The aim of this research was to investigate the blood coagulation function in the patients with avascular necrosis of the femoral head (ANFH) after severe acute respiratory syndrome (SARS). The expression of CD31, CD61, CD62p, CD63 and PAC-1 on platelet membrane was measured respectively by flowcytometry, and the plasma prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (Fbg) were measured by blood clotting instrument in 26 patients with ANFH after SARS and in 17 healthy adults. The expression of CD31, CD61, CD 62p, CD63 and PAC-1 on platelet membrane in 26 patients was all lower than that in 17 healthy subjects (P < 0.01). The levels of PT, APTT, TT and Fbg in 26 patients were all normal. There is no significant difference (P>> 0.05) in those markers between patients and 17 healthy adults. The blood may not be in hypercoagulable state in patients with ANFH after SARS.

Spinal cord injury (SCI) leads to severe dysfunction below injured segment and poses a great pressure to the individual and society. In this study, we applied ¹⁸F-alfatide II positron emission tomography/computed tomography (PET/CT) to monitor angiogenesis in an SCI model after estrogen (E2) treatment, as well as to evaluate the prognosis in a noninvasive manner. The SCI model was established with male rats and the rats were randomly divided into E2-treated group (SCI + E2) and E2-untreated group (SCI). Sham group was also used as control (Sham). The angiogenesis after SCI was monitored by ¹⁸F-alfatide II PET/CT and verified by immunofluorescence of CD31 and CD61. We also evaluated the level of E2 and growth-associated protein 43 (GAP43) by enzyme-linked immunosorbent assay. Finally, Basso, Beattie, and Bresnahan (BBB) scores were determined to evaluate the exercise capacity of the rats in all 3 groups. Our results showed that the BBB score of SCI + E2 group was significantly different from that of SCI group (P < .05) and Sham group (P < .01). The uptake of ¹⁸F-alfatide II was positively correlated with the expression level of GAP43, both of which reached the peak at day 7 after injury. CD31 and CD61 immunostaining further verified increased angiogenesis in E2-treated SCI lesions. We concluded that ¹⁸F-alfatide II PET/CT can monitor the angiogenesis status after SCI in vivo and it may help clinician predict the progression of patients with SCI. This may benefit the study of vascular repair after SCI and provide a tool for evaluation of SCI treatment in clinical practices.

Purpose: Mesenchymal stem cells (MSCs) release hematopoietic cytokines, growth factors, and Microvesicles (MVs) supporting the hematopoietic stem cells (HSCs). MVs released from various cells, playing a crucial role in biological functions of their parental cells. MSC-derived MVs contain microRNAs and proteins with key roles in the regulation of hematopoiesis. Umbilical cord blood (UCB) is a source for transplantation but the long-term recovery of platelets is a main problem. Therefore, we intend to show that MSC-MVs are able to improve the differentiation of UCB-derived CD34⁺ cells to megakaryocyte lineage. Methods: In this descriptive study, MSCs were cultured in DMEM to collect the culture supernatant, which was ultracentrifuged for the isolation of MVs. HSCs were isolated from UCB using MACS method and cultured in IMDM supplemented with cytokines and MVs in three different conditions. Megakaryocyte differentiation was evaluated through the expression of specific markers and genes after 72 hours, and the data was analyzed by t test (P<0.05). Results: The expression of specific megakaryocyte markers (CD41 and CD61) in the presence of different concentrations of MSC-MVs did not show any significant difference. Also, the expression of specific genes of megakaryocyte lineage was compared with control group. The expression of GATA2 and c-Mpl was significantly increased, GATA1 was not significantly decreased, and FLI1 was significantly decreased. Conclusion: MSC-MVs could improve the expression of specific megakaryocyte genes; however, there was no significant expression of CD markers. Further studies, including the evaluation of late stages of megakaryocyte differentiation, are required to evaluate platelet production and shedding.

UNASSIGNED

The main goal of this work was to develop haemocompatibile thin film materials dedicated to novel flexible mechanical heart valves intended for pulsatile ventricle assist devices.

