Safety and pharmacokinetics of the antisense oligonucleotide (ASO) LY2181308 as a single-agent or in combination with idarubicin and cytarabine in patients with refractory or relapsed acute myeloid leukemia (AML).

Survivin is expressed in tumor cells, including acute myeloid leukemia (AML), regulates mitosis, and prevents tumor cell death. The antisense oligonucleotide sodium LY2181308 (LY2181308) inhibits survivin expression and may cause cell cycle arrest and restore apoptosis in AML. In this study, the safety, pharmacokinetics, and pharmacodynamics/efficacy of LY2181308 was examined in AML patients, first in a cohort with monotherapy (n = 8) and then post-amendment in a cohort with the combination of cytarabine and idarubicin treatment (n = 16). LY2181308 was administered with a loading dosage of three consecutive daily infusions of 750 mg followed by weekly intravenous (IV) maintenance doses of 750 mg. Cytarabine 1.5 g/m(2) was administered as a 4-hour IV infusion on Days 3, 4, and 5 of Cycle 1, and idarubicin 12 mg/m(2) was administered as a 30-minute IV infusion on Days 3, 4, and 5 of Cycle 1. Cytarabine and idarubicin were administered on Days 1, 2, and 3 of each subsequent 28-day cycle. Reduction of survivin was evaluated in peripheral blasts and bone marrow. Single-agent LY2181308 was well tolerated and survivin was reduced only in patients with a high survivin expression. In combination with chemotherapy, 4/16 patients had complete responses, 1/16 patients had incomplete responses, and 4/16 patients had cytoreduction. Nine patients died on study: 6 (monotherapy), 3 (combination). LY2181308 alone is well tolerated in patients with AML. In combination with cytarabine and idarubicin, LY2181308 does not appear to cause additional toxicity, and has shown some clinical benefit needing confirmation in future clinical trials.

When hymenopteran males reinvented diploidy.

In most plants and animals, a consistent relationship exists between the DNA content of a cell and its metabolic activity. The male-haploid sex determination of Hymenoptera and other arthropods may therefore impose a particular selective pressure upon males, which must evolve adaptations to cope with a genomic DNA reduced by half compared with that of females. Here, we show that a nuclear DNA content similar to that of females is restored in muscles of males in all hymenopteran lineages tested except the most basal one (Xyelidae). This doubling of DNA content in males does not occur in other haplodiploid insects, such as thrips (Thysanoptera) and whiteflies (Sternorrhyncha). These results indicate that this adaptation probably occurred early in hymenopteran history, possibly because males acquired strong flying and dispersal abilities.

Quantification of circulating endothelial progenitor cells: a methodological comparison of six flow cytometric approaches.

OBJECTIVES: The validity of endothelial progenitor cells as biomarkers and their therapeutic potential depend on the accuracy of techniques used for enumeration. This study assessed the agreement between 6 flow cytometric methods and a CFU assay used for EPC quantification. METHODS: Two blood samples were obtained from 30 healthy volunteers (60 samples). CD34+/VEGFR2+ cells were analyzed with flow cytometry, starting from whole blood (A-C) or PBMC (D-F), using different gating strategies: A: lymphocyte gating; B and D: exclusion of autofluorescent cells (CD3 negative selection); C and E: exclusion of autofluorescence and cell aggregates (pulse shape analysis by FSCarea/FSCpeak); F: exclusion of autofluorescence, cell aggregates and non-nucleated cells (Draq 5). PBMC were cultured under endothelial cell conditions to assess CFU numbers. RESULTS: Moderate agreement was found between methods B-C and D-E (ICC 0.647 and 0.530). Comparison of methods B-D and C-E showed poor agreement (ICC 0.178 and 0.249). This was also the case for techniques that considerably differed with regard to gating strategies (A-B, A-F, B-F). CFU numbers did not correlate with flow cytometric quantification (all p>0.05). CONCLUSIONS: Agreement between methods for EPC quantification is moderate to poor, which may explain apparent controversies in literature. Although each protocol is highly reproducible, this study cautions against comparing study results gathered with different enumeration techniques.

Investigation of morphometric parameters for granulocytes and lymphocytes as applied to a solution of direct and inverse light-scattering problems.

