Protein synthesis and storage in human platelets: a defective storage of fibrinogen in platelets in Glanzmann's thrombasthenia.

In vivo metabolic labelling experiments were performed to investigate the ability of human platelets to synthesize and store fibrinogen and thrombospondin. Newly synthesized proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Results were compared with those obtained for the platelets of a patient with Glanzmann's thrombasthenia where endogenous fibrinogen levels were severely reduced. Normal human platelets were able to synthesize the different subunits of fibrinogen and thrombospondin and to assemble them into native fibrinogen and thrombospondin molecules. This synthesis was inhibited by cycloheximide. Synthesis of both fibrinogen and thrombospondin was observed in the platelets of the Glanzmann's thrombasthenia patient. However, radiolabelled fibrinogen was no longer detected after an 18-h non-radioactive chase, although it was retained in the control platelets. Neosynthesized thrombospondin of the patient was normally preserved during the same chase period. When the fate of the radioactive fibrinogen was studied, it was found to be degraded in Glanzmann's thrombasthenia platelets to the same extent as neosynthesized cytoplasmic proteins, whereas in control platelets less degradation had occurred. We conclude that human platelets maintain a residual capacity to synthesize fibrinogen and that its deficiency in Glanzmann's thrombasthenia results from a storage abnormality and not from a synthesis defect.

Bcr-abl translocation can occur during the induction of multidrug resistance and confers apoptosis resistance on myeloid leukemic cell lines.

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT-PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis.

Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia.

A flow cytometry method has been introduced into the routine investigation of whole bone marrow samples following red blood cell lysis on the basis of a primary CD45/side scatter (SSC) gating procedure. Blast cells were first identified by CD45/SSC gating in 74 cases of acute myeloid leukemia (AML) and the results were compared to a conventional FSC/SSC gating procedure and to MGG-staining smears. The percentages of blast cells in these samples as defined by the morphological analysis of MGG smears correlated better with the values determined by CD45/SSC gating (r = 0.94) than with the blast cell counts recorded with FSC/SSC gating (r = 0.76). These findings were not surprising because while CD45 expression was regularly lower on leukemic blasts than on normal lymphoid and monocytic cells, the FCS/SSC characteristics of these populations were overlapping. In 53 samples, the blast cell populations were also analyzed with a panel of FITC-conjugated monoclonal antibodies that were utilized in double labeling with CD45-PE. We show that the CD45/SSC gating procedure improved phenotypic determination of the blast cells in three ways: (1) by discriminating between leukemic blast cells and residual normal cells; (2) by excluding normal cells from the phenotypic analysis of leukemic blast cells; and (3) by identifying blast cell heterogeneity in many cases of leukemia on the basis of different CD45 display. Moreover, this immunophenotyping procedure on whole bone marrow samples also allowed an efficient discrimination between the various cell lineages and facilitated the analysis of leukemic blasts present in low proportions.

Flow cytometric study of the activation of polymorphonuclear cells.

The activation of human polymorphonuclear cells (PMN) by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or the protein kinase C activator, phorbol myristate acetate (PMA), was studied using flow cytometry. Two probes were used to evaluate PMN activation: 1) a monoclonal antibody (MoF11) directed against an antigen (Ag) expressed on the membrane of monocytes and of activated PMN; 2) rhodamine phalloidin was used at the cytoplasmic level to measure the F-actin content. The expression of MoF11 antigen was found to be 3 to 5 times greater on the membrane of PMN activated by either FMLP or PMN as compared with membrane expression of the same Ag on resting PMN. This increase was found to be dose dependent for the two activators. Kinetic studies showed that a maximum response was observed in 1 to 2 min at 37 degrees C when FMLP was used, whilst a similar response required 10 min when PMA was used. The same discrepancy with activators was observed when actin polymerization was measured by labelling with rhodamine phalloidin. However, pretreatment of PMN with cytochalasin B inhibited actin polymerization whilst MoF11 antigen expression was increased, suggesting that the MoF11 antigen could be stored in granules of resting PMN. The study of actin polymerization and of MoF11 antigen expression, separately or in combination, could be a useful tool for the detection of activated PMN in biological samples.

Veno-active drugs in the management of chronic venous disease. An international consensus statement: current medical position, prospective views and final resolution.

