This study aimed to determine the optimal growth factor combination for expansion of megakaryocyte (Mk) progenitors with clonogenic potential from CD34+-enriched mobilized peripheral blood stem cells (PBSC). Mobilized PBSC were monocyte depleted and CD34+ enriched, then cultured with various combinations of interleukin-3 (IL-3), IL-6, IL-11, Flt3 ligand (Flt3-L), stem cell factor (SCF), granulocyte-macrophage colonystimulating factor (GM-CSF), and erythropoietin (EPO), using a 2(7-3) IV fractional factorial design. Expansion of Mk committed progenitors (CD41+) and primitive precursors (CD61+ CD34+) was determined using FACS and colony-forming assays. Amplification of Mk progenitor production was attributed to IL-3 (p < 0.002), SCF (p < 0.001), and GM-CSF (p < 0.05). Flt3-L inhibited the production of total CD61+ cells (p < 0.05), CD61+CD34+ cells (p < 0.03), and total CD41a+ cells (p < 0.01). Addition of Flt3-L to the optimum growth factor combination of megakaryocyte growth and development factor (MGDF), SCF, IL-3, and GM-CSF caused the greatest increase in total nucleated cells but reduced Mk progenitor expansion. There was also a 20% reduction in Mk+ colonies from cells expanded in the presence of Flt3-L. Factorial analysis identified the optimal combination of growth factors required to expand Mk precursors with clonogenic potential. The addition of Flt3-L to the optimal combination of MGDF, SCF, IL-3, and GM-CSF reduced both the fold expansion of Mk progenitors and Mk colony numbers.

Platelets of patients with sickle cell disease (SCD) show evidence of mild activation in the non-crisis steady state and greater activation during vaso-occlusive crises (VOC). Prasugrel, a potent inhibitor of ADP-mediated platelet activation and aggregation, may be useful in attenuating VOC. We compared platelet responses to ADP stimulation in patients with SCD and healthy subjects before and after treatment with prasugrel. In a phase 1 study, platelet biomarker levels were assessed in 12 adult patients with SCD and 13 healthy subjects before and after 12 ± 2 days of 5.0 or 7.5 mg/day prasugrel. The following were determined in whole blood samples stimulated with 20 µM ADP: (i) percentages of monocytes and neutrophils with adherent platelets (cell-platelet aggregates); (ii) the relative number (mass) of platelets associated with each monocyte and neutrophil as reported by CD61 mean fluorescence intensity (MFI) of the monocyte-platelet and neutrophil-platelet aggregates; (iii) the percentages of platelets positive for surface expression of CD40 ligand (CD40L), P-selectin (CD62p) and activated glycoprotein IIb-IIIa (GPIIb-IIIa); and (iv) the percentages of platelets and monocyte-platelet aggregates positive for surface tissue factor (TF) expression. At baseline, there were no significant differences between cohorts in the percentages of platelets expressing activation biomarkers. Following 12 days of prasugrel administration, the percentages of platelets expressing activation biomarkers following ADP stimulation were reduced in both cohorts, and there were no significant differences between groups. Both patients with SCD and healthy subjects had significant reductions in the monocyte-platelet and neutrophil-platelet aggregate MFI and the percentage of platelets expressing P-selectin and activated GPIIb-IIIa (all p < 0.05). Healthy subjects also had significant reductions in monocyte-platelet aggregate percentages (p = 0.004), neutrophil-platelet aggregate percentages (p = 0.011) and the percentage of CD40L-positive platelets (p = 0.044) that were not observed in patients with SCD. Prasugrel administration to SCD patients attenuates ex vivo ADP-stimulated platelet activation as measured by the percentage of platelets positive for P-selectin and GPIIb-IIIa, thus reducing the proportion of platelets that may participate in aggregates. Furthermore, prasugrel decreases ex vivo ADP-stimulated platelet aggregation with monocytes and neutrophils as measured by the monocyte-platelet and neutrophil-platelet aggregate MFI. This implies that in the presence of prasugrel, fewer platelets adhere to monocytes and neutrophils, which may result in reducing cell-platelet aggregate size. Therefore, reduced platelet reactivity and decreased size of leukocyte-platelet aggregates suggest additional mechanisms by which prasugrel may provide benefit to patients with SCD and support further investigation of possible therapeutic benefits of prasugrel in this population.

