) is used in the treatment of several malignancies. Harvesting sufficient peripheral blood progenitor cells (PBPCs) for a potential second autotransplantation at the time of relapse several years after diagnosis is becoming an increasingly common practice. STUDY DESIGN AND METHODS: Cryopreserved PBPCs were prepared with different concentrations of dimethyl sulfoxide (DMSO; 2, 4, 5, and 10%) and stored for at least 5 years before the recovery of CD34+ cells and various T- and natural killer (NK)-cell subsets were analyzed by flow cytometry. Furthermore, clinical variables for myeloma patients having a second autotransplantation with long-term-stored autografts were evaluated. RESULTS: The number of viable CD34+ cells in long-term-stored grafts was higher when autografts were cryopreserved with 4 or 5% than with 2 and 10% DMSO. The number of viable CD34+ cells was reduced by 13.9% after 5 years of cryostorage in 5% DMSO. Lymphocyte viability was also higher with 4 or 5% DMSO. However, the frequencies of several T-cell subsets showed DMSO-dependent differences, whereas NK-cell subsets did not. Furthermore, after a second autotransplantation with long-term-stored PBPC grafts at the time of myeloma relapse (median storage time, 42 months) all 17 patients reached neutrophil counts exceeding 0.5 × 109/L and platelet counts exceeding 20 × 109/L within 15 days. There was no difference in engraftment between patients receiving autografts preserved with 5 and 10% DMSO. CONCLUSION: PBPC autografts can safely be stored for at least 5 years in 5% DMSO and used for ASCT.

Intercellular communication participates in the regulation of normal hematopoiesis and possibly in neoplastic transformations in the hematopoietic niches. The role of gap junctions (GJs) and their connexins (Cxs) on hematopoietic tissues have been studied since the 1970s. Clearly, GJs are involved in different functional characteristics and connexin expression seems to be thoroughly regulated in hematopoietic tissues, suggesting that these molecules participate in the physiology of hematopoiesis. However, the exact mechanisms of GJs and Cxs are not characterized in detail. In this review we present the major findings of GJs and Cxs in normal hematopoietic tissues, and briefly discuss possible pathways of action and the potential of these molecules in hematopoietic reconstitution after cancer chemotherapy.

In an attempt to explore T-cell functions shortly after allogeneic bone marrow transplantation more fully, IL2- and IL4-dependent proliferation was assessed on CD4+ TCRαβ+ T-cell clones derived 4–6 weeks after transplantation. Both allogeneic pooled peripheral blood mononuclear cells and Epstein-Barr virus-transformed B-cell lines (BCL) could function as accessory cells (AC) for PHA activation of T-cell clones. Although minimal clonal proliferation was seen when the T-cell activation signal was BCL + PHA + IL4, a majority of the clones could undergo IL4-dependent proliferation after previous activation with AC + PHA + IL2. For certain clones, IL4 also showed an additive effect with IL2. Thus, IL4 was a growth factor for a majority of the investigated posttransplant T-cell clones, and in vivo modulation of IL4-dependent T-cell functions may thus become a future therapeutic possibility to enhance graft-versus-leukaemia effects in bone marrow transplant recipients.

The effects of insulin-like growth factor 1 (IGF-1) on autonomous proliferation, cytokine-dependent proliferation and constitutive cytokine secretion by acute myelogenous leukemia (AML) blasts were investigated using serum-free in vitro conditions. IGF-1 enhanced AML blast proliferation independent of the presence of other exogenous cytokines only for 2 of 21 patients, but for 10 additional patients IGF-1 altered blast proliferation in the presence of certain exogenous cytokines or cytokine combinations. IGF-1 had minor effects on AML blast cytokine secretion only for a subset of the patients (decreased levels for 1 patient, increased levels for 7 patients). Our in vitro observations indicate that IGF-1 can modulate AML blast proliferation and/or cytokine secretion for a subset of patients.

CD4+ and CD8+ TCR alpha beta+ T-cell clones were derived from 4 leukaemia patients early (4-6 weeks) after allogeneic bone marrow transplantation. Leukemia inhibitory factor (LIF) secretion in response to the activation signal accessory cells (AC) + phytohaemagglutinin (PHA) was investigated for each individual clone. Only a minority of CD4+ TCR alpha beta+ T-cell clones secreted LIF in response to AC + PHA, whereas most T-cell clones were capable of LIF secretion when exogenous interleukin 2 was added together with AC + PHA. LIF secretion could also be demonstrated for CD8+ TCR alpha beta+ posttransplant T-cell clones. Thus, posttransplant CD4+ and CD8+ TCR alpha beta+ clonogenic T-cells are capable of LIF secretion, and LIF secretion may be a T-cell effector mechanism in graft versus host disease, graft versus leukaemia effects or posttransplant haematopoietic reconstitution.