BACKGROUND: Veno-active drugs (VAD) have effects on edema and symptoms related to chronic venous disease (CVD), especially so-called venous pain. VAD's effectiveness, although well established, is regularly debated. OBJECTIVE: Our purpose was to select all randomized controlled trials (RCTs) and meta-analyses devoted to VAD and symptoms in CVD, to submit them to a group of international experts in CVD and to vote with secrete ballot to determine the level of efficacy of each drug, according to EBM (Evidence-Based Medicine) rules and critical analysis. METHODS: Publications in any language devoted to VAD and venous symptoms were searched for in different databanks and submitted to the experts prior to the meeting. RESULTS: 83 papers were analyzed, including 72 RCTs or meta-analyses. Experts determined the level of EBM of each drug, according to the literature and personal experience, using 3 levels of recommendation, A, B and C (from large RCTs to non-randomized trials). CONCLUSIONS: VAD are effective and may be applied in CVD when symptomatic, from C0s to C6s. However, etiological treatment of venous reflux and venous hypertension has always priority. In some cases VAD may replace compression and/or complement its effects. If respecting these prerequisites, VAD are safe and effective.

[The management of lung transplantation candidates. A case series]. / Préparation des insuffisants respiratoires à la transplantation. Un état des lieux.

INTRODUCTION: Lung transplantation (LT) is associated with an increased risk of infection, cancer, chronic renal failure, cardiovascular disease and osteoporosis. Some risk factors precede transplantation and could benefit for early diagnosis and optimised care. METHODS: The incidence of comorbidities and their treatment before referral were assessed in 157 consecutive lung transplant candidates between 2008 and 2011. RESULTS: The median age was 37years [25; 51]. Fifty-six percent had a body mass index below 19kg/m(2). In the COPD group, only 50 % had undergone a pulmonary rehabilitation program in the preceding 2 years. Osteoporosis was present in 42 %, of whom 36 % were on bisphophonate therapy. Vitamin D deficiency was present in 65 %. Previously undiagnosed cardiovascular risk factors were discovered during LT assessment: hypertension in one patient, hypercholesterolemia in 6 % and diabetes in 4 %. Poor dental condition necessitating extractions were found in 41 % of patients. Protective anti-HBs antibodies levels were present in 50 % of the patients at the time of referral. CONCLUSION: The assessment and early treatment of nutritional disorders, osteoporosis and risk factors for infection as well as addressing associated cardiovascular risk factors should be optimised in the care of patients with chronic respiratory insufficiency. The potential for becoming a lung transplant candidate in the future should be kept in mind early in the global management of those patients.

Megakaryopoiesis disturbances in atherosclerotic rabbits.

Rabbits given a hypercholesterolemic diet (500 mg/day) for 6 months and then maintained for another 6 months on a normal diet were found to have developed fibrous lipidic lesions in the aorta. Although circulating platelet levels in these animals were normal there was a reduction in mean megakaryocyte ploidy. The high concentrations of megakaryoblasts in all the sedimentation fractions collected by the 'STAPUT' system suggested an increase in megakaryocyte turnover with activation of committed stem cells. In addition, other defects in maturation of megakaryocytes were observed, such as abnormalities in the demarcation membrane system and granule number. These data reveal that defects in megakaryocyte maturation and turnover may occur during the process of reparative fibrosis of the arterial tree following a period of moderate hypercholesterolemic diet in the rabbit.

Endothelial dysfunction during acute methionine load in hyperhomocysteinaemic patients.

Hyperhomocysteinaemia has been associated with arterial and venous thrombosis possibly by causing damage to the endothelium. We hypothesised that an oral load in methionine, that increases plasma homocysteine, would also result in an increase in biological markers of endothelial or platelet dysfunction. Then we investigated two groups of patients with arterial or venous occlusive disease: 17 with hyperhomocysteinemia and 12 without hyperhomocysteinemia. We measured in both groups plasma soluble thrombomodulin, von Willebrand factor, P-selectin and tissue factor plasma inhibitor before and 6 hours after a load with 100 mg/kg oral methionine. Methionine load resulted in a significant increase in von Willebrand factor in both groups (P<0.02), suggesting that endothelial dysfunction occurs during the load.

Influence of selected heparins on human neutrophil functions in vitro.

