[Four years of circulatory support with the INCOR axial pump from Berlin Heart]. / Quatre ans d'assistance circulatoire avec la pompe axiale INCOR Berlin Heart.

Implantation of definitive left ventricular support is now a therapeutic option for certain patients in refractory heart failure who are not candidates for transplantation. Here we report the case of a patient assisted for more than 4 years with an INCOR axial pump from Berlin Heart. This case shows the feasibility of long term assistance with a continuous flow pump, and an innovative anti-thrombotic strategy relying on the combination of low molecular weight heparin with platelet anti-aggregants.

[Blood activation during long-term mechanical circulatory support]. / L'activation sanguine au cours de l'assistance circulatoire mécanique prolongée.

Mechanical circulatory support has become an approved treatment option for patients with cardiogenic shock or end-stage heart failure. However, recipients of heart assist devices are prone to high incidences of bleeding, thrombo-embolic and infectious complications. The occurrence of these complications is favoured by systemic alterations of coagulation and fibrinolysis, inflammation and immune responses. Several studies have evaluated these pathophysiological changes in patients undergoing long term circulatory support with pulsatile devices. However, the systemic consequences of the more recently introduced rotary blood pumps remain largely unknown. The present review focuses on the systemic consequences of long term circulatory support with pulsatile and non-pulsatile devices.

An in vitro system for testing leucocyte and leukaemic cell line adhesion to synthetic fibres.

Leucocyte adhesion is an important phenomenon in antimicrobial defence, inflammation and immunological mechanisms and has been shown to be dependent upon specialized adhesion molecules. To prevent side-effects related to blood transfusion (e.g. anti-human leucocyte antigen immunization and transmission of infectious agents) leucocyte reduction of blood products is now systematically performed in various countries. The most common system used for leucoreduction is blood filtration. For further understanding of the mechanisms responsible for the interaction between leucocytes and the fibres present in filters we used a flow chamber to study the adhesion of leucocytes and leukaemic cell lines to different types of fibre. Adhesion was quantified using video-microscopy and computer image analysis. Our results demonstrate that adhesion to filter fibres was dependent on the expression of beta2-integrins CD11--CD18 and was inhibited by anti-CD18. The amount of fibres present, their spatial arrangement and the physicochemical characteristics of the fibres were important factors in leucocyte adhesion. Leucocyte adhesion was the highest to polyethylene terephthalate (PET) and polyimide fibres. Lymphocytes or lymphocytic cell lines were poorly adherent to PET fibres. The retaining capacity of leucocyte filters can be improved by taking into account the different parameters for the design of new filters

Human promyelocytic cell line: a convenient tool for studying the molecular basis of WBC filtration.

BACKGROUND: Blood filtration is a technique widely used to reduce the levels of WBCs in blood components. Several studies have been conducted to define the factors that are involved in WBC reduction, but the various mechanisms are not clearly delineated. This study explored the role of WBC adhesion molecules in WBC reduction during filtration. STUDY DESIGN AND METHODS: A minifilter has been developed that has properties similar to those of the standard filter (Sepacell, Asahi Medical) but that allows a smaller volume of blood to be used (15 mL). WBC reduction was achieved to a similar extent in the standard filter and the minifilter (4.15 log and 4.18 log, respectively). Samples of human promyelocytic cell line (HL60) were filtered before and after differentiation induced by vitamin D3 (D3-HL60). Flow cytometry was used to characterize the D3-HL60 filtrates and to count the WBCs after filtration. RESULTS: HL60 was retained in the filter to the same extent as all other WBCs. A higher level of integrin receptors (CD11b/CD18; CD11c/CD18) was expressed by D3-HL60 than by HL60. When the blood was incubated with anti-CD11b, anti-CD11c, or anti-CD18, fewer D3-HL60 cells were trapped by the filter, while only anti-CD11b alters HL60 retention in the filter. CONCLUSION: The receptors CD11b/CD18 and CD11c/CD18 appear to bind to the filter fibers and to be one of the mechanisms responsible for WBC retention.

Evaluation of fluorescein-labeled autologous leukocytes for examination of retinal circulation in humans.