T lymphocyte functions in acute leukaemia patients with severe chemotherapy-induced leucopenia were investigated using 3 different approaches: (i) analysis of serum concentrations of the T cell cytokine interleukin 4 (IL4) demonstrated that serum IL4 levels increased during complicating bacterial infections. However, this response was modulated by a concomitant increase in serum levels of the potential IL4 antagonist soluble IL4 receptor alpha chain (sIL4R alpha). (ii) Even during leucopenia a subset of T lymphocytes derived from leucopenic patients expressed the activation markers CD25 (IL2 receptor), CD71 (transferrin receptor) and HLA-DR. (iii) Subsets of circulating CD4+ and CD8+ T lymphocytes could undergo clonogenic proliferation in vitro, and a majority of these clones secreted IL4. CD4+ clones showed higher IL4 levels than CD8+ clones. Our results indicate that T lymphocytes can be activated and contribute to cytokine responses in acute leukaemia patients with severe chemotherapy-induced cytopenia.

Blast cells derived from peripheral blood of patients with acute myelogenous leukaemia (AML) were cultured in vitro and interleukin 1 receptor antagonist (IL1RA) concentrations determined in culture supernatants. AML blasts derived from patients classified as AML-M4 and AML-M5 subtype showed an increased release of IL1RA. IL1 alpha and IL1 beta caused a similar increase in AML blast release of IL1RA, and addition of anti-IL1 antibodies decreased IL1RA release. IL1RA release from AML blasts was also increased by stem cell factor, tumour necrosis factor alpha (TNF alpha), granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor, whereas interleukin 3, interleukin 6, leukaemia inhibitory factor and granulocyte colony-stimulating factor did not significantly alter IL1RA release. When investigating IL1RA serum levels, serum concentrations were decreased in acute leukaemia patients with chemotherapy-induced cytopenia compared with healthy controls. Serum levels of both IL1RA as well as IL1 beta and soluble TNF alpha receptors increased when the leucopenic patients developed complicating bacterial infections.

Secretion of the potentially antileukaemic cytokines IFN-gamma and TNF-alpha was investigated for CD4+ and CD8+ TCR alpha beta + T-cell clones derived from 4 leukaemia patients 3-6 weeks after allogeneic BMT. We investigated cytokine secretion in response to the activation signal accessory cells+phytohaemagglutinin+Interleukin 2. All clones derived after BMT were capable of IFN-gamma and TNF-alpha secretion, and both for CD4+ (n = 96) and CD8+ (n = 8) T cells quantities of IFN-gamma and TNF-alpha were significantly correlated with one another. When comparing the overall results for posttransplant and normal T-cell clones derived from 2 bone marrow donors (n = 65), both CD4+ and CD8+ TCR alpha beta + T-cell clones showed increased IFN-gamma production, and CD4+ but not CD8+ clones showed a decreased TNF-alpha secretion. The results suggest that noncytotoxic T cells derived after allogeneic BMT can produce IFN-gamma and TNF-alpha and may thus be capable of mediating antileukaemic effects.

BACKGROUND: Autologous stem cell transplantation with cryopreserved autografts is a prerequisite for high-dose chemotherapy in treatment of several malignancies. Adverse effects due to the cryoprotectant dimethyl sulfoxide (DMSO) vary from mild to severe. DMSO-associated adverse effects can be reduced by DMSO depletion before autograft infusion. The aim was to investigate whether DMSO depletion by manual single wash reduced frequency of adverse effects or had detrimental effects on the engraftment potential of peripheral blood progenitor cell (PBPC) autografts. STUDY DESIGN AND METHODS: Ten percent DMSO was used to cryopreserve PBPC autografts for a total of 53 patients with multiple myeloma (n = 41), non-Hodgkin's lymphoma (n = 8), amyloidosis (n = 3), and polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes (POEMS) syndrome (n = 1). After high-dose chemotherapy, 34 patients received unmanipulated autografts, whereas for 19 patients the autografts were manually washed before stem cell infusion. Adverse effects after the infusion as well as neutrophil (neutrophil count >0.5 × 109/L) and platelet (PLT) engraftment (PLT count >20 × 109/L) for these two groups were compared. RESULTS: DMSO depletion reduced the frequency of adverse effects significantly. Patients transplanted with DMSO-depleted autografts had similar neutrophil engraftment time as patients receiving unmanipulated autografts. PLT engraftment time, however, was significantly prolonged and PLT transfusion requirements significantly increased for patients receiving DMSO-depleted autografts, even though the numbers of infused CD34+ cells per kg did not differ between the groups. CONCLUSIONS: DMSO depletion through a manual single wash is a time-consuming procedure that reduces adverse effects. Although the procedure leads to an increase of 2 days in PLT engraftment time, it can be recommended for selected patients with high risk of serious DMSO toxicity.

