Acute response in circulating microRNAs following a single bout of short-sprint and heavy strength training in well-trained cyclists.

Background: Heavy strength (HS) and short-sprint (SS) are commonly used training methods for competitive road cyclists, with the aim to improve the anaerobic power and short time cycling performance. Knowledge of how such training methods affects biochemical as well as molecular factors, are particularly important for determining individual recovery and long-term adaptations. The primary aim of the current study was to investigate the expression levels of small non-coding RNAs in response to HS and SS training in elite cyclists as potential biomarkers for individual optimal restitution time. Methods: Eleven well trained cyclists performed one session of HS training and one session of SS training on separate days. Blood samples were taken at baseline and 5 min, 1 h and 21 h post training. Along with physiological measurements and biochemical factors (serum creatine kinase, myoglobin, human growth hormone and plasma lactate), real-time quantitative PCR was used to explore whether HS and/or SS training influenced the abundance of 24 circulating miRNAs, in serum, associated with muscle development, angiogenesis, and/or inflammation. Results: Based on complete miRNA profiles from nine cyclists, the miRNAs showing most altered expression after both training sessions included the three striated muscle-specific miRNAs (myomiRs) miR-1-3p, 133a-3p and 133b-3p. While all three miRNAs showed significantly highest expression at 1 h post HS session, the acute effect of the SS session included a significantly higher level of miR-1-3p alone, at 5 min (highest), as well as at 1 h and 21 h post session. Correlation (negative) with biochemical markers was only shown for miR-133a-3p and CK (r = -0.786, p = 0.041) and between miR-133b-3p and [La-] (r = -0.711, p = .032), at 21 h post SS session. Conclusion: Our findings support that unique myomiRs are regulated by HS and SS training. Such knowledge may be important for individually adjusted restitution times.

Vitamin B12 status in infancy and the effect of a vitamin B12 injection in infants with subclinical vitamin B12 deficiency: study protocol for a register-based randomised controlled trial.

INTRODUCTION: Vitamin B12 (cobalamin) is crucial for optimal child development and growth, yet deficiency is common worldwide. The aim of this study is twofold; (1) to describe vitamin B12 status and the status of other micronutrients in Norwegian infants, and (2) in a randomised controlled trial (RCT), investigate the effect of vitamin B12 supplementation on neurodevelopment in infants with subclinical vitamin B12 deficiency. METHODS AND ANALYSIS: Infant blood samples, collected at public healthcare clinics, are analysed for plasma cobalamin levels. Infants with plasma cobalamin <148 pmol/L are immediately treated with hydroxocobalamin and excluded from the RCT. Remaining infants (cobalamin ≥148 pmol/L) are randomly assigned (in a 1:1 ratio) to either a screening or a control group. In the screening group, baseline samples are immediately analysed for total homocysteine (tHcy), while in the control group, the baseline samples will be analysed after 12 months. Screening group infants with plasma tHcy >6.5 µmol/L, are given an intramuscular injection of hydroxocobalamin (400 µg). The primary outcomes are cognitive, language and motor development assessed using the Bayley Scales of Infant and Toddler Development at 12 months of age. ETHICS AND DISSEMINATION: The study has been approved by the Regional Committee for Medical and Health Research Ethics (ref: 186505). Investigators who meet the Vancouver requirements will be eligible for authorship and be responsible for dissemination of study findings. Results will extend current knowledge on consequences of subclinical vitamin B12 deficiency during infancy and may inform future infant feeding recommendations. TRIAL REGISTRATION NUMBER: NCT05005897.

IL-6 Responsiveness of CD4+ and CD8+ T Cells after Allogeneic Stem Cell Transplantation Differs between Patients and Is Associated with Previous Acute Graft versus Host Disease and Pretransplant Antithymocyte Globulin Therapy.