PURPOSE: Increased leukocyte-endothelium interaction have been suggested as a phenomenon contributing to capillary occlusion and/or rupture of the blood-retina barrier during human retinal vascular diseases. This study was performed to evaluate if fluorescein-labeled autologous leukocytes (FLALs) can be used for examination of leukocyte transit in the human retina. METHODS: The preparation consisted of human dextran-separated leukocytes mixed with fluorescein. After reinjection in normal subjects and in one diabetic patient, a confocal scanning laser ophthalmoscope was used to visualize them in the retinal circulation. The changes between FLALs and control leukocytes in the expression of leukocytes adhesion molecules CD11b and CD62L were evaluated by flow cytometry. RESULTS: The circulating FLALs were clearly visible in retinal vessels. The mean (+/- SD) capillaries velocity was 1.43 (+/- 1.3) mm/s in the macula and 1.82 (+/- 1.4) mm/s in the peripapillary area. No leukostasis was detected in the normal subjects, while it was detected in te diabetic patient. Flow cytometry revealed an increase in CD11b and a decrease in CD62L expression of leukocytes after labeling, suggesting that compared to normal leukocytes FLALs are more susceptible to interact with vascular endothelium. CONCLUSIONS: The use of FLAL is presently the only technique applicable in humans for study of leukocyte transit in the retina. Their preparation is technically simple and unexpensive. Precise measurement of the velocity of leukocytes in small vessels can be obtained. Despite evidence of a certain degree of leukocyte activation after the labeling procedure, no leukostasis was detected in vivo in normal subjects. Potential applications for this technique may include the detection of leukostasis in the human retina during severe forms of diabetes and retinal phlebitis.

[Flow cytometry: applications in transfusion medicine]. / Cytométrie en flux: applications en médecine transfusionnelle.

In transfusion medicine, flow cytometry (FCM) is a methodology combining laser radiation, optics and a computerized treatment of numerous results. We can measure size, cellularity and fluorescence intensity of cells or particles in suspension after the binding of appropriate fluorescent antibodies or fluorescent dyes. The main utilisation of FCM in transfusion medicine is for quality control of the process of leukocyte reduction in red cell concentrates or in platelet units, using commercial kits. In addition, it is used for the enumeration of CD 34 positive cells before bone marrow transplantation and for control of platelet function in platelet units. For clinical investigations, FCM may be used for red cell phenotyping, essentially to detect minor populations (chimerism), for the estimation of red cell survival, or for the detection of fetal erythrocytes. In the field of platelet immunology, FCM is an essential tool for detecting platelet antibodies (auto or allo), for platelet phenotyping or for cross-matching. In the future perhaps, FCM will permit us to detect bacterial contamination or prion protein in transfused blood cells.

Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes.

Although it has been well established that the drug efflux pump P-glycoprotein (P-gp) protects the brain against the entry of cytotoxic drugs, its real in situ localization, i.e., at brain capillary endothelial cells or on astrocyte foot processes, is still controversial. The aim of this study was to compare the expression of P-gp and of multidrug resistance-associated protein (Mrp1), another drug efflux pump, in cultured neonatal rat brain astrocytes and in cultured brain capillary endothelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the mdr1b gene was preferentially expressed in astrocytes, whereas both mdr1a and mdr1b mRNA were detected in endothelial cells. Moreover, the mrp1 gene encoding Mrp1 was expressed in both cell types. Western blotting analysis revealed higher expression of P-gp in endothelial cells as compared with astrocytes, but higher expression of Mrp1 in astrocytes. Moreover, P-gp and Mrp1 expression was not modified in more differentiated astrocytes obtained when cultured with db-cAMP for 48 hr. Our functional analysis of P-gp showed a modest effect of P-gp modulators (CsA, verapamil, PSC 833) on the uptake of colchicine (a substrate of P-gp) by astrocytes, whereas they increased by about 50% the uptake of vincristine (a common substrate of P-gp and MRP) by astrocytes. MRP modulators (genistein, probenecid, and sulfinpyrazone) did not modify the uptake of colchicine but increased that of vincristine with a major effect found for sulfinpyrazone. Moreover, indomethacin, probenecid, and sulfinpyrazone increased the uptake of fluorescein (a substrate of MRP but not of P-gp). Taken together, our results provide the first biochemical and functional evidence supporting the expression of P-gp and Mrp1 in rat cultured astrocytes.