Quantitative data on cell structure, shape, and size distribution are obtained by optical measurement of normal peripheral blood granulocytes and lymphocytes in a cell suspension. The cell nuclei are measured in situ. The distribution laws of the cell and nuclei sizes are estimated. The data gained are synthesized to construct morphometric models of a segmented neutrophilic granulocyte and a lymphocyte. Models of interrelation between the cell and nucleus metric characteristics for granulocyte and lymphocyte are obtained. The discovered interrelation decreases the amount of cell-nucleus size combinations that have to be considered under simulation of cell scattering patterns. It allows faster analysis of light scattering to discriminate cells in a real-time scale. Our morphometric data meet the requirements of scanning flow cytometry dealing with the high rate analysis of cells in suspension. Our findings can be used as input parameters for the solution of the direct and inverse light-scattering problems in scanning flow cytometry, dispensing with a costly and time-consuming immunophenotyping of the cells, as well as in turbidimetry and nephelometry. The cell models developed can ensure better interpretations of scattering patterns for an improvement of discriminating capabilities of immunophenotyping-free scanning flow cytometry.

Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple in vitro stimulations of CD8+ T cells with peptide-pulsed autologous dendritic cells.

BACKGROUND: Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity. RESULTS: In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711-20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-gamma ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-gamma-secreting CD8+ T cells in 5/5 healthy donors. CONCLUSION: We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death.

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H(2)O(2) resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H(2)O(2) was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 microM H(2)O(2) for 24h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.

Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease.

BACKGROUND: Dendritic cells are potent antigen-presenting and immune modulating cells that have been implicated in the development of atherosclerosis. In human blood, two distinct lineages are distinguished: plasmacytoid dendritic cells and myeloid dendritic cells. Although dendritic cells have been described in atherosclerotic plaques, no information exists concerning circulating blood dendritic cells in atherosclerosis. This study aims to evaluate the number of circulating dendritic cells in patients with coronary artery disease. The relation with the extent of coronary artery disease, the clinical syndrome and with a marker of inflammation will be documented. METHODS: Patients with angiographically proven coronary artery disease (n=18) and age and sex-matched controls (n=18) were included. Myeloid dendritic cells and plasmacytoid dendritic cells were detected with the specific blood dendritic cell antigens, blood dendritic cell antigen-1 and blood dendritic cell antigen-2, respectively. RESULTS: Absolute and relative numbers of circulating plasmacytoid dendritic cells were significantly lower in patients with coronary artery disease (5722+/-601/ml and 0.08+/-0.01%) than in controls (12,640+/-1289/ml and 0.21+/-0.02%). Plasmacytoid dendritic cells were more decreased in patients with troponin-positive unstable coronary syndromes than in patients with low troponin values, and tended to be lower in more extensive coronary artery disease. Absolute myeloid dendritic cells numbers tended to be reduced in patients, whereas relative numbers were significantly decreased: 11,857+/-1895/ml versus 15,226+/-928/ml and 0.17+/-0.03% versus 0.26+/-0.01% in controls. CONCLUSIONS: The present study shows a significant decrease of circulating blood dendritic cell antigen-2 positive plasmacytoid dendritic cells in patients with coronary artery disease. The decrease tended to be more pronounced in unstable coronary syndromes and extensive coronary artery disease, suggesting a possible role of dendritic cells in plaque progression and rupture.

Plant cytokinin analogues with inhibitory activity on cyclin-dependent kinases exert their antiproliferative effect through induction of apoptosis initiated by the mitochondrial pathway: determination by a multiparametric flow cytometric analysis.

OBJECTIVE: Regulation of the cell cycle by cyclin-dependent kinase (CDK) activity occurs at multiple levels and is often altered in human cancers. Therefore, CDK activity has been targeted for drug discovery, and a number of small molecules have now been identified as CDK inhibitors. Plant cytokinin analogues with CDK inhibitory activity and antiproliferative effects were studied to characterize the cellular basis of the cytotoxic effect. METHODS: The IC(50) value (concentration at which 50% of the cell proliferation is inhibited) and AC(50) value (concentration at which 50% of the cell population is apoptotic) were determined by flow cytometry and microscopy, respectively. A new multiparametric flow cytometric analysis was used to study the sequence of different apoptotic events. In this assay, analysis of phosphatidylserine exposure, mitochondrial membrane depolarization, activation of caspases and DNA condensation were combined. RESULTS: Treatment of Jurkat and KG1 cells with the CDK inhibitors results in a decrease of viable cells and a parallel increase in percentage of apoptotic cells. Apoptosis was accompanied by a rapid decrease of mitochondrial membrane potential, which precedes DNA condensation, exposure of phosphatidylserine and activation of caspases. CONCLUSIONS: The main cellular mechanism of the antiproliferative effect of plant cytokinin analogues with CDK inhibitory activity is the induction of apoptosis. The multiparametric flow cytometric technique allowed to follow the kinetics of various aspects of apoptotic cell changes and demonstrated that cytokinin analogue-induced apoptosis starts through the mitochondrial pathway. This technique could also become of value for the rapid screening of pro-apoptotic properties of chemotherapeutic compounds.