Graft-versus-host disease (GVHD), one of the most common and serious complications after allogeneic stem cell transplantation, is mediated by allocative T cells. IL-6 mediates both pro- and anti-inflammatory effects and modulates T cell response through classical signaling and trans-signaling. We investigated the effects on the mTOR and JAK/STAT pathways after various types of IL-6 signaling for circulating T cells were derived from 31 allotransplant recipients 90 days post-transplant. Cells were stimulated with IL-6 alone, hyper-IL-6 (trans-signaling), IL-6+IL-6 receptor (IL-6R; classical + trans-signaling) and IL-6+IL-6R+soluble gp130-Fc (classical signaling), and flow cytometry was used to investigate the effects on phosphorylation of AKT (Thr308), mTOR (Ser2442), STAT3 (Ser727) and STAT3 (Tyr705). CD3+CD4+ and CD3+C8+ T cells responded to classical and trans IL-6 stimulation with increased STAT3 (Tyr705) phosphorylation; these responses were generally stronger for CD3+CD4+ cells. STAT3 (Tyr705) responses were stronger for patients with previous acute GVHD; CD3+CD4+ cells from GVHD patients showed an additional STAT3 (Ser727) response, whereas patients without acute GVHD showed additional mTOR (Ser2448) responses. Furthermore, treatment with antithymocyte globulin as a part of GVHD prophylaxis was associated with generally weaker STAT3 (Tyr705) responses and altered STAT3 (Ser727) responsiveness of CD3+CD4+ cells together with increased mTOR (Ser2448) responses for the CD3+CD8+ cells. Thus, early post-transplant CD3+CD4+ and CD3+ CD8+ T cell subsets differ in their IL-6 responsiveness; this responsiveness is modulated by antithymocyte globulin and differs between patients with and without previous acute GVHD. These observations suggest that allotransplant recipients will be heterogeneous with regard to the effects of post-transplant IL-6 targeting.

The Female Menstrual Cycles Effect on Strength and Power Parameters in High-Level Female Team Athletes.

PURPOSE: The female menstrual cycle (MC) is characterized by hormonal fluctuations throughout its different phases. However, research regarding its effect on athletic performance in high level athletes is sparse. The aim of this study was to (i) investigate the female MCs effect on strength and power performance in highly trained female team athletes throughout the MC and (ii) examine whether eumenorrheic participants with natural hormonal fluctuations displayed enhanced performance in the follicular phase (FP) versus the luteal phase (LP), compared to controls using hormonal contraceptives. MATERIALS AND METHODS: A total of 29 athletes (Age 21.2 ± 3.3 years; weight 65.6 ± 8.7 kg; height 170.2 ± 8.0 cm; and fat free mass 52.7 ± 7.1) completed the study after a 6-week testing period (8 eumenorrheic participants and 21 hormonal contraceptive controls). Participants were recruited from the team sports soccer, handball and volleyball. Testing protocol consisted of maximal voluntary isometric grip strength, 20-m sprint, countermovement jump and pneumatic leg-press. Based on self-reported use of hormonal contraceptives, participants were divided into non-hormonal contraceptive group and hormonal contraceptive group, the latter working as a control group. Differences in performance between the FP and LP were investigated. MC phase was confirmed by serum hormonal levels through venous blood samples in the non-hormonal contraceptive group. RESULTS: There were no statistically significant changes for the two different phases of the MC, in terms of physical performance for the whole group. Further, there was no significant difference between groups during the MC for any of the outcome variables, maximal voluntary isometric grip strength F(3.29) = 0.362; 20-m sprint F(3.24) = 0.710; countermovement jump F(3.26) = 2.361; and leg-press F(3.26) = 1.746. CONCLUSION: In high level female team athletes, no difference in performance was observed based on hormonal contraceptive status. This suggests that the MC does not alter acute strength and power performance on a group level in high level team athletes.

Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation.

BACKGROUND: Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. METHODS: We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. RESULTS: We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. CONCLUSIONS: This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation.

Immunomodulatory Drugs Alter the Metabolism and the Extracellular Release of Soluble Mediators by Normal Monocytes.

