BACKGROUND

Microvesicles are small vesicles expressing specific antigens from their cells of origin. Elevated levels of microvesicles have been shown to be associated with coagulation disorders as well as with different types of malignancies. This study aims to evaluate a possible correlation of different microvesicle subpopulations with a positive history of venous thromboembolism (VTE) in patients with soft tissue sarcoma.

METHODS

Annexin V - positive microvesicles, leukocyte (CD45-positive), platelet (CD61-positive), activated platelet (CD62P-, CD63-positive), endothelium-derived (CD62E-positive) and tissue-factor (CD142-positive) microvesicles were identified in the peripheral blood of patients with soft tissue sarcoma (n = 39) and healthy controls (n = 17) using fluorescence-activated cell sorting (FACS).

RESULTS

Both the total amount of Annexin V-positive microvesicles and levels of endothelium-derived (CD62E-positive) microvesicles were shown to decrease significantly after tumor resection (n = 18, p = 0.0395 and p = 0.0109, respectively). Furthermore, the total amount of Annexin V - positive microvesicles as well as leukocyte (CD45-positive) and endothelium-derived (CD62E-positive) microvesicles were significantly higher in patients with grade 3 (G3) soft tissue sarcoma (n = 9) compared to healthy controls (n = 17) (p = 0.0304, p = 0.0254 and p = 0.0357, respectively). Moreover, patients with G3 soft tissue sarcoma (n = 9) presented higher levels of Annexin V-positive and endothelium-derived (CD62E-positive) microvesicles compared to patients with grade 2 (G2) soft tissue sarcoma (n = 8) (p = 0.0483 and p = 0.0045). Patients with grade 1 (G1) soft tissue sarcoma (n = 3) presented with significantly lower levels of platelet (CD61-positive) microvesicles than patients with G3 soft tissue sarcoma (n = 9) (p = 0.0150). In patients with a positive history of VTE (n = 11), significantly higher levels of activated platelet (CD62P- and CD63-positive) microvesicles (p = 0.0078 and p = 0.0450, respectively) were found compared to patients without a history of VTE (n = 28).

CONCLUSIONS

We found significantly higher levels of Annexin V-positive and endothelium-derived (CD62E-positive) microvesicles to be circulating in the peripheral blood of patients with G3 soft tissue sarcoma compared to patients with G2 soft tissue sarcoma. Furthermore, we showed that high counts of activated platelet-derived microvesicles correlate with the occurrence of VTE. Thus, the detection of these microvesicles might be an interesting new tool for early diagnosis of soft tissue sarcoma patients with increased risk for VTE, possibly facilitating VTE prevention by earlier use of thromboprophylaxis.

In vitro infection of CD61+ megakaryocytic cells with human herpesvirus-7 (HHV-7) induced a drastic increase of apoptosis. Moreover, cells surviving HHV-7 cytotoxicity showed enhanced megakaryocytic maturation with respect to control cultures. These data suggest that HHV-7 reactivation in the bone marrow of HIV-1 infected individuals may contribute to impair megakaryocytopoiesis.

Flow cytometry was used to screen a panel of 320 mAbs, submitted to the Animal Homologues Section of the HLDA8, for mAbs that recognize epitopes conserved on orthologous leukocyte differentiation antigens (LDA) in goats, lamas, and rabbits. Nineteen mAbs specific for CD11a (1), CD14 (3), CD18 (1), CD21 (1), CD29 (2), CD44 (2), CD47 (3), CD49d (1), CD172a (1), CD45RB (1), CD61 (1), RACT48A, and GBSP71A reacted with goat LDA. Twenty three mAbs specific for CD7 (1), CD9 (2), CD11a (1), CD14 (3), CD18 (4), CD29 (1), CD32 (1), CD44 (1), CD47 (4), CD49d (2), CD50 (1), CD80 (1), CD172a (1), and GBSP71A reacted with llama LDA. Eighteen mAbs specific for CD9 (2), CD11a (1), CD14 (2), CD18 (4), CD21 (1), CD44 (2), CD45RB (1), CD49d (1), CD209 (1), RACT48A, and GBSP71A reacted with rabbit LDA. The specificities of two cross reactive mAbs that recognize different conserved epitopes on all leukocytes in two species (RACT48A) and all three species (GBSP71A) have not been determined. The patterns of reactivity of most of the mAbs were consistent with patterns of reactivity noted on human leukocytes. The specificity of some cross reactive mAbs generated in non-human species were validated on human leukocytes. Further studies are needed to verify that CD7, CD32, CD45RB, CD50, and CD209 recognize orthologous molecules in the indicated species.