Extracellular nucleoside diphosphate kinase NM23/NDPK modulates normal hematopoietic differentiation.

OBJECTIVE: We previously demonstrated the presence of nucleoside diphosphate kinase NDPK/NM23 in normal human plasma. It also was reported that extracellular NM23 could inhibit differentiation of certain hematopoietic cell lines. We further investigated the extracellular effect of NM23 on hematopoiesis by adding recombinant NM23-H1, NM23-H2, and NM23-H3 proteins to in vitro differentiation assays of normal human hematopoietic progenitors. MATERIALS AND METHODS: To study the effect on the earlier stages of hematopoietic maturation, NM23 was added to serum-free pre-colony-forming unit (pre-CFU) assays starting from immature CD34++CD38- bone marrow cells. Serum-free CFU assays starting from CD34+ CD38+ bone marrow cells were used as a model for terminal hematopoietic differentiation. RESULTS: In pre-CFU assays, none of the NM23 isoforms used significantly changed the expansion of CD34++CD38- cells, nor did NM23 alter the CD34++ CD38- cell lineage commitment. In contrast, terminal differentiation of CD34+CD38+ progenitor cells in CFU assays was significantly altered by addition of NM23 protein. More erythroid burst-forming units and fewer macrophage colonies were observed in cultures containing any of the NM23 isoforms examined. Similar effects were observed using the enzymatically inactive H118N mutant of NM23-H1, strongly suggesting that the observed effect is independent of the nucleoside diphosphate kinase activity of NM23. CONCLUSION: We demonstrated a modulating effect of extracellular NM23 proteins on the terminal stages of normal hematopoietic differentiation. Therefore, the fairly high concentrations of NM23 constitutively present in plasma could have a physiologic role in supporting erythropoiesis and inhibiting excessive macrophage formation.

The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer.

The cell cycle is controlled by numerous mechanisms ensuring correct cell division. This review will focus on these mechanisms, i.e. regulation of cyclin-dependent kinases (CDK) by cyclins, CDK inhibitors and phosphorylating events. The quality checkpoints activated after DNA damage are also discussed. The complexity of the regulation of the cell cycle is also reflected in the different alterations leading to aberrant cell proliferation and development of cancer. Consequently, targeting the cell cycle in general and CDK in particular presents unique opportunities for drug discovery. This review provides an overview of deregulation of the cell cycle in cancer. Different families of known CDK inhibitors acting by ATP competition are also discussed. Currently, at least three compounds with CDK inhibitory activity (flavopiridol, UCN-01, roscovitine) have entered clinical trials.

Cell cycle and apoptosis.

Apoptosis and proliferation are intimately coupled. Some cell cycle regulators can influence both cell division and programmed cell death. The linkage of cell cycle and apoptosis has been recognized for c-Myc, p53, pRb, Ras, PKA, PKC, Bcl-2, NF-kappa B, CDK, cyclins and CKI. This review summarizes the different functions of the proteins presently known to control both apoptosis and cell cycle progression. These proteins can influence apoptosis or proliferation but different variables, including cell type, cellular environment and genetic background, make it difficult to predict the outcome of cell proliferation, cell cycle arrest or cell death. These important decisions of cell proliferation or cell death are likely to be controlled by more than one signal and are necessary to ensure a proper cellular response.

Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro.

PURPOSE: The mechanism of radiosensitization by gemcitabine is still unclear. It has been hypothesized that the accumulation of cells in early S phase may play a role in enhancing radiosensitivity. METHODS AND MATERIALS: The schedule dependency of the radiosensitizing effect was studied in ECV304, human bladder cancer cells, and H292, human lung cancer cells, by varying the incubation time and time interval between gemcitabine and radiation treatment. To determine the role of cell cycle perturbations in the radiosensitization, the influence of gemcitabine on the cell cycle at the moment of radiation was investigated by flow cytometry. RESULTS: The radiosensitizing effect increased with a longer incubation period: Dose enhancement factors varied from 1.30 to 2.82 in ECV304 and from 1.04 to 1.78 in H292 after treatment during 8-32 h, respectively. Radiosensitization decreased with an increasing interval: Dose enhancement factors varied from 2.26 to 1.49 in ECV304 and from 1.45 to 1.11 in H292 after an interval 0-24 h, respectively. Cells were blocked in the early S phase of the cell cycle by gemcitabine. The highest percentage S-phase cells was observed after treatment with the schedules that resulted in the highest radiosensitizing effect. CONCLUSIONS: We observed a clear schedule-dependent radiosensitization by gemcitabine. Our findings demonstrated a correlation between gemcitabine-induced early S-phase block and the radiosensitizing effect.