The increasing use of cultured human cells in clinical trials is highlighting the need for alternatives to media containing animal sera that are typically used to support these cultures. Perfused cultures of BM mononuclear cells (MNC) were used to evaluate animal sera alternatives with respect to the output of primitive, progenitor, and stromal cells. A serum level of 20% was optimal, and this could be provided by FBS alone or by a mixture of horse serum (HoS) and FBS, but not by HoS alone. Allogeneic human plasma (20%) supported half the level of cell, CFU-GM, and LTC-IC output as compared with animal sera-containing control. Significant donor-to-donor variability in human plasma was observed, but this was mitigated by pooling of plasma samples. Autologous and allogeneic human plasma performed equivalently. The use of autologous or allogeneic human serum was found to be equivalent to the use of human plasma, but all were inferior to animal sera. Animal sera supported typical stroma and cobblestone formation, whereas stroma in human serum cultures was less dense. Eight commercial serum-free media were tested and found to support MNC expansion to varying degrees, but none approached the performance of the animal serum-containing control, particularly with respect to stromal (i.e., CFU-F) support. In fact, when MNC were cultured in parallel with CD34-enriched cells, output (from MNC) was higher only in control medium, apparently because serum-free media reduced accessory cell effects. Because of these results, a new serum-free medium was developed for MNC cultures. This formulation outperformed all commercial serum-free media, resulting in cell and LTC-IC output equivalent to that of control. However, CFU-GM and CFU-F output were 66% and 9% of control, respectively. Precoating the culture surface with collagen increased CFU-F (and Thy-1+ cell) output to control levels, although CFU-GM output was still lower than control. The addition of either fibronectin or PDGF had no measurable effect, nor did the use of 5-100-fold greater concentrations of growth factor supplementation. The serum-free medium also increased CD41+ and CD61+ cell output to 150%-220% of control levels. The development of this new serum-free medium has potential for use in the perfused BM MNC culture systems currently in clinical trials to test the efficacy of expanded cells after cytoablative chemotherapy.

Mammary stem cells (MSCs) expansion is associated with aggressive human breast cancer. The nuclear receptor peroxisome proliferator activated receptor γ (PPARγ) is a breast cancer tumor suppressor, but the mechanisms of this suppression are not completely characterized. To determine whether PPARγ regulates MSC expansion in mammary cancer, we deleted PPARγ expression in the mammary epithelium of an in vivo model of basal breast cancer. Loss of PPARγ expression reduced tumor latency, and expanded the CD24+/CD49f(hi) MSC population. PPARγ-null mammary tumors exhibited increased angiogenesis, which was detected in human breast cancer. In vivo inhibition of a PPARγ-regulated miR-15a/angiopoietin-1 pathway blocked increased angiogenesis and MSC expansion. PPARγ bound and activated a canonical response element in the miR-15a gene. PPARγ-null tumors were sensitive to the targeted anti-angiogenic drug sunitinib but resistant to cytotoxic chemotherapy. Normalization of tumor vasculature with sunitinib resulted in objective response to cytotoxic chemotherapy. Chemotherapy-treated PPARγ-null mammary tumors exhibited luminal phenotype and expansion of unipotent CD61+ luminal progenitor cells. Transplantation of chemotherapy-treated luminal progenitor cells recapitulated the luminal phenotype. These results have important implications for anti-angiogenic therapy in breast cancer patients.

BACKGROUND

Conventional automated cell counters cannot accurately count residual red blood cells (rRBCs) that are often present in various blood products. A two-color flow cytometric method (FC) was validated for detecting and enumerating rRBCs in platelets (PLTs) and mononuclear cell (MNC) products.

METHODS

PLT and MNC products for PLTs (CD61-fluorescein isothiocyanate) and rRBC (anti-glycophorin A-phycoerythrin) were double stained, and data were acquired with a flow cytometer. Assay linearity, accuracy, and precision were assessed with a standard-dilution series of rRBCs. This assay was used to determine the rRBCs of apheresis PLTs collected with Trima Accel (Gambro BCT) and MNC products collected with the COBE Spectra (Gambro BCT).

RESULTS

The linear range of this assay in PLT and MNC products is 10 to 2000 RBCs per microL (R2=0.994). FC had a mean intraassay coefficient of variation of 11.8 percent at 34 RBCs per microL. A standard clinical hematology analyzer overestimated rRBCs in MNC products by 1.59x10(5)+/-0.7x10(5) RBCs per microL. Apheresis PLTs had a median of 17.4 RBCs per microL, with 99.0 percent containing fewer than 90.0 RBCs per microL.

CONCLUSIONS

This method for determining rRBCs in blood products is accurate and repeatable with a lower limit of detection adequate to assess currently available blood products. FC should be considered for determining rRBCs in MNC products.