The effects of four heparin derivatives [unfractionated heparin (UFH) at 5-50 IU/ml, low molecular weight heparin (LMWH) at 2-20 IU/ml, pentasaccharide (Penta) at 5 IU/ml and a synthetic heparinoid (PPS) at 10(-6)-10(-5) M] on various polymorphonuclear (PMN) leukocyte end-functions (aggregation, chemotaxis, phagocytosis and burst) were examined. CR3 expression, actin polymerization and membrane surface charge were also studied to gain more insight on the mechanisms of the action of heparins on PMN. The different heparins were found to have rather different actions. PMN were found to be hyperreactive to PPS. Pentasaccharide hat no effect on PMN functions, while UFH and LMWH had intermediate reactivity, modulating responses in an adenosine-like manner. Interactions of heparins with PMN were attributed to biophysical properties of the molecules rather than to the presence of a specific sequence such as a pentasaccharide. Our results show that certain heparin derivatives, apart their well-known anticoagulant action, modulate polymorphonuclear leukocyte functions that may be involved in vascular injury.

Platelet dense bodies loaded with mepacrine. Study in chronic idiopathic thrombocytopenic purpura (ITP).

In 22 cases of chronic ITP, the platelet 5-HT storage organelles were counted by examination of platelets loaded with mepacrine and correlated with the size and volume of the platelets. Statistical analysis showed that the mean volume and the number of granules increased in ITP without increase in the mean number of granules per unit volume. A strong correlation was found between platelet long diameter and number of dense bodies in controls (44 healthy subjects) (r = 0.94; y = 2.826 x - 0.699) and in ITP (r = 0.92; y = 2.587 x + 0.06). This study demonstrated in chronic ITP the presence both of platelets without granules and others rich in granules. The anomalies were present no matter what the count of platelets and did not change the mean values for granules and for ADP in most cases. Most platelets remain morphologically normal.

Mepacrine labelling test and uranaffin cytochemical reaction in human megakaryocytes.

5-HT storage organelles were observed by electron microscope analysis in human megakaryocytes. They were less numerous per unit of surface than in platelets. Their number depended on the visualization technique employed. Thus after fixation with calcium-enriched glutaraldehyde a higher number of very opaque organelles was observed than of uranaffin-positive organelles after the cytochemical uranaffin reaction. With conventional electron microscopy deep black granules characteristic of dense bodies were not observed. Fluorescent microscopy showed greenish-yellow granules distributed throughout the whole cytoplasm in 96 +/- 1.4% of normal megakaryocytes incubated with mepacrine. In 85.6 +/- 5%, 5.28 +/- 1.28 granules per 10 microns 2 were observed. With the mepacrine labelling test, 74% of the megakaryocytes of a patient with Hermansky-Pudlak syndrome contained no granules. A similar finding was made in the platelets of the same patient. This suggests that mepacrine also stains the dense bodies in the megakaryocytes and that in the Hermansky-Pudlak syndrome the platelet anomaly is secondary to a megakaryocyte anomaly.

Hemorheological changes in elderly subjects--effect of pentosan polysulfate and possible role of leucocyte arachidonic acid metabolism.

The effects of pentosan polysulfate (PPS) on various hemorheological parameters were studied in a group of very elderly subjects in good general health. Alterations in blood viscosity and filterability were detected in these patients, without any concomitant changes in factors which are known to affect these parameters: notably hematocrit, fibrinogen and plasma lipid levels. The hemorheological abnormalities were considerably improved by twice daily treatment with 50 mg of PPS (i.m.). Apart from its anticoagulant activity, PPS has been shown to have an anti-inflammatory action. We were interested to investigate its effects on metabolism of exogenous arachidonic acid (AA) by both platelets and leucocytes. It is becoming increasingly recognized that metabolites of AA via the 5 LO pathway appear to play a role in inflammatory processes. In this study, PPS was found to inhibit leucocyte 5 LO activity. Reduction in the levels of these metabolites may therefore have an effect on whole blood rheology.

Megakaryocytes from the marrow of a patient with Glanzmann's thrombasthenia lacked GP IIb-IIIa complexes.

Although it is recognized that glycoprotein (GP) IIb-IIIa complexes are deficient in platelets in Glanzmann's thrombasthenia, little is known of the origin of the defect. We have examined the megakaryocytes in a bone marrow aspirate obtained from a thrombasthenia patient during surgery. Analysis of platelet proteins by SDS-polyacrylamide gel electrophoresis confirmed the patient to be of the type I subgroup. The megakaryocytes were examined by immunofluorescence or by immunocytochemical procedures combined with electron microscopy. Antibodies used included the murine monoclonal antibody, AP-2 and the human allo-antibody, IgG L, both of which recognize determinants on GP IIb-IIIa complexes. Bound antibody was detected by anti-IgG antibodies coupled to fluorescein isothiocyanate or absorbed on gold particles. In the immunofluorescence studies, permeabilized megakaryocytes were identified by double staining using an antibody to von Willebrand factor (vWF). Whereas mature megakaryocytes and their small precursor cells from normal individuals were strongly fluorescent with AP-2 and IgG L, most vWF positive cells from the Glanzmann's thrombasthenia patient were negative and the remainder gave but a weak background fluorescence. Immunogold staining on the surface of marrow cells was severely reduced. Our results confirm a deficiency of GP IIb-IIIa complexes in megakaryocytes in thrombasthenia.