UNASSIGNED

The studies performed have led to the selection of a material for the surface modification of the metallic scaffold. Haemocompatible, biofunctional, ultra-elastic, thin carbon-based coatings were proposed. The surface was designed to eliminate thrombogenic and microbial construction by a reduction in turbulence and sufficient washing of the biofunctional-adapted surfaces, thus allowing for extended use for temporary heart support. The article presents the influence of the mechanical properties of coatings and their influence on the haemocompatibility. In this study, we investigated a simplified model of the whole blood shear stress based on a cone and plate rotational viscometer. Several indices of platelet activation were analysed, including platelet and granulocyte-platelet aggregates, platelet activation markers and platelet-derived microparticles.

UNASSIGNED

The shear stress induced a platelet aggregate count in the range from 2% to 30% of the CD61 positive cells. For polyurethane (PU), the average value of platelet aggregates was on the level of 7%. The analyses have demonstrated that the cytometric methods of the direct determination of platelet-derived microparticles in plasma are reproducible and reliable. Considering the generation of microparticles on the tested coatings under hydrodynamic conditions, the best properties were observed for the coating a-C:H,N.

UNASSIGNED

The results indicate that a-C:H-based coatings with the thickness of 110 nm do not induce an immune response and do not influence the origin of platelet microparticle formation; thus, these type of coatings are the most promising for the parts which are planned to withstand blood contact under the high value of shear stress.

: Mycophenolate mofetil (MMF) raises platelet counts in patients with primary immune thrombocytopenia. However, studies indicate that MMF inhibits collagen-induced platelet aggregation, potentially increasing bleeding risk following MMF therapy. The study evaluates the in-vitro effect of MMF on platelet function. Blood samples (n = 6) from healthy donors were incubated with vehicle, MMF or mycophenolic acid (MPA) at clinically relevant concentrations. Platelet aggregation was measured with flow cytometry and 96-well light transmission aggregometry (LTA). Using flow cytometry, we measured the expression of platelet CD49b, CD42b, CD42a, CD61 and CD41. Platelet activation was measured as the expression of P-selectin and the active form of the GPIIb/IIIa receptor following agonist stimulation. Agonists were: ADP, thrombin receptor-activating peptide, collagen, collagen-related peptide and U46619. The Platelet Function Analyzer-200 was used to measure global platelet function. MMF and MPA did not change platelet aggregation regardless of the agonist used. An exception was a significant, but minor decrease in collagen-induced platelet aggregation in samples with MMF (6 ± 3%, P = 0.02) and MPA (8 ± 4%, P = 0.01) compared with vehicle (22 ± 11%). However, this was not observed using the lesser sensitive LTA method. Compared with vehicle, MPA led to a significantly lower relative disposition of the surface collagen-receptor GPVI (7.8 ± 1.8 versus 8.8 ± 2.1 mean fluorescence intensity, P < 0.001). In all other platelet-related tests, neither MMF nor MPA showed any effect. In conclusion, MMF and MPA only had a minor effect on collagen-induced platelet aggregation, with MPA reducing the relative disposition of surface GPVI receptors.

Genetic defects of the blood platelet membrane glycoproteins, GPIIb-IIIa (alpha IIb/beta 3; CD41/CD61) and GPIb-V-IX (CD42) are the origin of several rare bleeding disorders, the best known of which are Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand's disease. In Glanzmann's thrombasthenia, GPIIb-IIIa are missing or defective and platelet aggregation is lacking or reduced. Either gene can be affected and mutations leading to lack of expression or to expression of poorly functional forms have been described. In Bernard-Soulier syndrome, GPIb-V-IX are missing or defective, leading to poor platelet adhesion at high-shear stress to damaged vessel wall and reduced platelet response to thrombin. Mutations in both GPIb alpha (CD42b) and GPIX (CD42a) have been described. Mutations in GPIb alpha can also lead to platelet-type von Willebrand's disease in which GPIb-V-IX are expressed normally but bind von Willebrand's factor spontaneously, which leads to platelet aggregation and thrombocytopenia.

Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34⁺/CD41⁺ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.

OBJECTIVE

To investigate the role of antiapoptotic Bcl-x(L) protein in megakaryocyte differentiation and maturation.

METHODS

RNA interference was used to block the expression of Bcl-x(L) when K562 cells were induced to differentiate into megakaryocyte (CD61 + cells) by PDBu, and the expression of Bcl-x(L) was evaluated with flow cytometry and reverse transcription polymerase chain reaction (RT-PCR). The CD34 + cell fraction was positively isolated by using the MiniMACS system from normal bone marrow. Immunochemical staining and flow cytometry were used to detect the expression of Bcl-x(L) in the differentiation (CD41 + cells) of CD34 + cells induced by trombopoietin (TPO).

RESULTS

Among K562 cells induced by PDBu, the percentage of CD6L + cells rapidly increased in 24 hours and maintained at a high positive level in 72 hours. When exposured to si-Bcl-x(L), the percentage of CD6 1 + cells only slightly increased in 72 hours. The expression of Bcl-x(L) mRNA was significantly decreased after transfection compared with that of control group, and Bcl-x(L) protein expression decreased correspondingly. After the CD34 + bone marrow cells having been treated with TPO for 5 days to 20 days, the Bcl-x(L)-megakaryocytes increased as the culture time prolonged, and there was a strong expression of Bcl-x(L) in immature megakaryocyte and an obviously decreased expression in degenerating megakaryocytes maturation.

CONCLUSIONS

Increased expression of antiapoptotic Bcl-x(L) may be essential to mature megakaryocyte. The down-regulation of antiapoptotic Bcl-x(L) in mature megakaryocyte may be crucial to platelets formation.

OBJECTIVE

The transplantation of mobilised peripheral progenitor cells has resulted in shortening of neutrophil and platelet engrafment times following high-dose chemotherapy. Since reticulated platelet percentage (PR%) has been established as a measure of bone marrow platelet production, we performed this type of analysis on the thrombopoietic compartment during transplant-related chemotherapy.

METHODS

Kinetics of thrombopoiesis of 19 patients with solid tumors undergoing a single or double autologous peripheral blood progenitor cell transplant was characterized by evaluating the level of RP. The correlation between CD34(+) cell subsets and the time of highest percentage of RP was also evaluated.

RESULTS

The percentage of RP increases since day +8 after single transplant reaching the peak (3.4%) at day +10. In the group of patients receiving double transplant, the RP value of peak observed after second transplant is not significantly different from that one observed after the first transplant (3 vs 3.7%). In a subgroup of patients both the number of CD34(+) cells/Kg infused and the percentage of CD34(+) CD61(+) cell subsets correlate with the day of RP peak.

CONCLUSIONS

These results suggest that RP measurement is an early indicator of engraftment. Additionally, the observation that RP percentage is high at the time of platelet transfusion in 13 out of 20 cases of transfusions (the 7 cases with low RP value being transfused during the period of obligate thrombocytopenia) suggests that the evaluation of this parameter, together with the platelet count, can be used to monitor the need for platelet transfusion.

Increasing the number of megakaryocytic cells in stem cell transplants by ex vivo expansion culture may provide an approach to accelerate platelet engraftment after high-dose chemotherapy. However, it is unknown if a relationship exists between the expansion potential of progenitor cells and the time to platelet engraftment in vivo. Therefore, we questioned if those patients who potentially would benefit most from expanded cell supplements are able to generate megakaryocytic cells efficiently in vitro. The in vitro megakaryocyte proliferation was analyzed from 19 leukapheresis samples from a group of multiple myeloma patients who all showed rapid neutrophil engraftment, but varied from 7 to 115 days post-transplant to achieve platelet levels >20x10(9)/l. CD34+ cells were isolated and analyzed for their potential to form megakaryocytic colonies (CFU-Mk) in colony assays and megakaryocytic (CD61+) cells in suspension cultures. The frequency and size of CFU-Mk and the expansion potential of CD61+ cells varied eightfold between individual patients. A similar range was found with CD34+ cells isolated from normal bone marrow (n=9). Rapid platelet engraftment occurred in patients receiving both high or low CFU-Mk doses and with high and low expansion of CD61+ cells. Four patients who experienced prolonged (>3 weeks) thrombocytopenia received low CFU-Mk doses, but the expansion potential was around median values or higher. Therefore, we conclude that the megakaryocyte proliferation is not impaired and that in vitro expansion could increase the number of megakaryocytic cells, although other factors could be more relevant in platelet engraftment in this group of patients.