Immunophenotypic studies have a limited role in the diagnosis of chronic myelogenous leukemia (CML) but are increasingly being used in CML blast transformation (BT). Determination of the cell lineage of CML blasts is clinically important because patients with lymphoid blast transformation have a better response to chemotherapy and longer survival than those with other lineages. We studied the morphologic, cytochemical, immunophenotypic, cytogenetic, and molecular features of 20 patients with Philadelphia chromosome-positive CML and more than 10% blast cells in peripheral blood or bone marrow. The blasts were morphologically heterogeneous. CD33 was expressed in 19 cases (95%), followed by CD13 (85%), CD11c (80%), CD36 (60%), CD117 (40%), and CD15 (30%). Seven cases (35%) had a precursor-B lymphoid immunophenotype, and 13 (65%) had a predominantly myeloid immunophenotype. Of the former group, of which only one had a pure lymphoid phenotype, terminal deoxynucleotidyl transferase (TdT) and CD19 were expressed in 100%, CD10 in 85.7%, and CD20 in 14.3%. Of the latter group, all 13 expressed from 3 to 6 myeloid antigens, with 46.2% myeloperoxidase positive and 69.2% CD61 positive. No cases were interpreted as T lineage, but the T-cell antigens CD3, CD4, CD5, and CD7 were expressed in 5.0, 40.0, 5.3. and 30.0% of all cases, respectively. In most cases, the immunophenotype of the CML blasts could not be predicted from their morphologic features. Polymerase chain reaction showed that 80.0% of the lymphoid group and 37.5% of the myeloid group had immunoglobulin heavy-chain gene rearrangements. The frequent lineage infidelity of the blast cells in CML BT seems to be related to the stem cell origin of this disorder. Such lineage infidelity, however, makes classification of many cases difficult and the significance of and criteria for biphenotypic blast crisis of CML is yet to be determined.

Megakaryocytopoiesis involves the process of the development of hematopoietic stem cells into megakaryocytes (MKs), which are the specialized cells responsible for the production of blood platelets. Platelets are one of the crucial factors for hemostasis and thrombosis. In terminally differentiated MKs many molecular process such as caspase activation and a massive cytoskeletal rearrangement drive the formation of cytoplasmic extensions called proplatelets. These cytoplasmic extensions packed with granules and organelles are then released from the bone marrow into the blood circulation as platelets. Classically, caspase activation is associated with apoptosis and recent reports suggest their involvement in cell differentiation and maturation. There is no clear evidence demonstrating the stimulus for caspase activation during megakaryocyte development. In the current study, we attempted to understand the importance of endoplasmic reticulum stress in the caspase activation during megakaryocyte maturation. We used human megakaryoblstic cell line (Dami cells) as an experimental model. We induced these cells with PMA (Phorbol 12-myristate 13-acetate) a known inducer of megakaryocyte to understand the involvement of ER stress during maturation, with increased cell size in association with ER stress induced caspase activation. We used Thapsigargin, a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca²⁺ATPase (SERCA) as a positive control to induce ER stress.Upon its treatment, we observed larger and adherent cells with the increased expression of megakaryocytic markers (CD41 and CD61) and UPR markers in PMA or Thapsigargin treated cells as compared to control. Also, Thapsigargin treatment induced increased caspase activity and PARP cleavage and showed decrease in ER calcium levels leading to reduced activity of calcium-dependent ER chaperones and accumulation of unfolded protein. The increased megakaryocyte maturation markers alongside with ER stress and caspase activation suggests the importance of ER stress in caspase activation during MK maturation.

OBJECTIVE

Abnormal platelet counts have been noticed in acquired immune deficiency syndrome (AIDS) patients. However, the actual state of platelets in AIDS is unclear. We hypothesize that platelets are activated and platelet-derived microparticles increase in murine AIDS.

METHODS

To elucidate the ethanol effects on platelets in murine AIDS, we studied four groups: control, murine AIDS, ethanol, and ethanol plus murine AIDS. Platelet CD62p as a platelet activation marker and CD61(+) microparticles as platelet microparticles (PMPs) were measured by flow cytometry.

RESULTS

Platelets were significantly activated in mice with murine AIDS and chronic ethanol consumption. Increased platelet CD62p expression and increased PMPs were most pronounced in advanced stages of murine AIDS. Chronic ethanol consumption persistently enhanced platelet activation and PMP formation.

CONCLUSIONS

Elevated platelet CD62p and PMPs may represent a pro-thrombotic status that have important pathological consequences.