Bone marrow angiogenesis is suggested to play a role in the pathogenesis of acute myeloid leukaemia (AML) and endothelial cells may mediate chemosensitivity. This study investigated in vitro endothelial effects of coculture of microvascular endothelial cells (MVEC) with AML cells derived from 33 consecutive AML patients. A proliferation assay showed that (i) AML cells from the majority of patients examined increased endothelial cell proliferation, while cytokine neutralizing experiments had divergent effects on proliferation and (ii) the angiopoietin/Tie2 system was important for growth of AML cells, and angiopoietin-1 induced phosphorylation of signal transducers and activators of transcription (STAT) proteins in AML cells. Finally, gene expression profiling of MVEC cocultured with AML cells was conducted in non-contact cultures. Microarray analysis revealed that the majority of significantly expressed genes could be categorized into gene ontology terms involved in transcription, cellular organization and intracellular signalling. Our study indicates a role for the leukaemic-endothelium crosstalk in leukaemogenesis with enhancement of endothelial cell growth and increased AML cell proliferation possibly mediated by angiopoietin-1 and the STAT signalling pathway.

Normal T cells can mediate antileukemic reactivity after allogeneic stem cell transplantation and T cell targeting immunotherapy is now considered for patients receiving conventional chemotherapy. This antileukemic reactivity is most effective in patients with a low leukemia cell burden, and this burden is expected to be lowest early after transplantation/chemotherapy when patients are cytopenic. Local T cell recruitment will then be essential for the efficiency of the antileukemic response. In this context, the authors compared the chemokine receptor expression for T cells derived from healthy individuals and acute myelogenous leukemia patients with therapy-induced cytopenia after conventional chemotherapy or allogeneic stem cell transplantation. Circulating CD3(+) T cells showed the same chemokine receptor expression for all three groups: CCR1(low), CCR2(low), CCR3(low), CCR4(intermediate), CCR5(intermediate), CCR7(low/intermediate), CXCR2(low), CXCR3(intermediate), and CXCR4(high). Thus, only minor differences between the groups were observed when comparing individual receptors, and we therefore conclude that the chemokine receptor profiles of circulating CD3(+) T cells show no qualitative and only minor quantitative differences for these three groups.

Benzene, an aromatic hydrocarbon that is a natural component of crude oil and natural gas, is toxic to the blood and blood-forming organs. Epidemiological studies have established an association between benzene exposure and acute myeloid leukemia, and increasing evidence also indicates a possible association between benzene and multiple myeloma. A specific benzene-associated myelodysplastic syndrome has also been suggested. Chronic hematotoxic effects of benzene exposure, including reduced lymphocyte, neutrophil and platelet counts in peripheral blood, have been detected at occupational exposure below a level that had previously been considered not to cause any health effects. Whether these abnormalities represent bone marrow damage and/or initial events in the development of a true neoplastic disease is not known. Together with a reported nonlinear relationship between benzene exposure and the level of various metabolites, favoring production of biologically reactive quinones at exposure below 1 part per million, these observations suggest that benzene even at low exposure levels may contribute to the risk of acute myeloid leukemia or myelodysplastic syndrome, especially among genetically susceptible individuals. publishedVersion