Soluble adhesion molecules and endothelial cell damage in HIV infected patients.

Endothelial damage is present in HIV infection but our understanding of markers and mechanisms is incomplete. We found increased levels of markers of endothelial cell damage such as von Willebrand factor (vWf), soluble thrombomodulin (sTM) and adhesion molecule E-selectin in 90 subjects seropositive for HIV relative to healthy controls. sTM was strongly raised in those patients with the lowest CD4+ cell count (p < 0.001), but levels of vWf increased at each incremental fall in CD4+ cell count and the two indices correlated significantly (r = -0.485, p < 0.001). vWf correlated strongly with levels of the inflammatory cytokines tumor necrosis factor (TNF-alpha) and alpha interferon (IFN-alpha) but sTM correlated only weakly with IFN-alpha. We suggest that increased vWf is largely the result of inflammatory stimulus of the endothelium but that sTM is found only in those patients with more severe disease, and so truly represents endothelial damage.

Technical and biological conditions influencing the functional APC resistance test.

Poor anticoagulant response to APC is conveniently screened by a commercially available functional test (Coatest APC Resistance) allowing identification of APC-resistant patients. These patients may then be genotyped with respect to factor V, the Arg -> Gln mutation being the principle cause of APC resistance. However, determination of phenotype generally precedes that of genotype, and the need for an "abnormality threshold" prompted a study of inter-batch variations and the clinical conditions associated with an altered APC response. The response to APC was assessed twice in plasma from 111 patients using two of four successive kit batches. A modest but significant inter-batch variability was observed. At the same time, we also tested 130 patients with retinal venous occlusion (RVO), 28 patients with glaucoma and 24 normal volunteers. The APCaPTT/aPTT ratio was found to be lower in the presence of elevated thrombin-antithrombin complexes (r = 0.167, p < 0.02) and low blood viscosity (at high shear rate: r = 0.305, p < 0.0001) independently of any alteration in genotype.

Effect of the antioxidants selenium and beta-carotene on HIV-related endothelium dysfunction.

Patients infected with HIV are at increased risk of atherosclerosis, and have evidence of endothelium dysfunction. The hypothesis was tested that HIV-related endothelium dysfunction is related to loss of antioxidants. This was done by the supplementation of the antioxidants selenium and beta-carotene. We supplemented the diet of 10 HIV-seropositive subjects with 100 microg selenium daily, 11 subjects with 30 mg beta-carotene twice daily while 15 subjects were not supplemented. Plasma was obtained at outset and after a year, and tested by ELISA for endothelial cell, platelet and inflammatory markers. The non-supplemented patients experienced increases in von Willebrand factor and soluble thrombomodulin (both p <0.01). There were no changes in any of the indices in the patients taking selenium or beta-carotene. Increased von Willebrand factor and soluble thrombomodulin in the non-supplemented patients imply increased damage to the endothelium over the year of the study. Therefore we interpret the lack of increase in the patients taking antioxidants as evidence of the protection of the endothelium by these agents.

Acute promyelocytic leukemia with (15;17) translocation and chromosome no. 11 deletion (q23).

The patient, a 76-year-old man, was referred with fever, large ecchymotic lesions and ulcerative laryngitis. Blood counts showed a hemoglobin of 11 g/100 ml, hematocrit of 31%, red blood cell count of 3.5 X 10(12)/1, white blood cell count of 6.8 X 10(9)/1 and platelet count of 16.0 X 10(9)/1. The differential count showed 17% neutrophils, 4% lymphocytes, 40% promyelocytes and 39% myeloblasts. The sternal marrow sample showed a marked hypercellularity. Of the cells, 80-85% were hypergranular promyelocytes, some of them showing bundles of Auer rods. No granulocytic maturation was observed. A few erythroblasts were present. A disseminated intravascular coagulation was observed (fibrinogen 0.85 g/l, factor V 18%, fibrin degradation products 640 mg/l). The serum creatinin was at 217 micromol/1 and the urea at 16.8 mmol/1. The treatment (daunorubicin, heparin, platelet transfusion) was unsuccessful and the patient died three days after entering hospital. The bone marrow karyotype by direct examination showed only normal metaphases (32 photographed). All the metaphases from the unstimulated blood 48-h culture (25 photographed) were clonal, showing the pattern 47,XY,del(11) (q23),t(15;17) (q24? q22?), +mar. The marker was '16 like' in size but its origin could not be determined (Figs. 1 and 2).