The plasma-membrane integrin αIIbβ3 (CD41/CD61, GPIIbIIIa) is a major functional receptor in platelets during clotting. A common isoform of integrin β3, Leu33Pro is associated with enhanced platelet function and increased risk for coronary thrombosis and stroke, although these findings remain controversial. To better understand the molecular mechanisms by which this sequence variation modifies platelet function, we produced transgenic knockin mice expressing a Pro32Pro33 integrin β3. Consistent with reports utilizing human platelets, we found significantly reduced bleeding and clotting times, as well as increased in vivo thrombosis, in Pro32Pro33 homozygous mice. These alterations paralleled increases in platelet attachment and spreading onto fibrinogen resulting from enhanced integrin αIIbβ3 function. Activation with protease-activated receptor 4- activating peptide, the main thrombin signaling receptor in mice, showed no significant difference in activation of Pro32Pro33 mice as compared with controls, suggesting that inside-out signaling remains intact. However, under unstimulated conditions, the Pro32Pro33 mutation led to elevated Src phosphorylation, facilitated by increased talin interactions with the β3 cytoplasmic domain, indicating that the αIIbβ3 intracellular domains are primed for activation while the ligand-binding domain remains unchanged. Acute dosing of animals with a Src inhibitor was sufficient to rescue the clotting phenotype in knockin mice to wild-type levels. Together, our data establish that the Pro32Pro33 structural alteration modifies the function of integrin αIIbβ3, priming the integrin for outside-in signaling, ultimately leading to hypercoagulability. Furthermore, our data may support a novel approach to antiplatelet therapy by Src inhibition where hemostasis is maintained while reducing risk for cardiovascular disease.

OBJECTIVE

To provide an earlier diagnosis and efficiently treat acute rejection episodes (ARE) after renal transplantation, we studied its relationship to platelet activation.

METHODS

The peripheral blood levels of platelet surface glycoprotein (CD61), platelet activation-dependent granule membrane protein (CD62p), lysosomal enzyme glycoprotein (CD63), macula densa granule membrane glycoprotein (CD42a), and fibrinogen receptor monoclonal antibody (PAC-1) among 203 patients with uremia in various stages before and after transplantation were assayed by flow cytometry. The patients with ARE were prospectively and randomly assigned to either a treatment group with an antiplatelet activation agent or a control group.

RESULTS

The incidence of ARE was remarkably increased among patients with greater expression levels of platelet activation markers in peripheral blood preoperatively. The values of platelet activation markers were significantly higher among patients with ARE compared with those showing either normal graft function or acute tubular necrosis. The greater the increase in CD63, the worse the ARE. The expression levels of platelet activation markers decreased remarkably among the group treated with a platelet activation inhibitor in addition to antirejection therapy: the rejection reversal time shortened and the dose of antihuman thymocyte globulin (ATG) was lower. The sensitivity of platelet activation markers was better than its specificity.

CONCLUSIONS

Our studies demonstrated an association between platelet activation and ARE after renal transplantation. Platelet activation before transplantation can predict the occurrence of ARE. Platelet activation inhibitor therapy after transplantation improved ARE reversal.

Scant knowledge exists concerning lineage-restricted mixed chimerism (mCh) after allogeneic peripheral blood stem cell transplantation (PSCT) in patients with chronic idiopathic myelofibrosis (CIMF). Following a sex-mismatched PSCT, a combined immunopheno- and genotyping by fluorescence in-situ hybridization (FISH) was performed on sequential bone marrow (BM) biopsies at standardized intervals. Results were compared with PCR analysis of corresponding peripheral blood samples in five patients. According to FISH, pretransplant specimens revealed a gender congruence of more than 99%, while in the first three months the total BM exhibited a persistent fraction of host cells (30% to 40%) with a tendency to decline after about one year. It is noteworthy that the majority of endothelial cells maintained a recipient origin, whereas CD34+ progenitors and especially CD61+ megakaryocytes exhibited only very few host-derived cells. In keeping with the prevalence of donor cells in the hematopoietic compartment, PCR analysis of peripheral blood cells displayed a non-significant degree of mCh. In conclusion, according to FISH and PCR analysis, successful PSCT in CIMF results in an almost complete chimeric (donor-derived) state of the hematopoietic cell population. The non-transplantable stromal compartment includes the vascular endothelium with a predominance of recipient cells. The minimal mCh of this population implies probably a donor-derived origin (endothelial progenitor cells).