Background Previous in vitro studies have demonstrated decreased apoptosis and necrosis in peripheral blood progenitor cells (PBPCs) cryopreserved with 5 percent instead of 10 percent dimethyl sulfoxide (DMSO). This study was carried out to investigate whether these in vitro findings were supported by clinical data concerning hematopoietic engraftment after autologous stem cell transplantations with PBPCs cryopreserved with 5 and 10 percent DMSO. Study design and methods During a 6-year period, 103 consecutive patients with newly diagnosed multiple myeloma (MM; n = 58) and lymphoma (n = 45) were transplanted with autologous PBPCs. Throughout the first part of the period cells were cryopreserved with 10 percent DMSO and later with 5 percent. A retrospective comparison was carried out of the clinical results for these two groups. Results No significant difference in median time to neutrophil and platelet (PLT) engraftment was demonstrated for MM and lymphoma patients transplanted with PBPCs cryopreserved with 5 or 10 percent DMSO. Time until neutrophil counts of more than 0.5 x 10(9) per L was 10 days both for the 5 and 10 percent MM groups and 12 days for both the 5 and the 10 percent lymphoma patients. Median time until stable PLT counts of more than 20 x 10(9) per L was 11 days in all four groups. In addition, transfusion requirements and duration of days admitted to hospital did not differ between the groups. Conclusion The routines for cryopreservation of autografts vary considerably between transplantation centers, and this makes it difficult to compare different clinical studies. Our results suggest that cryopreservation with 5 percent DMSO alone followed by storage in nitrogen is a simple, highly standardized, and safe procedure for cryopreservation of autologous stem cell graft.

Peripheral blood stem cell transplantation (PBSCT) is used to reconstitute normal hematopoiesis after myeloablative chemotherapy. The hematopoietic stem cells are collected from the blood by apheresis machines using density gradient centrifugation. Because of density similarities the grafts contain high levels of leukocytes and platelets that release various mediators into the grafts. The collected hematopoietic stem cells are therefore exposed to platelet released mediators including platelet-derived growth factor, transforming growth factor-beta, vascular endothelial growth factor and platelet factor-4. To investigate whether platelet activation and secretion can affect hematopoietic stem cells during PBSCT, we cultured (i) normal cord blood stem cells and (ii) mobilized peripheral blood stem cells from autografts together with the total secretion product of thrombin activated platelets (i.e. platelet released supernatants). Platelet released supernatants enhanced the cell proliferation of both peripheral blood stem cell (PBSC) autograft and normal cord blood CD34+ cells. Our study shows that platelet secretion in PBSCT autografts affect the hematopoietic stem cell function and possibly thereby the hematopoietic reconstitution after PBSCT.

Characterization of epigenetic events in carcinogenesis has led to the discovery of a new class of oncogenes and thereby a new class of therapeutic targets. Among the new therapeutic approaches are modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs). HDACs deacetylate histones as well as transcription factors and can modulate gene expression through both these mechanisms in normal and malignant cells. Furthermore, acetylation is an important posttranslational modulation of several proteins involved in the regulation of cell proliferation, differentiation and apoptosis in normal as well as cancer cells. Even though several HDAC inhibitors have been characterized in vitro, only a limited number of these agents are in clinical trials. Various HDAC inhibitors differ in their toxicity profile when comparing the side effects described in the available clinical studies of HDAC inhibition in the treatment of cancer. These drugs may also affect normal hematopoiesis; hematologic toxicity is common to many drugs but stimulation of hematopoiesis seems to occur for others. HDAC inhibitors usually affect < 10% of the genes in cancer cells. Divergent effects of HDAC inhibition on the global gene expression profiles have been described when testing various cancer cells, and this is further complicated by altered HDAC expression induced by HDAC inhibitors. However, increased p21 expression seems to be a common characteristic for most studies, suggesting an important role of this molecule during HDAC inhibitory treatment. Even though the initial studies are encouraging, additional in vitro and in vivo pharmacological characterization is definitely needed.

The balance between proangiogenic Angiopoietin-1 (Ang-1) and the antagonistic Ang-2 is important both for leukemogenesis and chemosensitivity in human acute myelogenous leukemia (AML). We examined the release of Ang-1 and Ang-2 by AML cells cultured alone and in cocultures with stromal cells. Detectable Ang-1 release from AML cells was observed for most patients (62/91), whereas Ang-2 release was detected only for a minority (23/91). Coculture of AML and stromal cells led to increased Ang-1 levels. Furthermore, the role of the angiopoietin system was investigated by characterizing whether the differences in angiopoietin expression in AML patients can be related to nucleophosmin (NPM1) mutations. We compared the gene expression profiles of AML cells derived from 19 patients with FLT3 mutations and normal cytogenetics with and without NPM1 mutations and observed increased expression of Ang-1 in patients with NPM1 mutations. Finally, we found significantly higher Ang-2 levels in serum of AML patients compared with healthy controls. Our results suggest that AML cells are a major source of Ang-1 in leukemic bone marrow, especially in patients with NPM1 mutations, but the local levels are also influenced by stromal cells. Local Ang-2 release from AML cells is less common, but high systemic levels of Ang-2 may affect bone marrow angioregulation.