Immunotoxicology in wood mice along a heavy metal pollution gradient.

We carried out an immunotoxicological field study of wood mice in three populations along a heavy metal pollution gradient. Heavy metal concentrations in liver tissue indicated that exposure to silver, arsenic, cadmium, cobalt and lead decreased with increasing distance from a non-ferrous smelter. Host resistance to the endoparasite Heligmosomoides polygyrus decreased with increasing exposure, while the abundance of tick larvae and the nematode Syphacia stroma was unrelated to heavy metal exposure. Spleen mass was increased at the intermediate and the most polluted sites and was positively correlated with the number of H. polygyrus and tick larvae. Proportion of early apoptotic leukocytes increased towards the smelter and was positively related to cadmium exposure. Red and white blood cell counts and lysozyme activity showed no relationship with metal exposure. All together, our observations suggest negative effects of heavy metal exposure on the immune function of wood mice under field conditions.

Dose study of the multikinase inhibitor, LY2457546, in patients with relapsed acute myeloid leukemia to assess safety, pharmacokinetics, and pharmacodynamics.

BACKGROUND: Acute myeloid leukemia (AML) is a life-threatening malignancy with limited treatment options in chemotherapy-refractory patients. A first-in-human dose study was designed to investigate a safe and biologically effective dose range for LY2457546, a novel multikinase inhibitor, in patients with relapsed AML. METHODS: In this nonrandomized, open-label, dose escalation Phase I study, LY2457546 was administered orally once a day. Safety, pharmacokinetics, changes in phosphorylation of target kinases in AML blasts, and risk of drug-drug interactions (DDI) were assessed. RESULTS: Five patients were treated at the starting and predicted minimal biologically effective dose of 50 mg/day. The most commonly observed adverse events were febrile neutropenia, epistaxis, petechiae, and headache. The majority of adverse events (81%) were Grade 1 or 2. One patient had generalized muscle weakness (Grade 3), which was deemed to be a dose-limiting toxicity. Notably, the pharmacokinetic profile of LY2457546 showed virtually no elimination of LY2457546 within 24 hours, and thus prevented further dose escalation. No significant DDI were observed. Ex vivo flow cytometry studies showed downregulation of the phosphoproteins, pcKIT, pFLT3, and pS6, in AML blasts after LY2457546 administration. No medically relevant responses were observed in the five treated patients. CONCLUSION: No biologically effective dose could be established for LY2457546 in chemotherapy-resistant AML patients. Lack of drug clearance prevented safe dose escalation, and the study was terminated early. Future efforts should be made to develop derivatives with a more favorable pharmacokinetic profile.

Is there a difference between T- and B-lymphocyte morphology?

We characterize T- and B-lymphocytes from several donors, determining cell diameter, ratio of nucleus to cell diameter, and refractive index of the nucleus and cytoplasm for each individual cell. We measure light-scattering profiles with a scanning flow cytometer and invert the signals using a coated sphere as an optical model of the cell and by relying on a global optimization technique. The main difference in morphology of T- and B-lymphocytes is found to be the larger mean diameters of the latter. However, the difference is smaller than the natural biological variability of a single cell. We propose nuclear inhomogeneity as a possible reason for the deviation of measured light-scattering profiles from real lymphocytes from those obtained from the coated sphere model.

Rhodococcus fascians infection accelerates progression of tobacco BY-2 cells into mitosis through rapid changes in plant gene expression.

* To characterize plant cell cycle activation following Rhodococcus fascians infection, bacterial impact on cell cycle progression of tobacco BY-2 cells was investigated. * S-phase-synchronized BY-2 cells were cocultivated with R. fascians and cell cycle progression was monitored by measuring mitotic index, cell cycle gene expression and flow cytometry parameters. Cell cycle alteration was further investigated by cDNA-AFLP (amplified fragment length polymorphism). * It was shown that cell cycle progression of BY-2 cells was accelerated only upon infection with bacteria whose virulence gene expression was induced by a leafy gall extract. Thirty-eight BY-2 genes showed a differential expression within 6 h post-infection. Among these, seven were previously associated with specific plant cell cycle phases (in particular S and G2/M phases). Several genes also showed a differential expression during leafy gall formation. * R. fascians-infected BY-2 cells provide a simple model to identify plant genes related to leafy gall development. R. fascians can also be regarded as a useful biotic agent to alter cell cycle progression and, thereby, gain a better understanding of cell cycle regulation in plants.