Using sequential double-immunostaining and a newly-developed three-dimensional (3D-) reconstruction technique on serially cut sections from bone marrow trephines, we studied the transmural passage of megakaryocytes through the sinus wall. Biopsies derived from patients with primary (idiopathic) osteomyelofibrosis were exposed to monoclonal antibody against type IV collagen to delineate the sinus walls and also the frequently thickened basement membrane. Staining with the primary antibody was followed by Y2/51 (CD61) to identify all elements of megakaryopoiesis. In most instances serial sectioning and 3D-reconstruction revealed an amoeboid shape of megakaryocytes and a tandem-like arrangement in close spatial contact with the abluminal surface of the sinus wall. Preceded by formation of cytoplasmic processes, straight penetration of entire megakaryocytes through gaps in the sinus walls into the lumen was seen. Where collagen deposits apparently presented a barrier, a mole-like tunnelling through the basement membrane material (type IV collagen) was recognizable. Our findings are in keeping with the assumption that megakaryocyte locomotion is an essential requirement for normal thrombocytogenesis.

Polycythemia vera (PV) is a clonal disorder characterized by trilinear hematopoietic proliferation. PV progenitors are hypersensitive to several growth factors although their receptor expression is reduced or absent. In this study selected CD34+ peripheral blood (PB) cells from PV patients, PB healthy donors and patients with secondary polycythemia (SP) were investigated and compared concerning frequency, morphology, antigen expression, transcription of differentiation markers and proliferation as well as apoptosis rate following short-term culture. The highest amount of CD34+ cells was found in the SP group while the frequency was slightly lower but more variable in PV. Native PV CD34+ cells varied in shape and form and developed intracytoplasmatic organelles like mitochondria and a more extended Golgi apparatus while in the other groups they constituted a uniform phenotype. However, no premature transcripts for glycophorin A (GypA) and CD41b, both markers for advanced erythropoiesis and megakaryopoiesis, could be detected in sorted PV progenitors. Also, coexpression for early acting hematopoietic cytokine receptors IL-3Ralpha and c-Kit and for initial erythropoiesis (GypC) or megakaryopoiesis (CD61) was similar in the different groups. Performing 96 h cocultures with bone marrow (BM) fibroblasts the frequency of CD34+ cells was elevated, downregulation of IL-3Ralpha delayed, coexpression of GypC reduced and proliferative activity higher in the PV group. Our results suggest that primitive hematopoiesis is altered in PV because PB CD34+ cells in this disease are characterized by a maturation dissociation with increased activation in untreated populations and a delayed differentiation in short-term cultures.

Calcineurin inhibitor (CI) toxicity in renal allografts is frequently associated with arteriolar injury. Platelet activation occurs in response to vascular injury associated with CI therapy. Platelets are detectable in tissue sections by immunohistochemistry for CD61. This immunohistochemical study examines patterns of platelet deposition in CI-associated vascular toxicity of renal allografts. Renal allograft biopsies were grouped into (i) CI-associated thrombotic microangiopathy (CITMA, n = 28); (ii) vascular CI toxicity without thrombotic microangiopathy (VTox, n = 43); and (iii) allograft controls with minimal arteriolopathy abnormalities, both with CI exposure (MA, n = 10) and without CI exposure (NCI, n = 15). Two-micrometer paraffin sections were stained using a monoclonal antibody to CD61 by standard immunoperoxidase methods. Mural and luminal CD61 deposits in arterioles were graded from 0 to 3+, and proportions of arteriolar and glomerular profiles with CD61 deposits were determined for each biopsy. Granular CD61 deposits were detected in arterioles in biopsies with CITMA (92.9%) and VTox (81%) and less frequently in MA (30%) and NCI (33%). The proportion of arterioles with CD61 deposits was greater in CITMA than in VTox (46.3% +/- 28.5% vs 21.3% +/- 22.2%, P = .001) and more extensive than in controls (MA, 3.6% +/- 8.9%; NCI, 3.2% +/- 5.5%). Median arteriolar CD61 grades for CITMA exceeded grades for VTox (2 vs 0.5, P = .001), and CD61 grades in VTox were significantly greater than in controls with MA (0) and NCI (0) (P = .0001). In conclusion, arteriolar mural platelet CD61 deposition was observed in vascular CI toxicity and was most extensive and severe in CI-associated thrombotic microangiopathy. Identification of "insudative platelet arteriolopathy" in renal allograft biopsies, by immunohistochemical detection of CD61, may facilitate recognition of vascular CI toxicity.