Differentiation-inducing therapy with the DNA-methylation inhibitor Decitabine (5'-aza-deoxycytidine) and histone deacetylase (HDAC) inhibitors are now considered in acute myelogenous leukemia (AML). We investigated the in vitro effects of Decitabine and two structurally unrelated HDAC inhibitors (Sodium 4-phenyl butyrate, Tricostatin A) on clonogenic AML cells. Based on morphological criteria we identified four major colony types: (i) non-erythroid colonies, (ii) erythroid colonies that were detected only for a subset of patients and could be further sub classified into mature and immature forms, and (iii) intermediate colonies. Erythroid differentiation was associated with low CD34 expression. The colonies showed differences in morphology, viability, cell cycle distribution and expression of differentiation markers. Both Decitabine and the two HDAC inhibitors altered AML cell expression of differentiation markers, whereas the drugs did not have any major influence on cell cycle distribution. However, the pharmacological effects differed between the four colony subsets, and differences were also detected between the two HDAC inhibitors. We conclude that clonogenic AML cells can be classified into well-defined subsets based on their differentiation, and these subsets differ in their biological characteristics as well as their response to pharmacological targeting of epigenetic regulation.

T lymphocyte defects may contribute to the immune insufficiency seen in acute myelogenous leukemia (AML). We therefore characterized the T cell system for untreated AML patients. T lymphocyte subsets were analyzed by flow cytometry for 45 AML patients. The in vitro interferon-gamma (IFNgamma) release in response to stimulation with anti-CD3 plus anti-CD28 in the presence of autologous AML cells was examined for 31 patients. The majority of circulating lymphocytes were CD3(+)T cells, and CD19(+)B cells usually constituted10% of the lymphocytes. Most T cells expressed the alphabeta T cell receptor (TCRalphabeta(+)), and only a minority of the cells was TCRgammadelta(+). Both CD4(+) and CD8(+)T cells were detected, the CD4:CD8 ratio showed a wide variation but was generally1.0. The majority of CD4(+) and CD8(+)T cells were CD45RA(+) cells. The T cells could be stimulated to release IFNgamma in response to anti-CD3 plus anti-CD28 ligation even in the presence of excess autologous AML blasts, and for a subset of patients (6 of 27) these IFNgamma levels could be further increased by the novel protein kinase C (PKC) agonist PEP005. In conclusion, circulating T cells in patients with untreated AML are mainly CD4(+) or CD8(+) TCRalphabeta(+); both CD45RA(+) and CD45R0(+) can be detected, and these cells can be activated through the CD3/TCR complex even in the presence of excess AML cells. For a subset of patients T cell responsiveness can be further increased by targeting PKC and these data therefore suggest that T cell function is not inhibited in AML patients.

The use of gene expression arrays in the evaluation and classification of tumors is becoming increasingly important in a number of malignancies. This is a powerful technique able to disclose interpatient variance in gene expression. Such variation in gene expression may be the cause of different disease outcome and may reflect disease phenotypes or chemoresistance. Acute myeloid leukemia is a malignant disease of the bone marrow where overall long-term disease-free survival is less than 50%. The need for better disease classification and evaluation is consequently evident. Gene expression profiling in acute myeloid leukemia has, in recent years, proven able to distinguish acute myeloid leukemia subclasses and predict clinical outcome and is, as such, a promising technique for improved disease evaluation. The early detection of gene expression in response to chemotherapy may be a novel way of monitoring disease management. The immediate gene response may be an indication of whether the drug of choice is efficient in leukemic cell eradication and may early indicate the need for other therapeutic measures. Furthermore, these early alterations in gene expression could facilitate identification of new treatment targets, thereby enabling better patient care and follow-up in the future.

Background and Objectives Chemokines are soluble mediators involved in angiogenesis, cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells.Design and Methods Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation, colony formation), chemokine receptor expression, constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells.Results Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors, but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression, but, compared to CD34− AML cells, CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore, a broad constitutive chemokine release profile was detected for most patients, and the following chemokine clusters could be identified: CCL2-4/CXCL1/8, CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFκB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased.Interpretation and Conclusions Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile.