Immunomodulatory drugs (IMiDs) are used in the treatment of hematological malignancies, especially multiple myeloma. IMiDs have direct anticancer effects but also indirect effects via cancer-supporting stromal cells. Monocytes are a stromal cell subset whose metabolism is modulated by the microenvironment, and they communicate with neighboring cells through extracellular release of soluble mediators. Toll-like receptor 4 (TLR4) is then a common regulator of monocyte metabolism and mediator release. Our aim was to investigate IMiD effects on these two monocyte functions. We compared effects of thalidomide, lenalidomide, and pomalidomide on in vitro cultured normal monocytes. Cells were cultured in medium alone or activated by lipopolysaccharide (LPS), a TLR4 agonist. Metabolism was analyzed by the Seahorse XF 96 cell analyzer. Mediator release was measured as culture supernatant levels. TLR4 was a regulator of both monocyte metabolism and mediator release. All three IMiDs altered monocyte metabolism especially when cells were cultured with LPS; this effect was strongest for lenalidomide that increased glycolysis. Monocytes showed a broad soluble mediator release profile. IMiDs decreased TLR4-induced mediator release; this effect was stronger for pomalidomide than for lenalidomide and especially thalidomide. To conclude, IMiDs can alter the metabolism and cell-cell communication of normal monocytes, and despite their common molecular target these effects differ among various IMiDs.

A Pilot Study of Circulating Monocyte Subsets in Patients Treated with Stem Cell Transplantation for High-Risk Hematological Malignancies.

Background and Objectives: Autologous and allogeneic stem cell transplantation is used in the treatment of high-risk hematological malignancies, and monocytes are probably involved in hematological reconstitution as well as posttransplant immunoregulation. The aim of our study was to investigate the levels of circulating monocyte subsets in allotransplant recipients. Materials and Methods: The levels of the classical, intermediate, and nonclassical monocyte subsets were determined by flow cytometry. Sixteen patients and 18 healthy controls were included, and the levels were analyzed during pretransplant remission (n = 13), early posttransplant during cytopenia (n = 9), and early reconstitution (n = 9). Results: Most patients in remission showed a majority of classical monocytes. The patients showed severe early posttransplant monocytopenia, but the total peripheral blood monocyte counts normalized very early on, and before neutrophil and platelet counts. During the first 7-10 days posttransplant (i.e., during cytopenia) a majority of the circulating monocytes showed a nonclassical phenotype, but later (i.e., 12-28 days posttransplant) the majority showed a classical phenotype. However, the variation range of classical monocytes was wider for patients in remission and during regeneration than for healthy controls. Conclusions: The total peripheral blood monocyte levels normalize at the very early stages and before neutrophil reconstitution after stem cell transplantation, and a dominance of classical monocytes is reached within 2-4 weeks posttransplant.

Myeloid-Derived Suppressor Cells in Hematologic Diseases: Promising Biomarkers and Treatment Targets.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells that exist at very low numbers in healthy subjects but can expand significantly in malignant, infectious, and chronic inflammatory diseases. These cells are characterized as early-MDSCs, monocytic-MDSCs, and polymorphonuclear-MDSCs and can be studied on the basis of their immunophenotypic characteristics and their functional properties to suppress T-cell activation and proliferation. MDSCs have emerged as important contributors to tumor expansion and chronic inflammation progression by inducing immunosuppressive mechanisms, angiogenesis and drug resistance. Most experimental and clinical studies concerning MDSCs have been mainly focused on solid tumors. In recent years, however, the implication of MDSCs in the immune dysregulation associated with hematologic malignancies, immune-mediated cytopenias and allogeneic hemopoietic stem cell transplantation has been documented and the potential role of these cells as biomarkers and therapeutic targets has started to attract a particular interest in hematology. The elucidation of the molecular and signaling pathways associated with the generation, expansion and function of MDSCs in malignant and immune-mediated hematologic diseases and the clarification of mechanisms related to the circulation and the crosstalk of MDSCs with malignant cells and other components of the immune system are anticipated to lead to novel therapeutic strategies. This review summarizes all available evidence on the implication of MDSCs in hematologic diseases highlighting the challenges and perspectives arising from this novel field of research.

Circulating monocyte subsets in multiple myeloma patients receiving autologous stem cell transplantation - a study of the preconditioning status and the course until posttransplant reconstitution for a consecutive group of patients.