Expression and localization of CHODLDeltaE/CHODLfDeltaE, the soluble isoform of chondrolectin.

The C-type lectin family is a group of animal proteins which can be distinguished from other lectins by the presence of a Ca2+-dependent carbohydrate recognition domain (CRD) in their protein sequence. They are classified into 17 groups according to their domain architecture and have a wide variety of functions. The human chondrolectin gene encodes transmembrane (CHODL, CHODLf) and soluble proteins (CHODLDeltaE, CHODLfDeltaE) belonging to the family of C-type lectins because of the presence of one CRD domain in their N-terminal region. The CHODL splice variants (CHODLf, CHODLDeltaE and CHODLfDeltaE) are differentially expressed in T lymphocytes. The transmembrane-containing isoform CHODLf is localized in the ER-Golgi apparatus. CHODLDeltaE and CHODLfDeltaE are devoid of the transmembrane domain and terminate in QDEL, an ER retention signal. In this paper we have investigated the expression of the CHODLDeltaE/CHODLfDeltaE protein. This variant localizes in the late endoplasmic reticulum. We detected the protein in spleen and tonsils in a small population of lymphocytes. Moreover, the isoform seems to be differentially expressed in thymocytes and lymphocytes suggesting an important biological function during T cell development.

The 2001 World Health Organization and updated European clinical and pathological criteria for the diagnosis, classification, and staging of the Philadelphia chromosome-negative chronic myeloproliferative disorders.

The clinical criteria according to the Polycythemia Vera Study Group (PVSG) do not distinguish between essential thrombocythemia (ET), thrombocythemia associated with early-stage polycythemia vera (PV) and prefibrotic chronic idiopathic myelofibrosis (CIMF). The criteria only classify the advanced stage of PV with increased red cell mass. The classification of myeloproliferative disorders (MPDs), proposed by the World Health Organization (WHO) in 2001, is a compromise of the clinical PVSG and WHO bone marrow criteria, and excludes early stages of ET and PV. The updated European clinical and pathological criteria combine the WHO bone marrow criteria with established and new clinical, laboratory, biological, and molecular MPD markers. This allows clinicians and pathologists to diagnose early-stage MPD and to differentiate ET, PV, and prefibrotic chronic idiopathic myelofibrosis (CIMF). Depending on laboratory tests and diagnostic criteria used, the population of the MPD patients defined as ET, PV, and CIMF are heterogeneous at the clinical, laboratory, and biological and pathological levels. The recent discovery of the JAK2 V617F mutation, which is the cause of a distinct trilinear MPD in its manifold clinical manifestations during long-term follow-up, increases the specificity of a positive JAK2 V617F polymerase chain reaction (PCR) test for the diagnosis of MPD (near 100%), but only half of the ET and CIMF patients according to the PVSG (sensitivity 50%) and the majority of PV patients (sensitivity 95%) are JAK2 V617F positive. A comparison of the laboratory features of JAK2 V617-positive and JAK2 wild-type ET patients clearly showed that JAK2 V617-positive ET is characterized by higher values for hemoglobin, hematocrit, and neutrophil counts; lower values for serum erythropoietin (EPO) levels, serum ferritin, and mean corpuscular volume; and by increased cellularity of the bone marrow in biopsy material. This indicates that JAK2 V617-positive ET patients, diagnosed according to the PVSG criteria, represent a "forme fruste of PV" consistent with early PV mimicking ET (JAK2 V617F trilinear MPD). In contrast, the JAK2 wild-type ET patients had significantly higher platelet counts and usually had a clinical picture of ET with normal serum EPO levels, PRV-1 expression, and leukocyte alkaline phosphatase score, and a typical WHO ET bone marrow picture. The clinical and pathological data on JAK2 V617F-positive MPD patients suggest that the JAK2 V617F mutation defines one disease entity with several sequential steps of ET, PV, and secondary myelofibrosis during long-term follow-up, and that the wild-type JAK2 MPDs may represent another distinct entity with a related but different molecular etiology. MPD-specific markers such as serum EPO, endogenous erythroid colony formation (EEC), and JAK2 V617F have high specificities, but the sensitivities are not high enough to detect the early stages of the MPDs, ET, PV, and prefibrotic CIMF. Bone marrow histopathology in addition to clinical, laboratory, biological, and molecular markers, including the JAK2 V617 PCR test, serum EPO, PRV-1, EEC, LAP score, peripheral blood parameters, and spleen size on echogram will detect the early stages of MPD and allows diagnostic differentiation of the three primary MPDs (ET, PV, and CIMF) in both JAK2 V617F-positive and JAK2 wild-type MPD patients.