Accurate and precise platelet counts are important for patients with severe thrombocytopenia or who are receiving chemotherapy. We developed a novel flow cytometric analysis of platelets that may be particularly valuable for assessing the necessity for platelet transfusions. This ImmunoPlt (CD61) assay is based in part on CD61 monoclonal antibody labeling and has been automated and implemented on the CELL-DYN 4000 hematology analyzer. It is well suited for thrombocytopenic specimens, since it reduces interference by nonplatelet particles. It takes less than 5 minutes from closed-tube aspiration to report. Data for more than 350 thrombocytopenic specimens demonstrate that the ImmunoPlt (CD61) assay is more accurate than the optical scatter or the impedance count for specimens with platelet counts between 1 and 60 x 10(3)/microL (1 and 60 x 10(9)/L). The ImmunoPlt (CD61) assay is more precise than the optical scatter or the impedance count for specimens with platelet counts between 1 and 50 x 10(3)/microL (1 and 50 x 10(9)/L).

Acute megakaryoblastic leukaemia (AML-M7) is a rare myeloproliferative disorder in domestic animals. Recently, thanks to the greater availability of immunophenotype techniques, precise diagnosis is more easily made. The morphological evaluation has its limitations, especially in the study of poorly differentiated cells. Few reports have described AML-M7 in dogs using flow cytometry. This clinical case points out the utility of flow cytometry in the characterization of AML-M7 in a 3-year-old German Shepherd dog. Flow cytometry investigation has established megakaryocytic lineage involvement by showing the presence of two megakaryocyte/platelet associated antigens (CD9 and CD61). In human medicine CD9 may be used as a platelet and megakaryocyte marker. There is an evidence of cross-reactivity of human anti-CD9 monoclonal antibody with canine samples. To our knowledge, the use of CD9 has never been described before, for this purpose in the dog.

The megakaryopoietic potential in the bone marrow (BM) of patients in first remission after treatment for acute myelogenous leukaemia (AML) was investigated using long-term bone marrow cultures (LTC) stimulated with megakaryocyte growth and development factor (MGDF). The baseline number of megakaryocyte colony-forming cells (Meg-CFC) was very low. However, there was a 10 to 100-fold increase of Meg-CFC in cultures treated with 10 ng/ml MGDF with mean numbers within the normal range for the first 4 weeks of culture with a 24-fold increase in their cumulative numbers. Similarly, a 12-fold increase in the numbers of megakaryocytes (MKs) was found by CD61 immunostaining. These effects were lost at the dose of 100 ng/ml. In contrast, the cumulative mean numbers of Meg-CFC in the control cultures from normal bone marrow (NBM) were not significantly different from those in cultures treated with 10 or 100 ng/ml MGDF. These results demonstrate that MGDF stimulates megakaryocytopoiesis in patients with AML in first remission, restoring the Meg-CFC compartment to normal values, a result with potential clinical implications for their treatment with autologous transplantation.

OBJECTIVE

To study the changes of platelet in May-Hegglin anomaly (MHA) and the molecular pathogenesis mechanism.