BACKGROUND: Induction therapy of multiple myeloma patients prior to autologous stem cell transplantation has changed from conventional chemotherapy to treatment based on proteasome inhibitors or immunomodulatory drugs. We used flow cytometry to analyze total monocyte and monocyte subset (classical, intermediate and non-classical monocytes) peripheral blood levels before and following auto-transplantation for a consecutive group of myeloma patients who had received the presently used induction therapy. RESULTS: The patients showed normal total monocyte concentrations after induction/stem cell mobilization, but the concentrations of classical monocytes were increased compared with healthy controls. Melphalan conditioning reduced the levels of total CD14+ as well as classical and non-classical monocytes, whereas intermediate monocytes were not affected. Thus, melphalan has a non-random effect on monocyte subsets. Melphalan had a stronger effect on total and classical monocyte concentrations for those patients who had received induction therapy including immunomodulatory drugs. Total monocytes and monocyte subset concentrations decreased during the period of pancytopenia, but monocyte reconstitution occurred before hematopoietic reconstitution. However, the fractions of various monocyte subsets varied considerably between patients. CONCLUSIONS: The total level of circulating monocytes is normalized early after auto-transplantation for multiple myeloma, but pre- and post-transplant levels of various monocyte subsets show considerable variation between patients.

Standardization of sampling and sample preparation for analysis of human monocyte subsets in peripheral blood.

INTRODUCTION: Monocytes are important for innate immunity and include the classical (CD14brightCD16negative), intermediate (CD14brightCD16dim) and non-classical (CD14dimCD16bright) monocyte subsets. The quantification of these functionally different subsets in peripheral blood may become useful for diagnosis and follow-up in human diseases. The aim of the present study was to investigate how different pre-analytical parameters influence analysis of monocyte subsets in peripheral blood samples. METHODS: We determined relative levels of monocytes and monocyte subsets by flow cytometry of peripheral blood samples derived from healthy individuals. A gating strategy exclusively extracting viable CD14+ monocytes and focusing on the three monocyte subsets was applied. We investigated the effects of (i) various anticoagulants (i.e. Li-Heparin, ACD-A, K2EDTA), (ii) insufficient filling of blood sampling tubes, (iii) cryopreservation. In addition, we analysed expression of the CCR2 chemokine receptor. RESULTS: The relative numbers of CD14+ monocytes depended on the anticoagulant used, whereas the fraction of the three monocyte subsets did not. Insufficient filling of blood sampling tubes altered the relative levels of monocytes out of leukocytes, but not the relative levels of the monocyte subsets. Finally, the fraction of CD14+ monocytes out of isolated peripheral blood mononuclear cells was not significantly altered by cryopreservation, but the relative percentages of monocyte subsets was altered (similar effects for ACD-A and K2EDTA samples) and this was observed in correlation to a decreased CD16 expression. CONCLUDING REMARKS: Analysis of the monocyte subsets (i.e. classical, intermediate, non-classical) in peripheral blood samples requires a careful standardization of peripheral blood sampling and pre-analytic handling of the samples with respect to the anticoagulant used, filling of sample tubes, and cryopreservation of cells prior to analysis.

Peripheral Blood Stem Cell Mobilization in Healthy Donors by Granulocyte Colony-Stimulating Factor Causes Preferential Mobilization of Lymphocyte Subsets.