Bone marrow transplantation in severe Glanzmann's thrombasthenia with antiplatelet alloimmunization.

Glanzmann's thrombasthenia is an autosomal recessive disorder characterized by a lack of platelet aggregation due to the absence of platelet glycoprotein IIb and IIIa. Usually, the disease leads to mild hemorrhage but sometimes bleeding is severe enough to be life-threatening. We report the case of a 16-year-old girl, presenting with very severe type 1 Glanzmann's thrombasthenia, successfully treated with an HLA-identical sibling bone marrow transplant (BMT). We also update the clinical and laboratory data of her brother, who had received a BMT 16 years ago for the same disease. In the light of these two cases and two others published in the literature, we discuss the indications for BMT from HLA-identical sibling donors in Glanzmann's thrombasthenia. Alloimmunization against the missing platelet GPIIb/IIIa complex and severity of bleeding episodes may constitute sufficient criteria for allogeneic BMT after careful assessment of the risk-benefit of such a procedure, although this remains exceptional in this disease. Bone Marrow Transplantation (2000) 25, 327-330.

Cultured endothelial cells from human arteriovenous malformations have defective growth regulation.

Vascular malformations are frequent in newborns, and they persist throughout life, which differentiates them from vascular tumors (eg, hemangiomas). Arteriovenous malformations are high-flow vascular malformations. They are considered nonmalignant but can expand and become a significant clinical risk when extensive. To characterize endothelial cells from arteriovenous malformations (AMEC), we cultured cells obtained from surgical specimens and studied their properties. After selection, the cells that grew out from explants had phenotypic and antigenic features (platelet endothelial cell adhesion molecule, von Willebrand factor) of human endothelial cells. Their spontaneous proliferation rate was higher (1.8 to 6.4 times) than that of human umbilical vein, arterial, or microvascular endothelial cells. The proliferation rate of AMEC was not sensitive to the inhibitory activity of various cytokines (interleukin-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, Interferon-gamma). In basal conditions, intercellular adhesion molecule (ICAM-1) was detected at a higher level of expression (6- to 10-fold) on AMEC, but these cells failed to express E-selectin or the vascular cell adhesion molecule (VCAM-1) after cytokine stimulation. Expression of c-ets-1 proto-oncogene was shown by in situ hybridization. The low response to cytokines, the higher propensity to proliferate, and the ets-1 expression suggest that AMEC have a defective regulation of proliferation that may be due to a reduced apoptotic process.

Studies of platelet volume, chemistry and function in patients with essential thrombocythaemia treated with Anagrelide.

Anagrelide (imidazoquinazolin derivative) is a new compound proposed for the treatment of myeloproliferative disorders. In this study, Anagrelide was given to patients with essential thrombocythaemia (ET) in a compassionate-use protocol. The aim of this study was to test the effect of this drug not only on the platelet count but also on platelet volume, chemistry and function, which has not previously been reported. Thus, in ET, different functional or structural platelet abnormalities were reported: a shortening of the bleeding time, hypoaggregation to several agonists, and in particular a lack of response to adrenalin, an increase in the amount of total platelet glycoprotein IV (or CD36), and an abnormal migration of thrombospondin on electrophoresis. These different parameters were studied before and during therapy with Anagrelide. Although the platelet count was corrected, no functional or chemical abnormality was improved. Furthermore, platelet volume was shown to be constantly increased under Anagrelide. Thus, Anagrelide, in reducing the platelet count, may possibly decrease the risk of thrombosis and haemorrhage. Nevertheless, if the risk of thromboses and/or myelofibrosis is related not only to the platelet count but also to the platelet abnormalities, the persistence of a thrombocytopathy in patients treated with Anagrelide must be taken in consideration. Our data suggest that thromboses and myelofibrosis are clinical end-points which should be included in future large-scale use of Anagrelide.

P-glycoprotein is more efficient at limiting uptake than inducing efflux of colchicine and vinblastine in HL-60 cells.