Proteasome inhibitors represent a new class of antineoplastic drugs that are considered in the treatment of haematological malignancies. We compared the effects of the reversible proteasome inhibitor bortezomib (Velcade) and the epoxomicin derivative PR-171, an irreversible inhibitor, on primary human acute myeloid leukaemia (AML) cells. Both drugs inhibited autocrine- and cytokine-dependent proliferation of primary AML blasts when tested at nanomolar levels (0.1-100 nmol/l). The antiproliferative effect was independent of basal chymotrypsin-like proteasome activity (showing a 20-fold variation between patients), genetic abnormalities, morphological differentiation and CD34 expression when testing a large group of consecutive patients (n = 54). The effect was retained in cocultures with bone marrow stromal cells. In addition, both drugs enhanced apoptosis. The effect of PR-171 could be detected at lower concentrations than for bortezomib, especially when testing the influence on clonogenic AML cell proliferation. Both drugs had divergent effects on AML cells' constitutive cytokine release. Furthermore, both drugs caused a decrease in proliferation and viability when tested in combination with idarubicin or cytarabine. An antiproliferative effect on primary human acute lymphoblastic leukaemia cells was also detected. We conclude that nanomolar levels of the proteasome inhibitors tested had dose-dependent antiproliferative and proapoptotic effects on primary AML cells in vitro.

Allogeneic stem cell transplantation is used in the treatment of younger patients with severe hematological diseases, especially hematological malignancies, and acute graft versus host disease (GVHD) is then an important immune-mediated posttransplant complication. Several risk factors for acute GVHD have been identified, including pretransplant factors that possibly influence the posttranspant course through their effects on host immunocompetent cells. Metabolic regulation is important for immunoregulation, and we therefore investigated whether the pretransplant metabolic status of allotransplant recipients was associated with later acute GVHD. In our population-based study we investigated the systemic (serum) metabolic profile for 86 consecutive allotransplant recipients. The samples were collected before start of the pretransplant conditioning therapy. Patients who developed later acute GVHD especially showed altered pretransplant amino acid metabolism, including (1) altered metabolism of immunoregulatory branched chain amino acids (leucine, isoleucine and valine); and (2) altered levels of potentially proinflammatory tyrosine metabolites (p-cresol sulphate, 3-phenylpropionate) formed by the gastrointestinal microbial flora. However, isobutyrylcarnitine and propyonylcarnitine levels were also altered; the carnitines are important for the transport of fatty acids and may also be important for the release of immunoregulatory cytokines in allotransplant recipients. These metabolic alterations were associated with an ongoing pretransplant acute phase reaction or early hematopoietic/immune reconstitution. Thus, allotransplant recipients developing acute GVHD showed altered preconditioning/pretransplant levels of several immunoregulatory metabolites. Our hypothesis is that these metabolites alter or activate recipient immunocompetent cells and thereby enhance or initiate anti-recipient immune reactivity. Electronic supplementary material The online version of this article (doi:10.1007/s11306-015-0880-x) contains supplementary material, which is available to authorized users.

A range of cytokines are altered during sepsis. Multiplex analysis of inflammatory mediators makes it easier to study a larger range of mediators, and combined with newer bioinformatical tools (unsupervised hierarchical clustering), this has the potential to bring new knowledge of the inflammatory process during sepsis.

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow disorders, with dysplasia, cytopenias and increased risk of progression to acute myeloid leukemia. A dysregulated immune system precipitates MDS, and to gain insights into the relevance of cytotoxic T lymphocyte (CTL) in this process, we examined the frequency and function of CX3CR1- and CD57-positive T lymphocytes from MDS patients. Peripheral blood and/or bone marrow samples from 31 MDS patients and 12 healthy controls were examined by flow cytometry. Expression of cytotoxic granule constituents, immunological co-receptors, adhesion molecules and markers of activation were quantified on unstimulated lymphocytes. Degranulation, cytotoxicity and conjugate formation with target cells following co-culture of CTL with target cell lines or autologous bone marrow-derived CD34+ cells were quantified by flow cytometry. CX3CR1 expression was increased in bone marrow from high-risk MDS patients compared to healthy controls. Expression of CD57 and CX3CR1 was closely correlated, identifying a CTL subset with high cytotoxic capacity. In vitro, TCR-induced redirected cytotoxicity was markedly decreased for high-risk MDS patients compared to controls. CTL from MDS patients with the lowest target cell cytotoxicity had reduced expression of adhesion molecules and formed fewer conjugates with target cells. Although phenotypically defined CTL numbers were increased in the bone marrow of MDS patients, we found that CTL from high-risk MDS patients exhibited a lower TCR-induced redirected cytotoxic capacity. Thus, decreased T cell cytotoxicity seems related to reduced adhesion to target cells and may contribute to impaired anti-leukemic immune surveillance in MDS.