OBJECTIVE

Umbilical cord blood (UCB) is a rich source of hematopoietic cells. Here, for the first time, we surveyed the effects of different concentrations of platelet growth factors and cytokine cocktail (CC) on the expansion and differentiation of UCB CD133(+) stem cells into megakaryocyte progenitors.

METHODS

UCB CD133(+) cells were separated by magnetic cell sorting and cultured in different concentrations of platelet growth factors in combination with a CC containing interleukins 3 and 6, stem cell factor, and thrombopoietin. Cell expansion and differentiation were assessed using mononuclear cell count and flow cytometry.

RESULTS

The results show that either activated platelet-rich plasma or the platelet supernatant, when added in the first day of culture, significantly suppress the expansion of CD133(+) cells after 7 days in culture (p < 0.05). By contrast, the expression of CD41, CD61, and CD42b markers in the presence of all platelet growth factors increased compared with that of the control (p < 0.05).

CONCLUSIONS

Taken together, platelet growth factors in the presence of CC suppress ex vivo expansion of UCB CD133(+) cells and enhance their differentiation into megakaryocytic progenitor cells in a dose- and time-dependent manner.

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 +/- 0.9% CD34+ cells, 2.6 +/- 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 +/- 1.8-fold, but the number of viable CD34+ cells decreased by 46 +/- 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 +/- 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 +/- 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.

The distribution of cell adhesion molecules (CAMs) and matrix proteins in the normal synovium of four subjects was studied by immunohistology in order to determine the factors governing the cellular and tissue organization of the intimal and subintimal compartments. Basement membrane proteins, laminin, and collagen type IV, as well as vitronectin and fibronectin, were identified in the intima and there was corresponding expression of integrin and non-integrin receptors (e.g., CD29, CD49b, CD49d, CD49e, CD49f, CD51, CD61, CD44) for these matrix proteins. There were notable differences in CAM expression between intimal, subintimal, and vascular compartments of the synovial membrane. Phenotypic heterogeneity for CAMs involved in cell-cell interactions, particularly CD11a, CD11b, ICAM-1, and HLA-DR, was also present. The range of CAMs expressed by synovial and endothelial cells not only indicates a structural role for these antigens, but also suggests that they may control leucocyte traffic into the membrane, including recruitment of cells into the synovial lining.

Research on normal human megakaryopoiesis has been limited by technical problems in obtaining megakaryocytic cells in sufficient quantities for experimental purposes. We describe here an ex vivo serum-free liquid culture system to expand normal human megakaryoblasts from purified bone marrow-, cord blood- or peripheral blood-derived CD34(+) cells. The early megakaryocytic cells are expanded in the presence of recombinant thrombopoietin (TpO) and interleukin-3 (IL-3), and if necessary further purified by employing anti-CD61 immunomagnetic beads. Our expansion system generates normal human megakaryoblasts in quantities sufficient to perform various functional studies on these cells as well as to isolate from them proteins and mRNA for molecular analysis. Megakaryocytic cells isolated from these cultures (i) express several markers characteristic of this lineage (CD41, CD61, CD62 P, CXCR-4, PAR-1, etc.), (ii) respond by calcium flux and phosphorylation of various intracellular proteins to stimulation by thrombin and (iii) adhere to fibrinogen and vitronectin. However, human megakaryoblasts derived from the cultures supplemented with TpO + IL-3, in contrast to murine megakaryocytic cells cultured under similar conditions, display poor polyploidization and do not release platelets. Since IL-3 has been reported to inhibit final maturation of megakaryocytic cells, we recently modified our expansion strategy. In this new approach CD34(+) cells are first expanded for 11 days in the presence of TpO + IL-3. Then megakaryoblasts derived are expanded for an additional 7 days supplemented with TpO only. We found that megakaryocytic cells expanded in this 'two step culture' model are more differentiated, are polyploid and release platelets. The model described here provides normal human megakaryoblasts in adequate numbers, to study megakaryopoiesis and megakaryocyte function.