METHODS

Peripheral blood was drawn from the MHA proband, her father and her uncle. Platelet count and morphology were examined by automatic blood cell counter and microscopy, respectively. The platelet membrane protein was examined by flow cytometry. Membrane antibodies were determined by ELISA. PCR was used to amplify the exons 25, 31 approximately 32, 38 and 40 of the MYH 9 gene in the MHA patient and her diseased father. Furthermore, PCR products were sequenced, a specific point mutation was identified and inclusions (Dohle's body) in the neutrophil was detected by indirect immunofluorescence technique.

RESULTS

It was proved that in MHA patients, platelet count was higher by cell counter than by microscope (P < 0.01). Giant platelet was 94% but platelet membrane proteins (CD41, CD61, CD42A, CD42b) were in normal range. Membrane antibodies was undetectable. An A5521G mutation (GAG->>AAG) in the exon 38 was found in the proband and her diseased father, resulting in a characteristic change of NMMHC-A1841 (Glutamic acid->>Arginine), which was not found in other members of the family and in normal controls. Spindle-like inclusions with fluorescence were clearly displayed in neutrophil cytoplasm.

CONCLUSIONS

The molecular pathogenesis mechanism of May-Hegglin anomaly is the mutation in MYH 9 gene.

Application of ex vivo expanded megakaryocytic progenitor cells (MKPC) is a strategy for the treatment of thrombocytopenia after hematopoietic stem cell transplantation. Some growth factors including thrombopoietin (TPO), megakaryocyte growth and development factor (MGDF), interleukin (IL)-1, IL-3, IL-6, IL-11, platelet-derived growth factor (PDGF), and serotonin (5-HT) have been demonstrated to play an important role on the regulation of megakaryocyte/platelet development, the efficient conditions for the expansion of the megakaryocyte (MK) progenitors from hematopoietic stem/progenitor cells were discussed in this review article. TPO alone produced a high proportion of MK progenitors but a low total cell count. The addition of IL-1 beta, IL-3, IL-6 and Flt-3L improved the expansion outcome. The combination of three to five cytokines produced more efficient expansions of hematopoietic stem and MK progenitors. PDGF also enhanced the ex vivo expansion of CD61+ CD41+ cells and CD34+ cells in combination with TPO, IL-1 beta, IL-3, IL-6 and Flt-3L. PDGF is a suitable growth factor to improve the ex vivo expansion of MKPC without promoting their in vitro maturation. More importantly, PDGF also enhanced the engraftment of human stem and progenitor cells in NOD/SCID mice. It has been reported that MKPC can be safely administered to autologous peripheral blood progenitor cell transplant recipients. In short, MKPC can be expanded ex vivo and safely applied to autologous transplant.

Circulating microvesicles (cMV) are small phospholipid-rich blebs shed from the membrane of activated vascular cells that contribute to vascular disease progression. We aimed to investigate whether the quality of the Nordic diet is associated with the degree of blood and vascular cell activation measured by MV shedding in elderly patients after an acute myocardial infarction (AMI). One-hundred and seventy-four patients aged 70-82 years were included in this cross-sectional study. Fasting blood samples were taken within 2 to 8 weeks after an AMI. Annexin V (AV)⁺ cMV derived from blood and vascular cells were measured through flow cytometry. A patient's usual diet was recorded with the SmartDiet® questionnaire. Patients with higher adherence to the Nordic diet (highest diet score) had lower levels of total AV⁺ and platelet-derived (CD61⁺/AV⁺ and CD31⁺/AV⁺) cMV. Dietary habits influence cellular activation. A high adherence to the Nordic diet (assessed by the SmartDiet® score) in elderly post-AMI patients was associated with lower levels of platelet activation, which was reflected by a lesser release of MV carrying platelet-derived epitopes, potentially contributing to an explanation of the cardioprotective effects of the Nordic diet.