Background Cytarabine is used in the treatment of acute myeloid leukemia (AML). Low-dose cytarabine can be combined with valproic acid and all-trans retinoic acid (ATRA) as AML-stabilizing treatment. We have investigated the possible risk of immunotoxicity by this combination. We examined the effects of cytarabine combined with valproic acid and ATRA on in vitro activated human T cells, and we tested cytarabine at concentrations reached during in vivo treatment with high doses, conventional doses and low doses. Methods T cells derived from blood donors were activated in vitro in cell culture medium alone or supplemented with ATRA (1 μM), valproic acid (500 or 1000 μM) or cytarabine (0.01-44 μM). Cell characteristics were assessed by flow cytometry. Supernatants were analyzed for cytokines by ELISA or Luminex. Effects on primary human AML cell viability and proliferation of low-dose cytarabine (0.01-0.5 μM) were also assessed. Statistical tests include ANOVA and Cluster analyses. Results Only cytarabine 44 μM had both antiproliferative and proapoptotic effects. Additionally, this concentration increased the CD4:CD8 T cell ratio, prolonged the expression of the CD69 activation marker, inhibited CD95L and heat shock protein (HSP) 90 release, and decreased the release of several cytokines. In contrast, the lowest concentrations (0.35 and 0.01 μM) did not have or showed minor antiproliferative or cytotoxic effects, did not alter activation marker expression (CD38, CD69) or the release of CD95L and HSP90, but inhibited the release of certain T cell cytokines. Even when these lower cytarabine concentrations were combined with ATRA and/or valproic acid there was still no or minor effects on T cell viability. However, these combinations had strong antiproliferative effects, the expression of both CD38 and CD69 was altered and there was a stronger inhibition of the release of FasL, HSP90 as well as several cytokines. Cytarabine (0.01-0.05 μM) showed a dose-dependent antiproliferative effect on AML cells, and in contrast to the T cells this effect reached statistical significance even at 0.01 μM. Conclusions Even low levels of cytarabine, and especially when combined with ATRA and valproic acid, can decrease T cell viability, alter activation-induced membrane-molecule expression and decrease the cytokine release. Electronic supplementary material The online version of this article (doi:10.1186/s40360-015-0012-2) contains supplementary material, which is available to authorized users.

Even though malnutrition is associated with an adverse prognosis after allogeneic stem cell transplantation, few studies have addressed the question what is the optimal nutritional support for these patients. There is a general agreement that the body weight, body mass index and nutritional intake can be used to guide the post-transplant nutritional support; enteral nutrition may then be tried but most patients will require parenteral nutrition. There is no scientific basis for further standardization of post-transplant nutritional support. The nutritional status with regard to amino acid as well as fatty acid metabolism and vitamin levels are important for immunoregulation. Several amino acids and their metabolites function as signaling molecules through their binding to specific receptors, and they are thereby become important both in dendritic cell differentiation and T cell activation and the metabolic switch that often occurs during the activation of immunocompetent cells. We review previous studies of nutritional support in allotransplant recipients and discuss possible molecular mechanisms involved in metabolic immunoregulation and the development of post-transplant immune-mediated complications.

Human acute myeloid leukemia is a heterogeneous disease and the effect of therapeutic targeting of specific molecular mechanisms will probably vary between patient subsets. Cell cycle regulators are among the emerging targets (e.g., aurora and polo-like kinases, cyclin-dependent kinases). Inhibition of communication between acute myeloid leukemia and stromal cells is also considered; among the most promising of these strategies are inhibition of hedgehog-initiated, CXCR4-CXCL12 and Axl-Gas6 signaling. Finally, targeting of energy and protein metabolism is considered, the most promising strategy being inhibition of isocitrate dehydrogenase in patients with IDH mutations. Thus, several strategies are now considered, and a major common challenge for all of them is to clarify how they should be combined with each other or with conventional chemotherapy, and whether their use should be limited to certain subsets of patients.