The goal of the present paper was to define the immunophenotype of bone marrow mast cells (BMMC) from healthy controls and patients with hematologic malignancies (HM) based on the use of multiple stainings with monoclonal antibodies analyzed by flow cytometry. Our results show that BMMC from both groups of individuals display a similar but heterogeneous immunophenotype. The overall numbers of BMMC are higher in the HM group of individuals (p = 0.08). Three patterns of antigen expression were detected: (1) markers constantly positive in all cases analyzed (CD9, CD29, CD33, CD43, CD44, CD49d, CD49e, CD51, CD71, CD117, and Fc(epsilon)RI), (2) antigens that were constantly negative (CD1a, CD2, CD3, CD5, CD6, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD25, CD30, CD34, CD38, CD41a, CD42b, CD65, CD66b, HLA-DR, and CD138), and (3) markers that were positive in a variable proportion of cases--CD11b (50%), CD11c (77%), CD13 (40%), CD18 (20%), CD22 (68%), CD35 (27%), CD40 (67%), CD54 (88%) and CD61 (40%). In addition, BMMC from all cases explored were CD45+, and this antigen was expressed at an intensity similar to that of mature granulocytes. In summary, our results show that BMMC from both healthy controls and HM patients display a relatively heterogeneous immunophenotype. Interestingly, we have observed clear differences between the immunophenotype of BMMC and MC from other tissues. This could be due either to the heterogeneity of human MC according to their tissue localization or to the sensitivity of the method used for antigen detection.

In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34+ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells (NC), CD34+, CD34+/38(-)/33(-), CD34+/61+, CD61+ cells and the clonogenic potential. After 2 weeks the median fold expansion of NC, CD34+ and CD34+/38(-)/33(-) cells was around two log both with fresh and cryopreserved CB and the expansion continued similarly until week 5. Our data suggest that this serum free protocol induces similar ex vivo expansion of HSCs and their committed progenitors from both fresh and cryopreserved CB. Our findings can be useful in view of clinical applications, since CB used for transplantation is stored in the cryopreserved state.

Two novel cell lines (JURL-MK1 and JURL-MK2) have been established from the peripheral blood of a patient in the blastic phase of chronic myelogenous leukemia. The cells grow in a single cell suspension with doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2). Cytogenetic analysis has shown that JURL-MK1 is hypodiploid whereas JURL-MK2 is near triploid and that both cell lines retain t(9;22). Moreover, JURL-MK1 and JURL-MK2 have a bcr/abl-fused gene with the same junction found in the patient's fresh cells, and both cell lines express the b3/a2 type of hybrid bcr/abl mRNA. The morphology and immunophenotype of these cell lines are reminiscent of megakaryoblasts. In both lines, a limited but consistent percentage of cells expresses gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gplb (CD42b), glycophorin A, hemoglobin and CD34. Both cell lines are clearly positive for CD33, CD43, CD45RO and CD63, while CD13, CD44, CD54, CD30 and CD40 are specific features of JURL-MK2. Among cytokine receptors, CD117/SCF-R is strongly displayed by a large fraction of JURL-MK1 cells but is hardly detectable on about 20% JURL-MK2 cells. Both cell lines are clearly positive for CD25/IL2R alpha, while a marked expression of CD116/GM-CSF-R and CDw123/IL3R alpha is restricted to JURL-MK2. Induction of cell differentiation in vitro has demonstrated that TPA is able to modulate the JURL-MK1 phenotype, causing an increased expression of platelet-associated antigens. The JURL-MK2 phenotype is easily modulated by both TPA and DMSO, which cause an increased expression of CD41a and CD117 accompanied by a decreased expression of CD30. Proliferation studies demonstrated that JURL-MK1 cell growth is enhanced by stem cell factor, while JURL-MK2 proliferation is unaffected by this cytokine. JURL-MK1 and JURL-MK2 are two novel cell lines with divergent biological features, representing a 'two-sided' model for investigating new aspects of megakaryocytopoiesis.

Based on the morphological resemblance to megakaryocytes and upon the expression f the platelet specific glycoprotein antigen, CD61, it is highly l y that MOLM-1 is a clonal leukemic cell population of megakaryoblastic origin .