Using thrombopoietin (TPO), as selective pressure, several TPO-dependent clones were isolated from the murine multipotential IL-3-dependent cell line 32D. Four of them were fully characterized. They depended on TPO for survival and proliferation and, although retaining the capacity to grow in IL-3, did not respond to either EPO, G-CSF or GM-CSF. 32D TPO cells were heterogeneous in morphology and ranged from small cells, with a DNA content nearly tetraploid and a modal chromosome no. 66, to cells 50-75 microm in diameter containing multiple (up to 5-6) interconnected nuclei with a clear megakaryocyte (Mk) morphology by electron microscopy. Cell sorter isolation and single cell cloning experiments indicated that the small cells were those capable to proliferate in TPO and to generate the larger ones over time. 32D TPO cells expressed Mk-specific markers by FACS (CD41, CD61 and 2D5) and RT-PCR (acetyl cholinesterase E and platelet factor 4) and their unique profile, by gene array analysis, included expression of urokinase plasminogen activator surface receptor (CD87 or uPAR), plasminogen activator inhibitor and coagulation factor II (thrombin) receptor (Cf2r). In addition, by quantitative RT-PCR, 32D TPO clones expressed levels of Gata1 similar to those expressed by freshly isolated Mks (DeltaCt approximately 4.7 in both cases). In conclusion, the 32D TPO subclones described here are among the few pure Mk cell lines isolated so far and, for their unique properties, may prove themselves as a useful model to study Mk differentiation.

Platelet engraftment, the time course and magnitude of platelet recovery (PR) post-transplant, is imprecisely defined but is most often reported as the time to transfusion (tx) independence and/or a platelet count>> or = 20,000/microl. While correlations between engraftment time for granulocytes (PMN) and the dose of CD34-positive cells per kilogram are established, such associations have not been established for platelet engraftment. The objective of this study was to quantify subpopulations of CD34-positive cells in peripheral blood stem cell (PBSC) collections of normal, colony-stimulating factor-granulocyte) (G-CSF) primed donors that might represent megakaryocyte (MK) precursors, and to determine whether there is a statistical association between the dose transfused and the time course of the recovery. Based on previously published data of the sequential expression of CD34, HLA-DR, and CD61, among others, during MK maturation, a combination of corresponding antibodies for the detection of various antigen coexpressions by flow cytometry fluorescence-activated cell sorting [FACS] was chosen. CD34-positive cells were further subdivided into CD34++ (bright) and + (dim). Ploidy of density-gradient separated cells was examined in subsequent donor samples by FACS. For the entire group of patients, there was no strong correlation between any of the studied subpopulations and time to PR. Only in a selected groups of patients whose platelet counts showed a sustained increase during the first 6 days after engraftment, there was a weak correlation between the time to PR and the quantity of CD34+/+CD61+ (r = -0.57) and CD34++HLA-DR-CD61+ (r = -0.62) cells infused. The magnitude of platelet production in these pt., a product of the peripheral blood platelet count and the patient's blood volume, was correlated with the time to PR (r = -0.73). We conclude from this study that subpopulations within CD34+ cells are making some contribution to PR in allogeneic peripheral blood stem cell transplantation, but the correlations are not sufficiently strong because there are probably too many unpredictable and unknown variables in the allogeneic setting that influence the pattern of engraftment.

BACKGROUND

Climacteric increases the risk of thrombotic events by alteration of plasmatic coagulation. Up to now, less is known about changes in platelet- (PMP) and endothelial cell-derived microparticles (EMP).

METHODS

In this prospective study, plasma levels of microparticles (MP) were compared in 21 premenopausal and 19 postmenopausal women.

RESULTS

No altered numbers of total MP or EMP were measured within the study groups. However, the plasma values of CD61-exposing MP from platelets/megakaryocytes were higher in premenopausal women (5,364 × 10(6)/l, range 4,384-17,167) as compared to postmenopausal women (3,808 × 10(6)/l, range 2,009-8,850; p = 0.020). This differentiation was also significant for the subgroup of premenopausal women without hormonal contraceptives (5,364 × 10(6)/l, range 4,223-15,916; p = 0.047; n = 15). Furthermore, in premenopausal women, higher plasma levels of PMP exposing CD62P were also present as compared to postmenopausal women (288 × 10(6)/l, range 139-462, vs. 121 × 10(6)/l, range 74-284; p = 0.024). This difference was also true for CD63+ PMP levels (281 × 10(6)/l, range 182-551, vs. 137 × 10(6)/l, range 64-432; p = 0.015).

CONCLUSIONS

Climacteric lowers the level of PMP but has no impact on the number of EMP in women. These data suggest that PMP and EMP do not play a significant role in enhancing the risk of thrombotic events in healthy, postmenopausal women.

BACKGROUND

The mechanism of platelet recovery after hematopoietic stem cell transplantation and the factors that influence its time-course are not fully understood. Rapid hematopoietic recovery results in a reduction of transplantation-related complications. In the present study, we questioned and analyzed whether there were important factors predicting the speed of platelet engraftment.