PURPOSE: To investigate the role of the P-glycoprotein (P-gp) drug efflux pump in the intracellular disposition of colchicine and vinblastine. METHODS: Uptake and efflux kinetics were studied in vitro in human lymphocytes and in HL-60 cells with or without the P-gp modulator, verapamil. RESULTS: In human lymphocytes, colchicine was slowly taken up (uptake half-life was 18.9+/-1.1 hr.) and verapamil increased colchicine uptake by 37%, whereas it did not modify colchicine efflux from cells. In HL-60 cells, colchicine uptake was non-linear and slower than that of vinblastine, the colchicine uptake half-life (11.1+/-0.5 hr.) being 25-fold longer than that of vinblastine at 25 nM. Verapamil did not significantly modify colchicine uptake half-life, but increased its intracellular accumulation by 23% and that of vinblastine by 81%. Immuno-flow cytometry showed that P-gp expression in HL-60 cells increased significantly from 24 hr. following colchicine or vinblastine exposure. The significant increase in colchicine uptake induced by verapamil at 24 hr. was correlated with this enhanced P-gp expression. The drug efflux half-life was 11.5-fold higher for colchicine (23+/-0.9 hr) than vinblastine, indicating a much slower elimination of colchicine from cells that could be related to its longer dissociation half-life from the tubulin receptor. Verapamil treatment did not modulate either colchicine or vinblastine efflux kinetics, suggesting that the intracellular drugs are not available to the transmembrane P-gp binding sites. CONCLUSIONS: P-gp may not be the main reason for the slowness of colchicine uptake. It may be more efficient at controlling entry of colchicine and vinblastine through the plasma membrane than at mediating their efflux from HL-60 cells.

Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line.

P-glycoprotein (P-gp), a product of the multidrug-resistant (mdr) genes, is expressed in the endothelial cells of the blood-brain barrier (BBB). Effects of glial factors and retinoic acid (RA) on P-gp activity and level were investigated in the immortalized rat brain endothelial cell line RBE4, which expressed immunodetectable P-gp associated with a decrease in accumulation of the P-gp substrates, vinblastine and colchicine. When RBE4 cells were cultured either in the presence of C6-conditioned medium or on C6- or astrocyte-extracellular matrix, intracellular vinblastine and colchicine concentrations were decreased. When the cells were treated with RA, increases in P-gp activities were correlated with increases in P-gp levels. Effects of simultaneous treatments with glial factors and RA were studied in RBE4 cells cultured on astrocyte-extracellular matrix and were shown to be additive on P-gp activity and level. RBE4 cells may serve as a useful in vitro model for basic research on P-gp regulation at the level of the BBB.

Platelet factor 4 and other CXC chemokines support the survival of normal hematopoietic cells and reduce the chemosensitivity of cells to cytotoxic agents.

The effects of platelet factor 4 (PF4) on the viability and chemosensitivity of normal hematopoietic cells and cancer cell lines were studied to determine the mechanisms whereby PF4 functions as either an inhibitor or a protector and to evaluate its clinical significance. Two other chemokines, interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2), were also studied in comparison to PF4. Using a tetrazolium salt assay for cell viability, we observed that PF4 at 1 to 50 microg/mL supported the viability of normal human bone marrow cells. Approximately 45% of cells cultured for 48 hours survived, whereas 80% or more survived in the presence of PF4 5 microg/mL. PF4 also supported the viability of CD34+ cord blood (CB) cells and protected them from apoptosis induced by transforming growth factor beta1 (TGFbeta1) and cytotoxic drugs. Pretreatment of CD34+ cells by PF4, but not by TGFbeta1, caused an increase in the number of megakaryocyte colonies after these cells were replated in secondary cultures. Flow cytometry analysis showed that when CD34+ cells were preincubated with PF4 or TGFbeta1 for 12 days in hematopoietic growth factor-rich medium, an increased number of remaining CD34+ cells was observed only for PF4-treated cells. Furthermore, PF4 significantly reduced the chemosensitivity of bone marrow cells, as shown by its ability to increase the 50% inhibition concentration (IC50) of several cytotoxic agents. Like PF4, IL-8 and NAP-2 at 0.1, 0.6, and 1 microg/mL supported the survival of myeloid progenitors, including colony-forming units granulocyte, erythroblast, monocyte, megakaryocyte (CFU-GEMM), CFU-megakaryocyte (CFU-MK), CFU-granulocyte/macrophage (CFU-GM), and burst-forming units-erythroblast (BFU-E), and reduced their sensitivity to the toxicity of etoposide (ETP). Protamine sulfate at 1 to 100 microg/mL showed no such activity of PF4. Interestingly, the three chemokines failed to affect significantly the viability and chemosensitivity of three leukemic and two other tumor cell lines. Based on these results, we conclude for the first time that PF4 and IL-8 and NAP-2 support the survival of normal hematopoietic precursors and protect them from the toxicity of chemotherapeutic agents. Because such activities are unique to normal hematopoietic cells but not to the cancer cell lines evaluated, a potential clinical application of these molecules in the treatment of cancer is suggested.