Clinical and laboratory features, pathobiology of platelet-mediated thrombosis and bleeding complications, and the molecular etiology of essential thrombocythemia and polycythemia vera: therapeutic implications.

Microvascular disturbances in essential thrombocythemia (ET) and polycythemia vera (PV), including erythromelalgia, and atypical and typical transient cerebral, ocular, and coronary ischemic attacks, are caused by platelet-mediated transient and occlusive thrombosis in the end-arterial circulation. ET patients with microvascular disturbances have shortened platelet survival, increased beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), and thrombomodulin (TM) levels, and increased urinary thromboxane B2 (TXB2) excretion, indicating platelet-mediated thrombotic processes. Inhibition of platelet cyclooxygenase-1 by aspirin is followed by relief of microvascular disturbances; correction of shortened platelet survival; correction of increased plasma beta-TG, PF4, and TM levels; and correction of increased TXB2 excretion to normal. In PV associated with thrombocythemia, increased hematocrit and whole blood viscosity aggravate the platelet-mediated microvascular syndrome of thrombocythemia to produce major arterial and venous thrombotic complications. Correction of hematocrit to normal by phlebotomy will reduce the major arterial and venous thrombotic complications, but fails to prevent the platelet-mediated microvascular circulation disturbances in PV patients because thrombocythemia persists. Complete relief and prevention of microvascular and major thrombosis in ET and PV patients, in addition to phlebotomy, are obtained by treatment with aspirin and not with coumarin. The discovery of JAK2 V617F gain of function mutation in patients with myeloproliferative disorders (MPDs) expands our insights into the molecular etiology and biological features of ET, PV, and chronic idiopathic myelofibrosis (CIMF). The current concept is that heterozygous JAK2 V617F mutation with increased kinase activity is enough for megakaryocyte proliferation and increased hypersensitive platelets with no or slightly increased erythropoiesis in ET and in early PV mimicking ET. Homozygous JAK2 mutation with pronounced kinase activity is associated with trilinear megakaryocyte, erythroid, and granulocytic myeloproliferation, myeloid metaplasia, and secondary myelofibrosis (MF), with the most frequent clinical picture of classical PV complicated by major thrombosis in addition to the platelet-mediated microvascular thrombotic syndrome of thrombocythemia. The positive predictive value of a JAK2 V617F polymerase chain reaction test for the diagnosis of MPDs is high (near to 100%), but only half of ET and MF (sensitivity 50%) and the majority of PV (sensitivity 85 to 97%) are JAK2 V617F positive. Bone marrow histopathology, when used in combination with specific markers such as serum erythropoietin, PRV-1, endogenous erythroid colony formation, peripheral blood parameters and red cell mass, has a high sensitivity and specificity (near 100%) to detect the early and overt stages of the MPDs and to differentiate between ET, PV, and CIMF in both JAK2 V617F-positive and -negative MPDs.

Cellular immunotherapy for cytomegalovirus and HIV-1 infection.

Current antiviral drugs do not fully reconstitute the specific antiviral immune control in chronically human immunodeficiency virus (HIV)-1-infected patients or in cytomegalovirus (CMV)-infected patients after hematopoietic stem cell transplantation. Therefore, immunotherapy in which the patient's immune system is manipulated to enhance antiviral immune responses has become a promising area of viral immunology research. In this review, an overview is provided on the cellular immunotherapy strategies that have been developed for HIV infection and CMV reactivation in immunocompromised patients. As an introduction, the mechanisms behind the cellular immune system and their importance for the development of a workable immunotherapy approach are discussed. Next, the focus is shifted to the immunopathogenesis of CMV and HIV-1 infections to correlate these findings with the concepts and ideas behind the viral-specific immunotherapies discussed. Current and future perspectives of active and passive cellular immunotherapy for the treatment of CMV and HIV-1 infections are reviewed. Finally, pitfalls and key issues with regard to the development of immunotherapy protocols that can be applied in a clinical setting are addressed.