Background: Allogeneic hematopoietic stem cell transplantation is associated with a high risk of immune-mediated post-transplant complications. Graft depletion of immunocompetent cell subsets is regarded as a possible strategy to reduce this risk without reducing antileukemic immune reactivity. Study design and methods: We investigated the effect of hematopoietic stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) on peripheral blood and stem cell graft levels of various T, B, and NK cell subsets in healthy donors. The results from flow cytometric cell quantification were examined by bioinformatics analyses. Results: The G-CSF-induced mobilization of lymphocytes was a non-random process with preferential mobilization of naïve CD4+ and CD8+ T cells together with T cell receptor αß+ T cells, naïve T regulatory cells, type 1 T regulatory cells, mature and memory B cells, and cytokine-producing NK cells. Analysis of circulating lymphoid cell capacity to release various cytokines (IFNγ, IL10, TGFß, IL4, IL9, IL17, and IL22) showed preferential mobilization of IL10 releasing CD4+ T cells and CD3-19- cells. During G-CSF treatment, the healthy donors formed two subsets with generally strong and weaker mobilization of immunocompetent cells, respectively; hence the donors differed in their G-CSF responsiveness with regard to mobilization of immunocompetent cells. The different responsiveness was not reflected in the graft levels of various immunocompetent cell subsets. Furthermore, differences in donor G-CSF responsiveness were associated with time until platelet engraftment. Finally, strong G-CSF-induced mobilization of various T cell subsets seemed to increase the risk of recipient acute graft versus host disease, and this was independent of the graft T cell levels. Conclusion: Healthy donors differ in their G-CSF responsiveness and preferential mobilization of immunocompetent cells. This difference seems to influence post-transplant recipient outcomes.

Power Production and Biochemical Markers of Metabolic Stress and Muscle Damage Following a Single Bout of Short-Sprint and Heavy Strength Exercise in Well-Trained Cyclists.

Purpose: Although strength and sprint training are widely used methods in competitive cycling, no previous studies have compared the acute responses and recovery rates following such sessions among highly trained cyclists. The primary aim of the current study was to compare power production and biochemical markers of metabolic stress and muscle damage following a session of heavy strength (HS) and short-sprint training (SS). Methods: Eleven well-trained male cyclists (18 ± 2 years with maximal oxygen uptake of 67.2 ± 5.0 mL·kg-1·min-1) completed one HS session and one SS session in a randomized order, separated by 48 h. Power production and biochemical variables were measured at baseline and at different time points during the first 45 h post exercise. Results: Lactate and human growth hormone were higher 5 min, 30 min and 1 h post the SS compared to the HS session (all p ≤ 0.019). Myoglobin was higher following the HS than the SS session 5 min, 30 min and 1 h post exercise (all p ≤ 0.005), while creatine kinase (CK) was higher following the HS session 21 and 45 h post exercise (p ≤ 0.038). Counter movement jump and power production during 4 sec sprint returned to baseline levels at 23 and 47 h with no difference between the HS and SS session, whereas the delayed muscle soreness score was higher 45 h following the HS compared to the SS session (p = 0.010). Conclusion: Our findings indicate that SS training provides greater metabolic stress than HS training, whereas HS training leads to more muscle damage compared to that caused by SS training. The ability to produce power remained back to baseline already 23 h after both training sessions, indicating maintained performance levels although higher CK level and muscle soreness were present 45 h post the HS training session.

The healthy donor profile of immunoregulatory soluble mediators is altered by stem cell mobilization and apheresis.

BACKGROUND: Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and thereafter harvested by leukapheresis are commonly used for allogeneic stem cell transplantation. METHODS: Plasma levels of 38 soluble mediators (cytokines, soluble adhesion molecules, proteases, protease inhibitors) were analyzed in samples derived from healthy stem cell donors before G-CSF treatment and after 4 days, both immediately before and after leukapheresis. RESULTS: Donors could be classified into two main subsets based on their plasma mediator profile before G-CSF treatment. Seventeen of 36 detectable mediators were significantly altered by G-CSF; generally an increase in mediator levels was seen, including pro-inflammatory cytokines, soluble adhesion molecules and proteases. Several leukocyte- and platelet-released mediators were increased during apheresis. Both plasma and graft mediator profiles were thus altered and showed correlations to graft concentrations of leukocytes and platelets; these concentrations were influenced by the apheresis device used. Finally, the mediator profile of the allotransplant recipients was altered by graft infusion, and based on their day +1 post-transplantation plasma profile our recipients could be divided into two major subsets that differed in overall survival. DISCUSSION: G-CSF alters the short-term plasma mediator profile of healthy stem cell donors. These effects together with the leukocyte and platelet levels in the graft determine the mediator profile of the stem cell grafts. Graft infusion also alters the systemic mediator profile of the recipients, but further studies are required to clarify whether such graft-induced alterations have a prognostic impact.