METHODS

Thirty-seven patients with various hematologic diseases transplanted with allogeneic BM between January 2002 and December 2005 were included. We investigated the differences in mononuclear cell counts (MNC), numbers of infused CD34(+), CD34(+) CD41(+) and CD34(+) CD61(+) cells and phenotypic analysis of homing-associated cell adhesion molecules (CXCR4, CD49d and CD49e). The number of megakaryocytes formed in vitro (colony-forming unit-megakaryocytes; CFU-Mk) was also measured.

RESULTS

Median days of ANC>>/=0.5x10(9)/L and platelet count>>/=20x10(9)/L were 14.8 and 17.3, respectively. The number of infused CD34(+) CD41(+) and CD34(+) CD61(+) cells correlated much better with the time to platelet engraftment than that of infused CD34(+)cells (P<0.05 each). Rapid platelet recovery also occurred in patients receiving both higher homing-associated cell adhesion molecule doses and CFU-Mk (P<0.05 each).

CONCLUSIONS

Rapid platelet recovery has several advantages, including reducing the cost of supportive therapy and reducing the risk of fatal bleeding as a result of severe thrombocytopenia. Our findings suggest that phenotypic and clonogenic assessment of infused progenitor cells can identify patients in whom platelet engraftment is likely to be significantly delayed, and new strategies to overcome related problems might be employed in the very near future.

BACKGROUND

Dengue infection is a major public health problem. The mechanisms underlying severe bleeding in dengue DHF are not completely understood. It was proposed that the dengue virus nonstructural-1 protein (DNS1) generated antibodies to common epitopes on human blood clotting and integrin/adhesin proteins on the platelet. The proof on the functional correlation between DNS1 and platelet integrin/adhesin proteins is needed.

METHODS

Here, the author used a new gene ontology technology to predict the molecular function and biological process of DNS1 and platelet integrin/adhesin protein, CD61.

RESULTS

According to this study, the author can identify no function and biological process of DNS1. Also, there is no existence of functional similarity between DNS1 and CD61.

CONCLUSIONS

The hypothesis on role of molecular mimicry between DNS1 and CD61 is not supported in this study.

Endoreplication and maturation of the megakaryocyte (MK) may be retarded or delayed during ontogenesis. In this study, CD34+ cells were isolated from both human fetal liver and adult bone marrow and incubated with thrombopoietin (TPO). The cell number, morphological characteristics, platelet-associated antigen phenotype, maturation stage and DNA ploidy of CD41+ cells were examined from day 0 to day 12 in culture. 1) TPO stimulated the proliferation of fetal liver (FL)-derived CD34+ cells with a mean 73.14-fold increase of CD41+ cells after 12 d in culture. Adult BM-derived CD34+ cells increased only slightly, with a mean 8.18-fold increase of CD41+ cells. 2) Although the membrane phenotype of both FL CD34+-derived MKs and BM CD34+ -derived MKs analyzed with CD41a, CD42a, CD61 and CD34 were similar, all FL CD34+-derived MKs were in maturation stage I and II and in low ploidy (<4N) class. By comparison, BM CD34+ MKs possessed 15% MKs in maturation stage III and IV and with 23% MKs in high ploidy class (>> 4N). 3) Most of cultured FL-derived CD34+ cells did not have a well developed demarcation system (DM) and numerous alpha-granules after 12 d incubation. von Willebrand factor (vWF) appeared earlier on the cultured BM-derived CD34+ cells than on FL-derived CD34+ cells. 4) The expression of both cyclin E and cyclin B1 progressively increased in FL CD34+ cells induced by TPO during 12 d in culture. 5) The expression of cyclin D1 gradually decreased in FL CD34+ cells induced by TPO over 12 d incubation. 6) Immunocytochemical analysis showed that cyclin D3 was detected only in cytoplasm of cultured FL-derived CD34+ cells, whereas in both cytoplasm and nuclei of cultured BM-derived CD34+ cells. These data suggest that FL-derived CD34+ cells contain a high proportion of immature megakaryocytic progenitor cells. It further suggests that TPO can push these progenitor cells into proliferation by upregulating the expression of cyclins B1 and E, and drive a high proportion of cells into megakaryocytic lineage.