Acute erythroblastic leukemia presenting as acute undifferentiated leukemia: a report of two cases with ultrastructural features.

This report describes two elderly patients with acute leukemia in which blast cells were undifferentiated with conventional light microscopy (L.M.) and cytochemistry. Blast cells were identified as belonging to the erythroblastic line by their ultrastructural features: glycogen deposits, lipidic vacuoles, cytoplasmic ferritin molecules and rhopheocytotic invagination. Moreover, blast cells were surrounding a central macrophage. Thus, these two patients had acute erythroblastic leukemia which differs from erythroleukemia (M6 of FAB classification) in which blast cells present myeloblastic characteristics.

Study of the apoptosis induced in vitro by antitumoral drugs on leukaemic cells.

A flow cytometric method for simultaneous apoptotic cell detection and cell cycle analysis was applied on the U937 cell line. Four antitumoral drugs currently used in the treatment of acute myeloid leukaemia were studied in vitro: DNR, IDR, MITO and Ara-C. Our results show a dissociation between the cytostatic effect (the block in the cell cycle observed for low drug concentrations) and the cytotoxic effect (the induction of apoptosis induced by higher concentrations) for all the tested molecules. Low concentrations of Ara-C induced a block in the S phase while higher concentrations (>10(-7) M) induced apoptosis at the G1-S boundary. Low concentrations of anthracyclines (<40 nM DNR and <20nM IDR) induced a block in G2 without apoptosis. Apoptosis was induced in G1 and/or early S phases by higher concentrations of anthracyclines. The concentration inducing 50% apoptosis (IC50) was found to be, respectively, 200 and 40 nM for DNR and IDR. Analysis of MITO-treated cells showed a parallel increase in the percentages of S phase and apoptotic cells. However, the bivariate analysis showed that apoptosis did occur in a population with G1 DNA content. For two other drugs (CAM and COLC), apoptosis occurred for the same concentrations and in the same phase as the block (in S and G2M, respectively). The IC50 of MITO was found to be 100 nM. Cotreatment of the cells with colchicin and either Ara-C or IDR showed that the passage through mitosis was not necessary for the completion of apoptosis at the G1-S boundary. Short incubations of U937 cells with high concentrations of anthracyclines were found to be efficient in inducing further apoptosis. We conclude that, for all the assayed molecules, the cytotoxic and/or cytostatic effects of the antitumoral drugs tested greatly depend on the concentrations used and that, depending on their in vivo pharmacokinetics, the induction of apoptosis could be an important mechanism of action for some of these drugs.

Influence of rhGM-CSF on Ara-C sensitivity of patients with acute myeloid leukemia in relapse: a flow cytometry study.

Twenty-three patients with acute myelogenous leukemia (AML) in first relapse were treated with high-dose cytosine-arabinoside (Ara-C) and amsacrine or idarubicin. To prime the cells, the patients were given rhGM-CSF. We studied the influence of 48-h infusion of rhGM-CSF on proliferation and Ara-C sensitivity of leukemic cells both ex vivo and in vitro. We found that a 48-h infusion of rhGM-CSF increased both white blood cell counts and peripheral blood blast cell percentages. Using a Bromodeoxyuridine/DNA (BrdUrd/DNA) staining in flow cytometry, we found an non-constant increase in cells in the S-phase. Ex vivo 48-h culture of leukemic cells with or without rhGM-CSF, with or without other hematopoietic growth factors (HGFs), showed a greater increase of the cells in the S-phase with GF but no correlation with the ex vivo results. We used a method of quantitation of the DNA synthesis previously described (Lacombe F., et al. (1992) Cytometry 13, 730) to monitor the Ara-C sensitivity of the cells in S-phase before and after 48-h infusion with rhGM-CSF. We observed a great variation in the Ara-C sensitivity of the leukemic cells before and after infusion with rhGM-CSF from one patient to another. The BrdUrd/DNA method seems a convenient method to study the influence of HGFs on Ara-C sensitivity of the patients.