Increasing the number of megakaryocyte progenitors in stem cell transplants by ex vivo expansion culture may be an approach to accelerate platelet recovery in patients undergoing high-dose chemotherapy. We evaluated the effect of three different cytokine combinations on expansion, with special emphasis on the type of colony formation and migration of megakaryocytic cells. The number of clonogenic megakaryocyte progenitors (colony-forming units-megakaryocyte; CFU-Mk) with high- >> 20 cells/colony) and low-proliferative capacity (5-20 cells/colony) and the number of megakaryocytic (CD61+) cells were significantly increased by including interleukin 3 (IL-3) or IL-3 + IL-6 + IL-11 + Flt3-ligand to cultures containing megakaryocyte growth and development factor (MGDF) plus stem cell factor (SCF). No difference in the maturation of megakaryocytes from all three cytokine combinations to platelets were observed, as demonstrated by electron microscopy. In chemotaxis experiments, the migration towards stromal cell-derived factor 1 (SDF-1) was shown to be reduced for CD61+ cells and megakaryocyte progenitors cultured in other cytokines besides MGDF + SCF. The reduced migration was related to a lower expression of CXCR4, the receptor for SDF-1, on megakaryocytes from the proliferating cultures. These in vitro results demonstrate that expansion in IL-3 and other cytokines besides MGDF + SCF significantly impair the capacity of megakaryocytic cells to migrate.

Recent data provide evidence for an important role of thrombocytes in lymphangiogenesis within human malignant disease. The aim of this study was to investigate the role of thrombocytes in lymphangiogenesis in human esophageal cancer. Perioperative peripheral blood platelet counts (PBPC) were evaluated retrospectively in 320 patients with esophageal cancer, comprising 184 adenocarcinomas (AC), and 136 squamous cell carcinomas (SCC). Data on lymphangiogenesis evaluated by anti-podoplanin immunostaining were available from previous studies, platelets within the tumor tissue were assessed by CD61 immunostaining. For in vitro studies, human lymphatic endothelial cells (LECs) were isolated and co-cultured with peripheral blood platelets. Stromal thrombocytic clusters (STC) were evident in 82 samples (25.6%), and vascular thrombocytic clusters (VTC) in 56 (17.5%). STC and VTC were associated with a significantly higher PBPC at investigation of all cases. The presence of STC was associated with higher lymphatic microvessel density (p<0.001), PBPC and STC were associated with lymphovascular invasion of tumor cells in a regression model. The presence of STCs was associated with shorter DFS of all patients (p = 0.036, Breslow test), and VTC with shorter DFS in in SCC (p = 0.025, Breslow test). In cell culture, LEC proliferation was enhanced by co-culture with human platelets in a dose- and time-dependent manner mediated by the release of PDGF-BB and VEGF-C. Platelets play an important role in lymphangiogenesis and lymphovascular invasion in esophageal cancer, influencing prognosis. So the disruption of signaling pathways between platelets, tumor cells and lymphatic endothelium might be of benefit for patients.

The thrombocytopenia of Wiskott-Aldrich syndrome (WAS) is thought to be due to both reduced platelet production and accelerated platelet consumption. We have previously demonstrated that platelets from WASP-deficient mice are consumed more rapidly in vivo than are WT platelets, and that opsonization accelerates their uptake by bone marrow- derived macrophages more than it does that of WT platelets. Here we asked whether platelets from WAS patients show similar features. We show that ex vivo phagocytosis by activated THP-1 cells of DIO-labeled platelets from a series of WAS or XLT patients is increased in comparison to that of normal control platelets. Using a numerical analysis method, we distinguish this effect from a concurrent effect on the amount of detectable fluorescent signal transferred to the macrophage per phagocytosed platelet. We show that the latter quantity is reduced by platelet WASP deficiency, as might be expected if the fluorescence transferred from these smaller platelets is more rapidly quenched. We are unable to detect a differential effect of opsonization with anti-CD61 antibody on the uptake of WASP(-) vs. WT platelets. However, the high probability of phagocytosis per adsorbed WASP(-) platelet could limit the sensitivity of the assay in this case. We also see no effect of sera from WAS patients on the uptake of normal control platelets, suggesting that in vivo opsonization is not the cause of increased uptake of WASP(-) platelets. Finally, we show little, if any, increase in the reticulated platelet fraction in WAS patients, suggesting that impaired production of reticulated platelets contributes to the thrombocytopenia. Our findings suggest that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. They also demonstrate the feasibility of routinely performing functional assays of phagocytosis of small numbers of platelets obtained at remote locations, a method which should be applicable to the study of other types of thrombocytopenia such as ITP.