Interleukin-6 in Allogeneic Stem Cell Transplantation: Its Possible Importance for Immunoregulation and As a Therapeutic Target.

Allogeneic stem cell transplantation is associated with a high risk of treatment-related mortality mainly caused by infections and graft-versus-host disease (GVHD). GVHD is characterized by severe immune dysregulation and impaired regeneration of different tissues, i.e., epithelial barriers and the liver. The balance between pro- and anti-inflammatory cytokine influences the risk of GVHD. Interleukin-6 (IL-6) is a cytokine that previously has been associated with pro-inflammatory effects. However, more recent evidence from various autoimmune diseases (e.g., inflammatory bowel disease, rheumatoid arthritis) has shown that the IL-6 activity is more complex with important effects also on tissue homeostasis, regeneration, and metabolism. This review summarizes the current understanding of how pro-inflammatory IL-6 effects exerted during the peritransplant period shapes T-cell polarization with enhancement of Th17 differentiation and suppression of regulatory T cells, and in addition we also review and discuss the results from trials exploring non-selective IL-6 inhibition in prophylaxis and treatment of GVHD. Emerging evidence suggests that the molecular strategy for targeting of IL-6-initiated intracellular signaling is important for the effect on GVHD. It will therefore be important to further characterize the role of IL-6 in the pathogenesis of GVHD to clarify whether combined IL-6 inhibition of both trans- (i.e., binding of the soluble IL-6/IL-6 receptor complex to cell surface gp130) and cis-signaling (i.e., IL-6 ligation of the IL-6 receptor/gp130 complex) or selective inhibition of trans-signaling should be tried in the prophylaxis and/or treatment of GVHD in allotransplant patients.

Immunomodulation Induced by Stem Cell Mobilization and Harvesting in Healthy Donors: Increased Systemic Osteopontin Levels after Treatment with Granulocyte Colony-Stimulating Factor.

Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and harvested by leukapheresis are commonly used for allogeneic stem cell transplantation. The frequency of severe graft versus host disease is similar for patients receiving peripheral blood and bone marrow allografts, even though the blood grafts contain more T cells, indicating mobilization-related immunoregulatory effects. The regulatory phosphoprotein osteopontin was quantified in plasma samples from healthy donors before G-CSF treatment, after four days of treatment immediately before and after leukapheresis, and 18-24 h after apheresis. Myeloma patients received chemotherapy, combined with G-CSF, for stem cell mobilization and plasma samples were prepared immediately before, immediately after, and 18-24 h after leukapheresis. G-CSF treatment of healthy stem cell donors increased plasma osteopontin levels, and a further increase was seen immediately after leukapheresis. The pre-apheresis levels were also increased in myeloma patients compared to healthy individuals. Finally, in vivo G-CSF exposure did not alter T cell expression of osteopontin ligand CD44, and in vitro osteopontin exposure induced only small increases in anti-CD3- and anti-CD28-stimulated T cell proliferation. G-CSF treatment, followed by leukapheresis, can increase systemic osteopontin levels, and this effect may contribute to the immunomodulatory effects of G-CSF treatment.

Effects of cytarabine on activation of human T cells - cytarabine has concentration-dependent effects that are modulated both by valproic acid and all-trans retinoic acid.

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.

Antileukaemic effect of PI3K-mTOR inhibitors in acute myeloid leukaemia-gene expression profiles reveal CDC25B expression as determinate of pharmacological effect.

Acute myeloid leukaemia (AML) is a heterogeneous malignancy. Intracellular signalling through the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway is important for regulation of cellular growth and metabolism, and inhibitors of this pathway is considered for AML treatment. Primary human AML cells, derived from 96 consecutive adult patients, were examined. The effects of two mTOR inhibitors (rapamycin, temsirolimus) and two PI3K inhibitors (GDC-0941, 3-methyladenine) were studied, and we investigated cytokine-dependent proliferation, regulation of apoptosis and global gene expression profiles. Only a subset of patients demonstrated strong antiproliferative effects of PI3K-mTOR inhibitors. Unsupervised hierarchical clustering analysis identified two main clusters of patients; one subset showing weak or absent antiproliferative effects (59%) and another group showing a strong growth inhibition for all drugs and concentrations examined (41%). Global gene expression analyses showed that patients with AML cell resistance against PI3K-mTOR inhibitors showed increased mRNA expression of the CDC25B gene that encodes the cell cycle regulator Cell Division Cycle 25B. The antileukaemic effect of PI3K-Akt-mTOR inhibition varies between patients, and resistance to these inhibitors is associated with the expression of the cell cycle regulator CDC25B, which is known to crosstalk with the PI3K-Akt-mTOR pathway and mediate rapamycin resistance in experimental models.