Monocytes express tissue factor (TF) as a result of cytokine stimulation or endothelial adherence. We evaluated monocyte-platelet interaction in vitro as another trigger for monocyte TF enhancement in human mononuclear cells isolated by density gradient centrifugation from peripheral blood. Cell TF procoagulant activity (TF-PCA) was quantitated by a one-stage recalcification clotting time assay. Platelets were counted and identified by whole blood flow cytometry as CD61 positive particles, activated platelets were characterized by P-Selectin (CD62) expression, and monocytes by surface CD14 expression. A significant correlation between normalized TF-PCA of isolated mononuclear cells and platelet count was shown (r = 0.43, P < 0.001). Percentage of activated platelets in baseline samples was 4.2 +/- 3.5 while adenosine diphosphate (ADP) increased platelet positivity to 34 +/- 17% (P < 0.001). After isolation, 52 +/- 12% of platelets within suspensions were activated (P < 0.0001). Percentage of CD62-positive monocytes (CD14+ particles) increased from baseline 5% to 13 +/- 6% in ADP-stimulated samples to 53 +/- 17% after isolation (P < 0.001). These findings suggest that density gradient centrifugation activates platelets and that an adhesive interaction between monocytes and platelets may promote TF-PCA expression in isolated mononuclear suspensions. Enhanced monocyte TF expression as a result of an activated platelet-monocyte interaction seems to be an important laboratory effect requiring consideration when utilizing this technique in an experimental setup.

Introduction: Accurate platelet counting is essential for risk assessment of bleeding and thrombosis. Abbott Alinity hq hematology analyzer was recently introduced, and its performance in platelet counting has yet to be evaluated comprehensively. In this study, we evaluated the performance of the optical platelet counting of Abbott Alinity hq (Alinity-PLT) and the impedance and fluorescent platelet counting of Sysmex XN-9000 (XN-PLT-I and XN-PLT-F) compared with the international reference method.

Methods: Blood samples were analyzed via Alinity hq and XN-9000 with PLT-F channel. Immuno-platelet (ImmnoPLT) reference method was performed with CD41/CD61 antibodies using FACSLyric^TM flow cytometer (BD). Precision was determined using 10 replicates in a single run, and the platelet counts of Alinity-PLT, XN-PLT-I, XN-PLT-F, and ImmnoPLT were compared.

Results: At a platelet count of 13 × 10⁹ /L, the CVs of Alinity-PLT, XN-PLT-I, and XN-PLT-F were 4.2%, 6.7%, and 4.3%, respectively, and at a platelet count of 44 × 10⁹ /L, all showed a CV of less than 3%. For the total 210 samples, all three methods showed a very strong correlation with ImmunoPLT (r > 0.99). For platelet levels below 20 × 10⁹ /L, XN-PLT-F showed the strongest correlation with ImmunoPLT (r = 0.975), and for platelet levels of 20-100 × 10⁹ /L, Alinity-PLT and XN-PLT-I were comparable to ImmunoPLT. For platelet levels of 100-450 × 10⁹ /L, XN-PLT-I was the most comparable to ImmunoPLT, and for platelet levels above 450 × 10⁹ /L, Alinity-PLT was comparable to ImmunoPLT.

Conclusions: All three methods were highly correlated with ImmunoPLT, and each method had different performance advantages according to the platelet levels.

Keywords: Sysmex XN-9000; abbott alinity hq; hematology analyzer; platelet counting.

Mammary glands develop through primary ductal elongation and side branching to maximize the spatial area. Although primary ducts are generated by bifurcation of terminal end buds, the mechanism through which side branching occurs is still largely unclear. Here, we show that inhibitor of DNA-binding 2 (ID2) drives side branch formation through the differentiation of K6+ bipotent progenitor cells (BPs) into CD61+ luminal progenitor cells (LPs). Id2-null mice had side-branching defects, along with developmental blockage of the differentiation of K6+ BPs into CD61+ LPs. Notably, CD61+ LPs were found in budding and side branches, but not in terminal end buds. Hormone reconstitution studies using ovariectomized MMTV-hemagglutinin-nuclear localized sequence-tagged Id2 transgenic mice revealed that ID2 is a key mediator of progesterone, which drives luminal lineage differentiation and side branching. Our results suggest that CD61 is a marker of side branches and that ID2 regulates side branch formation by inducing luminal lineage commitment from K6+ BPs to CD61+ LPs.