Niacin is a natural pyridine derivative, proven to favorably modulate the blood lipid profile by increasing levels of high-density lipoprotein (HDL) cholesterol, and by reducing total cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, and Lp (a) lipoprotein concentrations. Considering that platelet activity is important in predicting vascular outcomes, and that HDL heavily constitutes platelet cellular membranes, we sought to evaluate the effect of niacin on human platelet activity indices. The blood obtained from 30 aspirin-naïve volunteers was preincubated with escalating concentrations of niacin in vitro. Platelet tests included whole blood and plasma aggregometry, rapid cartridge-based analyser, expression of major surface receptors by flow cytometry, and plasma prostaglandins by ELISA. Preincubation of blood with niacin at 0.3, 1.0 and 3.0 mM resulted in significant inhibition of maximal adenosine diphosphate (ADP)- (p=0.03), and collagen-induced platelet aggregation (p=0.01), and reduced activity by VerifyNow (p=0.007) bedside analyser. Surface platelet PAR-1 (MoAb WEDE-15; p=0.04), and vitronectin (CD51/CD61; p=0.02) receptors were up-regulated. Niacin was associated with a two- to three-fold increase of thromboxane B2, prostaglandins D2, and E2. Formation of platelet-monocyte microparticles (CD14+CD151), and expression of PECAM-1 (CD31), thrombospondin (CD36), GP IIb/IIIa (CD41a) antigen, and activity with MoAb PAC-1, GPIb (CD42b), P-selectin (CD62p), LAMP-3 (CD63), LAMP-1 (CD107a), CD40-ligand (CD154), GP37 (CD165), were not affected by niacin, suggesting no effect on prostacyclin release. In conclusion, niacin in vitro affects platelet activity by mildly inhibiting aggregation, and stimulating significant prostaglandin release, with mostly intact major platelet receptor expression. The effect of niacin is unique, differs from other known antiplatelet agents, and suggests potential opportunities for therapeutic combination, particularly in patients with low levels of HDL-C. These preliminary data, while intriguing, require confirmation in subjects receiving orally dosed extended-release niacin in order to determine whether these findings are clinically relevant.

Platelet transfusions are life-saving treatments for many patients with thrombocytopenia; however, their use is generally discouraged in the autoimmune disorder known as immune thrombocytopenia (ITP). We examined whether allogeneic platelet major histocompatibility complex (MHC) class I transfusions affected antiplatelet CD61-induced ITP. BALB/c CD61 knockout mice (CD61(-)/H-2(d)) were immunized against platelets from wild-type syngeneic BALB/c (CD61(+)/H-2(d)), allogeneic C57BL/6 (CD61(+)/H-2(b)), or C57BL/6 CD61 KO (CD61(-)/H-2(b)) mice, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP. When nondepleted splenocytes were transferred to induce antibody-mediated ITP, both CD61(+) platelet immunizations generated immunity that caused thrombocytopenia independently of allogeneic MHC molecules. In contrast, when B-cell-depleted splenocytes were transferred to induce T-cell-mediated ITP, transfer of allogeneic MHC-immunized splenocytes completely prevented CD61-induced ITP development. In addition, allogeneic platelet transfusions into SCID mice with established CD61-induced ITP rescued the thrombocytopenia. Compared with thrombocytopenic mice, bone marrow histology in the rescued mice showed normalized megakaryocyte morphology, and in vitro CD61-specific T-cell cytotoxicity was significantly suppressed. These results indicate that antibody-mediated ITP is resistant to allogeneic platelet transfusions, while the T-cell-mediated form of the disease is susceptible, suggesting that transfusion therapy may be beneficial in antibody-negative ITP.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder with a complex pathogenesis, which includes both antibody- and T-cell-mediated effector mechanisms. Rituximab (an anti-human CD20 monoclonal antibody [mAb]) is one of the treatments for ITP and is known to deplete B cells but may also work by affecting the T-cell compartments. Here, we investigated the outcome of B-cell depletion (Bdep) therapy on CD8(+) T-cell-mediated ITP using a murine model. CD61 knockout (KO) mice were immunized with CD61(+) platelets, and T-cell-mediated ITP was initiated by transfer of their splenocytes into severe combined immunodeficiency (SCID) mice. The CD61 KO mice were administrated an anti-mouse CD20 mAb either before or after CD61(+) platelet immunization. This resulted in efficient Bdep in vivo, accompanied by significant increases in splenic and lymph node CD4(+) and CD8(+) T cells and proportional increases of FOXP3(+) in CD4(+)and CD8(+) T cells. Moreover, Bdep therapy resulted in significantly decreased splenic CD8(+) T-cell proliferation in vitro that could be rescued by interleukin-2. This correlated with normalization of in vivo platelet counts in the transferred SCID mice suggesting that anti-CD20 therapy significantly reduces the ability of CD8(+) T cells to activate and mediate ITP.