International audience; The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication. INTRODUCTION CXXC5 is a retinoid-responsive gene localized to the 5q31.3 chromosomal region [1] and encoding a retinoid-inducible nuclear factor (RINF) [2] that is a protein containing a CXXC-type zinc-finger domain and acting as a transcription regulator [3]. Expression studies as well as gene silencing experiments suggest that CXXC5 is important in normal myelopoiesis [2] and for differentiation of endothelial cells [3]. Furthermore, we recently described that CXXC5 is expressed in primary acute myeloid leukemia (AML) cells; this expression shows a wide variation between patients and high levels are associated with an adverse prognosis and resistance to chemotherapy-induced apoptosis [4]. Another study recently confirmed our observations and CXXC5 expression was then of independent prognostic significance in multivariate analyses after adjustment

Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leukemia (AML), suggesting that there is an alternate route for cell transformation. We investigated the hypothesis that previously observed Bcl-2 family member overexpression suppresses wild-type p53 activity in AML. We demonstrate that wild-type p53 protein is expressed in primary leukemic blasts from patients with de novo AML using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and phospho-specific flow cytometry. We found that p53 was heterogeneously expressed and phosphorylated in AML patient samples and could accumulate following DNA damage. Overexpression of antiapoptosis protein Bcl-2 in AML cells was directly correlated with p53 expression and phosphorylation on serine residues 15, 46, and 392. Within those patients with the highest levels of Bcl-2 expression, we identified a mutation in FLT3 that duplicated phosphorylation site Y591. The presence of this mutation correlated with greater than normal Bcl-2 expression and with previously observed profiles of potentiated STAT and MAPK signaling. These results support the hypothesis that Flt3-mediated signaling in AML enables accumulation of Bcl-2 and maintains a downstream block to p53 pathway apoptosis. Bcl-2 inhibition might therefore improve the efficacy of existing AML therapies by inactivating this suppression of wild-type p53 activity.

Interactions between acute myelogenous leukemia (AML) blasts and neighbouring endothelial cells in the bone marrow seem important both for disease development and susceptibility to chemotherapy. We investigated the effects of soluble mediators released by microvascular endothelial cells on native human AML cells. AML cells derived from 33 patients were cocultured with microvascular endothelial cells, separated by a semipermeable membrane. We investigated the effect of coculture on AML cell proliferation, viability/apoptosis and cytokine release. Coculture increased AML cell proliferation, and this growth enhancement included the clonogenic leukemia cell subset. Increased release of several soluble mediators was also detected (interleukin 3, interleukin 6, granulocyte-macrophage and granulocyte colony-stimulating factors) in cocultures. Our cytokine neutralization experiments suggest that an intercellular crosstalk involving several soluble mediators contribute to the increased leukemia cell proliferation. The presence of endothelial cells had an additional antiapoptotic effect on the AML cells. The endothelial cells did not have any growth-enhancing effect on native human acute lymphoblastic leukemia cells. Our in vitro results suggest that the release of soluble mediators by microvascular endothelial cells supports leukemic hematopoiesis through paracrine mechanisms by direct enhancement of AML blast proliferation and by inhibition of leukemic cell apoptosis.

Purpose: The antioncogene protein product p53 has not been studied previously in cancer patients during in vivo chemotherapy. This study examined the early p53 protein and gene expression during induction chemotherapy in acute myeloid leukemia (AML). Experimental Design: Leukemic cells were collected from five AML patients during their first 18 hours of induction chemotherapy and examined for p53 protein and gene expression by one- and two-dimensional gel immunoblot and high-density gene expression arrays. Results: Up-regulation of p53 protein expression was detected in AML patients posttreatment in vivo. One- and two-dimensional gel immunoblots showed two main forms of p53, denominated αp53 and Δp53, both recognized by various NH2-terminal directed antibodies. As a response to treatment, we detected rapid accumulation of αp53, with significantly altered protein expression levels already after 2 hours. The accumulation of αp53 was accompanied by increased transcription of putative p53 target genes and subsequent cytopenia in the patients. Conclusion: Up-regulation of the p53 protein and target genes seems to be a prominent feature in induction chemotherapy of AML. The rapid shift from a shorter p53 protein form (Δ) toward the full-length protein (α) underscores the complexity of p53 protein modulation in patients undergoing chemotherapy.

The role of CD8 (T8, Leu 2)-positive T lymphocytes in the proliferative T-lymphocyte response to mumps and Coxsackie B4 viral antigens in vitro was investigated. The frequency among enriched T-lymphocyte blasts of antigen-reactive T lymphocytes (ARTL) restricted by different DR-associated elements was investigated, using antigenic restimulation with allogeneic antigen-presenting cells in a limiting dilution assay. A decreased frequency of DR3-restricted and an increased frequency of DR4-restricted mumps and Coxsackie B4 ARTL were seen in the limiting dilution assay, whether or not HLA class I determinants were shared in the antigenic restimulation. Removal of CD8-positive cells did not increase the primary in vitro responsiveness to mumps and Coxsackie B4 viral antigens, and did not change the DR-associated differences in the frequency of ARTL seen in the limiting dilution assay.