ATP synthase is powered by the flow of protons through the molecular turbine composed of two α-helical integral membrane proteins, subunit a, which makes a stator, and a cylindrical rotor assembly made of multiple copies of subunit c. Transient protonation of a universally conserved carboxylate on subunit c (D61 in E. coli) gated by the electrostatic interaction with arginine on subunit a (R210 in E. coli) is believed to be a crucial step in proton transfer across the membrane. We used a fusion protein consisting of subunit a and the adjacent helices of subunit c to test by NMR spectroscopy if cD61 and aR210 are involved in an electrostatic interaction with each other, and found no evidence of such interaction. We have also determined that R140 does not form a salt bridge with either D44 or D124 as was suggested previously by mutation analysis. Our results demonstrate the potential of using arginines as NMR reporter groups for structural and functional studies of challenging membrane proteins.

Interleukin (IL)-1alpha and IL-1beta are encoded by two separate genes, but both function as comitogens for lymphocyte activation. In this study, we observed K-562 cells to express constitutively mRNA for IL-1alpha, although IL-1alpha was not detected in the growth-conditioned medium (GCM). However, IL-1beta mRNA was not expressed unless the cells had been treated with phorbol myristate acetate (PMA). Both IL-1alpha and IL-1beta were detected in the GCM after the cells had been cultured with PMA, suggesting that IL-1 elaboration required PMA treatment. The K-562 cells treated with PMA differentiated to the myeloblastic stage, as observed by nuclear morphologic properties by electron microscopy. PMA treatment induced de novo expression of CD61 or gpIIIa, a marker associated with megakaryoblasts. These results showed that although K-562 cells constitutively expressed IL-1alpha mRNA, PMA treatment was required for secretion. On the other hand, both the expression and secretion of IL-1beta required treatment with PMA. This study showed that K-562 cells treated with PMA differentiated to the myeloblastic stage and expressed and secreted IL-1alpha and IL-1beta.

To observe the efficacy of the platelet activation inhibitor Lipo PGE1 therapy in the recovery of graft function after an acute rejection episode after kidney transplantation. Forty patients with acute rejection after kidney transplantation were randomly assigned into groups treated with or without Lipo PGE1. The expression levels of CD61, CD63, and PAC-1 on platelet surfaces were assayed by flow cytometry. The recovery time for graft function and 1-year patient and graft survival rates were recorded. Compared with controls, the expression levels of CD61, CD63, and PAC-1 were lower among acute rejection patients who received Lipo PGE1 therapy. The recovery time for graft function was shorter and the 1-year patient and graft survival rates higher. Lipo PGE1 therapy in patients with acute rejection episodes may inhibit platelet activation thereby benefiting graft functional recovery. The 1-year survival rates of patients and grafts might be increased if the expression levels of CD61, CD63, and PAC-1 on the platelet surfaces was decreased by Lipo PGE1 therapy.

Acute megakaryoblastic leukemia is uncommon and comprises about 5% of acute nonlymphoid leukemias in the French-American-British classification. Cell lines from such leukemias are relatively rare with only about 8 reported in the literature. We established a cell line from a case of acute megakaryoblastic leukemia arising in a 2-year-old child. Surface marker studies of the cells confirmed their megakaryoblastic nature, with 54% of the cells being CD61 positive and peroxidase and esterase negative. The cells had a doubling time of 72 h. Emperipolesis (a phenomenon in which a cell, usually a lymphocyte or neutrophil, enters another cell, moves about and leaves without undergoing phagocytosis) of one blast into another, larger one, was occasionally seen, and a review of the original bone marrow specimen also showed emperipolesis of neutrophils into the megakaryoblasts. The cells responded to interleukin 3 and were inhibited with all-trans-retinoic acid. The karyotype of the cells was the 46,XX,-16 with a marker chromosome. The marker chromosome is possibly chromosome 16 with a small segment of a chromosome translocated to the terminal portion of chromosome 16.