The expression and function of surface TRAIL and TRAIL receptors were investigated in primary megakaryocytic cells, generated in serum-free liquid phase from peripheral human CD34(+) cells. The surface expression of both TRAIL and "death receptor" TRAIL-R2 became detectable starting from the early phase of megakaryocytic differentiation (day 6 of culture) and persisted at later (days10-14) culture times. On the other hand, "death receptor" TRAIL-R1, "decoy receptors" TRAIL-R3, and TRAIL-R4 were barely detectable or undetectable at any time point examined. Addition of recombinant TRAIL at day 6 of culture increased the rate of spontaneous apoptosis of CD34(+)/CD41(dim) megakaryoblasts and it significantly decreased the total output of mature megakaryocytic cells evaluated after additional 4-8 days of culture. Conversely, addition in culture of TRAIL-R2-Fc chimera, which blocked the interaction between endogenous TRAIL and TRAIL-R2 on the surface of cultured megakaryocytic cells, increased the total megakaryocytic cell count. In addition, recombinant TRAIL promoted a small but reproducible increase of maturation in the surviving megakaryocytic cell population, evaluated by both phenotypic analysis and morphology. A similar pro-maturation effect was observed when TRAIL was added to bone marrow-derived CD61(+) megakaryocytic cells. Thus, our data suggest a role of TRAIL as a regulator of megakaryocytopoiesis.

Transplantation of thrombopoietin (TPO)-expanded cord blood CD34(+) cells accelerates human platelet recovery in NOD/SCID mice. It is unknown which subpopulations of the TPO-expanded cells mediate accelerated platelet recovery and bone marrow (BM) engraftment. In this study, the contribution of these subpopulations to human platelet appearance in the blood and BM engraftment was studied in NOD/SCID mice. Following transplantation of CD34(-) /CD61(-)/lineage(-) cells (Lin(-)), human platelets were detected in the blood of recipient mice from day 4. Both time to platelet recovery and blood platelet counts at 6 weeks after transplantation showed Lin(-) dose dependence. The Lin(-) population was virtually negative for lineage marker expression and lacked CD42b expression but was heterogeneous with regard to CD36 and CD38 expression, reflecting a population in transit but not fully committed toward the megakaryocyte (MK) lineage. Although no definitive phenotype could be established of the cells generating prompt platelet production and cells generating platelets 6 weeks after transplantation, this relatively heterogeneous Lin(-) population is prerequisite to accelerate platelet recovery in vivo. The interval to platelet recovery after transplantation of the CD34(+) cells remaining after expansion (rCD34(+)) was similar to mice transplanted with nonexpanded CD34(+) cells, although the total platelet counts and the engraftment levels in the BM were lower. Cobblestone area-forming cell colony-forming cells resided mostly in the rCD34(+) population. The pro-MK CD61(+) cells did not contribute to human platelet recovery or engraftment in the BM. Our study shows that not all expanded cells appear critical for transplantation. These data support that functional characterization of the expanded cell populations is warranted to make future expansion protocols suitable for clinical application.

We investigated the expression of adhesion molecules including LFA-1 alpha (CD11a), Mac-1 (CD11b), LFA-1 beta (CD18), VLA-beta 1 (CD29), H-CAM (CD44), VLA-4 (CD49d), VLA-5 (CD49e), ICAM-1 (CD54), N-CAM (CD56), LFA-3 (CD58), VNR-beta (CD61), and LECAM-1 (CD62L) on fresh myeloma cells and human myeloma cell lines. By two-color flow cytometric analysis with anti-CD38 antibody, we demonstrated that myeloma cells were located in the strongly CD38-positive (CD38++) fractions. Fresh myeloma cells were obtained from 28 patients with multiple myeloma (MM) and 3 patients with plasma cell leukemia (PCL). All myeloma cells expressed VLA-4 on their surface. Most of the myeloma cells also expressed VLA-5, ICAM-1, and LFA-3, H-CAM was strongly expressed in 3 cases of PCL and 2 cases of aggressive myeloma, and moderately expressed in other MMs. N-CAM was expressed in 68% of MMs, but none of the 3 PCLs. LFA-1 was expressed in two cases of aggressive myeloma, but not expressed in other non-aggressive myelomas. Most of the myeloma cells did not express Mac-1, VNR-beta, or LECAM-1. These results suggest that VLA-4, VLA-5, ICAM-1, LFA-3, and H-CAM are involved in cellular interaction and migration in MM, and that the expression of N-CAM and LFA-1 varies with disease activity in MM.