Up-regulation of anti-apoptotic genes confers resistance to the novel anti-leukaemic compound PEP005 in primary AML cells.

We showed previously that PEP005 induced apoptosis in leukaemic cell lines and blasts from patients with acute myeloid leukaemia (AML). Here we assess the anti-leukeamic effects of PEP005 in vivo and determine the mechanism of resistance of PEP005 non-responsive cells. We used 2 human xenograft mouse models of AML to assess the anti-leukaemic effects of PEP005 in vivo. Expression microarray analysis of primary AML blasts following treatment with PEP005 was used to determine patterns of gene expression that conferred resistance. PEP005 significantly reduced tumour burden in two human leukaemia mouse xenograft models. We also assessed responsiveness of 33 AML samples to PEP005, with 78% of the samples entering apoptosis at 100nM. Resistance to PEP005 was not restricted to a particular AML subtype. Expression microarray analysis of resistant samples following treatment with PEP005 revealed a significant up regulation of the anti-apoptotic genes Bcl-2A1, Mcl-1, and PHLDA1 which was verified using RT-PCR. We conclude that PEP005 shows broad efficacy against AML subtypes and that up regulation of anti-apoptotic genes underlies resistance to this agent and could be used to screen for patients unlikely to benefit from a therapeutic regime involving PEP005.

The combination of valproic acid, all-trans retinoic acid and low-dose cytarabine as disease-stabilizing treatment in acute myeloid leukemia.

BACKGROUND: A large proportion of patients with acute myeloid leukemia (AML) are not fit for intensive and potentially curative therapy due to advanced age or comorbidity. Previous studies have demonstrated that a subset of these patients can benefit from disease-stabilizing therapy based on all-trans retinoic acid (ATRA) and valproic acid. Even though complete hematological remission is only achieved for exceptional patients, a relatively large subset of patients respond to this treatment with stabilization of normal peripheral blood cell counts. METHODS: In this clinical study we investigated the efficiency and safety of combining (i) continuous administration of valproic acid with (ii) intermittent oral ATRA treatment (21.5 mg/m2 twice daily) for 14 days and low-dose cytarabine (10 mg/m2 daily) for 10 days administered subcutaneously. If cytarabine could not control hyperleukocytosis it was replaced by hydroxyurea or 6-mercaptopurin to keep the peripheral blood blast count below 50 × 109/L. RESULTS: The study included 36 AML patients (median age 77 years, range 48 to 90 years) unfit for conventional intensive chemotherapy; 11 patients responded to the treatment according to the myelodysplastic syndrome (MDS) response criteria and two of these responders achieved complete hematological remission. The most common response to treatment was increased and stabilized platelet counts. The responder patients had a median survival of 171 days (range 102 to > 574 days) and they could spend most of this time outside hospital, whereas the nonresponders had a median survival of 33 days (range 8 to 149 days). The valproic acid serum levels did not differ between responder and nonresponder patients and the treatment was associated with a decrease in the level of circulating regulatory T cells. CONCLUSION: Treatment with continuous valproic acid and intermittent ATRA plus low-dose cytarabine has a low frequency of side effects and complete hematological remission is seen for a small minority of patients. However, disease stabilization is seen for a subset of AML patients unfit for conventional intensive chemotherapy.

Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine.

BACKGROUND: Amphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by (3)H-thymidine incorporation. RESULTS: Cathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p-Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p-Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p-Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death. CONCLUSIONS: Single-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced T-lymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.