Hemostatic disorders associated with chronic renal failure (CRF) include hemorrhagic and/or thrombotic manifestations, which were ascribed, in part, to uremic platelet dysfunction including abnormalities of expression of platelet glycoprotein receptors. There is, however, still no general agreement on the exact characterization of these platelet abnormalities. This study aims at characterizing the platelet glycoprotein abnormalities associated with CRF, by recording the effect of the three renal replacement therapies, hemodialysis (HD), chronic ambulatory peritoneal dialysis (CAPD), and renal transplantation, on these receptors. The study, which was mainly cross-sectional, included two groups: (i) Patient groups (n = 50): HD patients (n = 20), CAPD patients (n = 10) and successful renal transplant patients (n = 20); (ii) Healthy Controls (n = 34): 23 were men and 11 were women who were age- and sex-matched with the patients. Flow cytometric quantitation of CD41, CD42a, CD42b and CD61 was carried out using a Becton-Dickinson FACScan. The expression of CD41 levels showed a highly significant increase in HD and CAPD patients when compared with the normal control levels. However, levels in transplant patients were comparable to the normal control levels. On the other hand, the expression of CD42a, CD42b, and CD61 showed no significant change in HD and CAPD patients when compared with normal control levels, but there was a significant decrease in transplant patients when compared to the normal control levels. In conclusion, there was evidence of increased expression of one glycoprotein receptor (GpIIb-IIIa) pre-dialysis whether HD or CAPD. In transplant patients, no evidence of platelet activation could be demonstrated.

Chronic myeloprolifeative diseases (CMPD) are clonal hematopoietic stem cell disorders characterized by excessive proliferation and production of one or more of the myeloid cells and are subclassified according to the predominant cells, such as chronic myelogenous leukemia (CNL), chronic eosinophilic leukemia (CEL), polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF). This brief review focuses on the characteristic morphology of each clinical entity and the useful cytochemical (including leukocyte alkaline phosphatase, myeloperoxidase, butyrate esterase, chloroacetate esterase and cyanide-resistant peroxidase) and immunohistochemical (including von Willebrand factor/CD61, keratin, tryptase, CD117, CD68 (PGM-1), c-Mpl and bFGF) stains for differential diagnosis.

Whether hematopoietic stem cells (HSCs) express lineage markers is controversial. In this study, we highly purified HSCs from the adult bone marrow of C57BL/6 mice and examined their gene expression and reconstitution potential. We first focused on the integrin family. Single-cell reverse transcription polymerase chain reaction revealed that the expression of ItgaM/Itgb2 (Mac-1) and Itga2b/Itgb3 (CD41/CD61) gradually increased along HSC differentiation, whereas Itga4, Itga5, Itga6, and ItgaV (CD51) together with Itgb1 were highly expressed in both HSCs and hematopoietic progenitor cells (HPCs). We next fractionated HSCs based on their expression of Mac-1, CD41, and CD51 by flow cytometry. We detected Mac-negative and Mac-low, but not Mac-high cells, in the HSC population. We also detected CD41-negative, -low, and -high cells in the HSC population. Competitive repopulation revealed that Mac-1-negative and -low HSCs were functionally similar, and CD41-negative and -low HSCs were functionally similar, at the single-cell level, but CD41-high HSCs were not detectable. We then found that the selection of Mac-1-negative HSCs or CD41-negative HSCs had no advantage in HSC purification. We moreover found that HSCs expressed more CD51 than CD41, and HPCs expressed more CD41 than CD51, suggesting that CD51 expression was gradually replaced by CD41 expression during megakaryocyte differentiation. We concluded that low levels of Mac-1 and CD41 expression are irrelevant to the self-renewal and differentiation potentials in HSCs.

Evidence is presented that the mAb 25E11, directed against the platelet integrin alpha IIb beta 3 (glycoprotein IIbIIIa;CD41b/CD61) also binds the distinct myeloid cell integrin alpha M beta 2 (Mac-1;CDIIb/CD18). The Ab is shown to identify only the alpha IIb beta 3 integrin complex and not the individual subunits in crossed Ab immunoelectrophoresis of platelet lysate. From cultured human macrophages, sequential immunoprecipitation of labeled glycoproteins indicated that 25E11 also bound the Mac-1 (CD11b/CD18) complex. This was confirmed using COS-7 and WOP cells doubly transfected with alpha M (CD11b) and beta 2 (CD18) or with alpha L (CD11a) and beta 2 when it was found that the Ab bound only the alpha M beta 2 transfectants. Studies with these cells and the RC2A myeloid cell line stimulated with tetradecanoyl phorbol acetate or FMLP indicated that the 25E11 epitope on Mac-1 did not depend on cell activation for its expression. The rationale for this cross-reactivity is not known, but since the 25E11 Ab inhibits the function of both platelets and myeloid cells, it is likely that this shared epitope is important to integrin function. Given the expression of this epitope on IIbIIIa and Mac-1, the dominant integrins of platelets and granulocyte/macrophage cells, but not on other integrins, a role of this epitope in the early events of inflammation is suggested.