BACKGROUND

Oestrogen alters megakaryocyte number in humans and mice. In mice, high-dose oestrogen stimulates an initial increase in megakaryocyte number followed by a decrease. However, the cellular action, effect of physiologically circulating and smaller supra-physiological oestrogen doses and whether changes in megakaryocyte number alter platelet counts have not been studied.

METHODS

To further examine oestrogen's effect on megakaryocytes and platelets we administered intact or ovariectomised mice various doses of oestrogen and measured megakaryocyte and platelet counts. To determine the cellular mechanism by which oestrogen influences megakaryocytopoesis we also examined its effect on markers of megakaryocytic differentiation (CD41, CD61, CD34).

RESULTS

We found that large doses of oestrogen (500 microg/kg) increased mature CD41+ megakaryocyte number within 2 days, and this was associated with an increase in circulating platelets. Smaller supra-physiological doses (100 microg/kg) lacked this anabolic effect, but still suppressed megakaryocyte and platelet number by day 10 in intact and ovariectomised mice. This was preceded by a reduction in the number of CD61+ megakaryoblasts and CD34+ precursors available to form mature megakaryocytes. In contrast, ovariectomy had no effect on megakaryocyte or platelet number, indicating that circulating oestrogen concentrations do not influence megakaryocyte differentiation or activity.

CONCLUSIONS

Our data suggest that in mice at least platelet counts reflect changes in megakaryocyte number, and while both are independent of physiological hormone concentrations, they are sensitive to even small supra-physiological doses of oestrogen. Therefore, to ovoid disrupting platelet homeostasis the dose of oestrogen given should be no more than replacement.

Radiation proctitis is an inflammatory process associated with persistent and refractory lower gastrointestinal bleeding. Purinergic signaling regulates hemostasis, inflammation, and angiogenesis. For example, CD39, the vascular ectonucleotidase, blocks platelet activation and is required for angiogenesis. Whether CD39 expression is affected by radiation injury is unknown. The aim of this work was to study CD39 expression patterns after clinical radiation injury to the rectum. We prospectively enrolled eight patients with radiation proctitis and five gender-matched controls. Biopsies were taken from normal-appearing rectal mucosa of controls and from the normal sigmoid and abnormal rectum of patients. Expression patterns of CD39, P2Y2 receptor, CD31, CD61 integrin, and vascular endothelial growth factor receptor 2 were examined by immunostaining; levels of CD39 were further evaluated by Western blots. Chronic inflammatory lesions of radiation proctitis were associated with heightened levels of angiogenesis. Immunohistochemical stains showed increased vascular expression of CD39, as confirmed by Western blots. CD39 was co-localized with vascular endothelial markers CD31 and CD61 integrin, as well as expressed by stromal tissues. Development of neovasculature and associated CD39 expression in radiation proctitis may be associated with the chronic, refractory bleeding observed in this condition.

An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein IIb)/CD61 (beta(III) or glycoprotein IIIa) and anti-CD41 antibodies. Abnormal laboratory findings included prolonged oral mucosal bleeding time (>12 hours), prolonged closure time with collagen/ADP (>300 seconds), and absence of clot retraction after 60 minutes. TEG reaction times were similar with kaolin and tissue factor in the patient and a control horse. However, maximum amplitudes in the patient were decreased with both kaolin (43.7 mm; control, 63.9 mm) and tissue factor (37.7 mm; control, 57.8 mm). Platelet aggregation responses to ADP and collagen were profoundly reduced in the affected horse compared with a control. Flow cytometry showed an absence of CD41 and decreased expression of CD41/CD61-reacting antigen on the patient's platelets compared with those from a control horse. The laboratory findings supported a diagnosis of Glanzmann thrombasthenia, likely caused by a mutation in the gene encoding the GPIIb subunit.