Scanning laser ophthalmoscope imaging of fluorescein-labelled blood cells.

PURPOSE: To demonstrate the feasibility of a technique for the visualization by scanning laser ophthalmoscope (SLO) of fluorescein-labelled autologous leukocytes and platelets in retinal vessels. METHOD: Individual blood samples from rats and rabbits were centrifuged to isolate platelets and leukocytes, then passively labelled with fluorescein and reinjected into the same animal. An SLO was used to visualize and record cell displacement in the retinal circulation. Labelled platelets were analysed by flow cytometry. RESULTS: By SLO, platelets appeared as a heterogeneous particle flow, and individual leukocytes appearing as brighter spots could easily be traced. Flow cytometry showed that after labelling platelets were well individualized and their size was slightly increased. CONCLUSION: Circulating blood cells can be visualized in retinal vessels by a simple method consisting of passive labelling of autologous platelets and leukocytes by fluorescein. No platelet toxicity was detected. This method could be applied to the study of blood cell movement in human retinal vascular diseases.

Endothelial cells in culture: an experimental model for the study of vascular dysfunctions.

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [3H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor-alpha and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selection and vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also in rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.

Monocyte modulation of endothelial leukocyte adhesion molecules.

Leukocyte adhesion to endothelium is dependent on expression of specialized molecules. Several of these molecules are upregulated by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). We investigated the effect of medium conditioned by unstimulated (MCM) or stimulated monocytes and of recombinant cytokines on endothelial adhesion receptor expression. IL-1 beta, TNF-alpha, and MCM induced E-selectin similarly, whereas MCM induced VCAM-1 and ICAM-1 to a lesser extent than did TNF-alpha, and MCM induced VCAM-1 only weakly. The addition of pentoxifylline (10(-3) mol/L) to monocytes during MCM preparation blocked TNF-alpha production but not that of IL-1 beta or IL-6, and it reduced IL-1ra significantly (p < 0.05). When the MCM was devoid of TNF-alpha or when TNF-alpha was neutralized with a specific antibody, the action of MCM on E-selectin expression was significantly lower. Anti-IL-1 beta decreased the activity of MCM on endothelial E-selectin expression by about 50%. The effect of MCM on adhesion molecules was accompanied by an increase in monocyte adhesion. Inhibition of TNF-alpha production reduced monocytes adhesion slightly but significantly (18%, p < 0.05), whereas anti-IL-1 beta antibody decreased adhesion by 48% (p < 0.001). These results show that adherent monocytes released cytokines and antagonists that affect leukocyte adhesion receptors on endothelium differently from recombinant cytokines. E-selectin expression--and to a lesser extent ICAM expression--is modified, resulting in a modulation of leukocyte adhesion to endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological study of platelet glycoproteins and alloantigens in families of patients with Glanzmann's thrombasthenia.

To explore the genetic transmission of Glanzmann's thrombasthenia (GT) we conducted a study of platelet glycoproteins and antigens using monoclonal and polyclonal antibodies and cytofluorometry. Twenty-one members of 4 different families in which at least one member was affected with GT were explored. The reactivity with AP2 (anti-GP IIb-IIIa complex, CD41a), AP3 (anti-GP IIIa) and antibodies against HPA-1a (PLA1) and HPA-3a (Leka) was lower than 5% in the platelets of 5 patients with GT type I. Nine obligatory carriers of the GT trait were tested by the same technique. The mean values of reactivity expressed as per cent of normal with AP2 (anti-CD41a) and AP3 (anti-GPIIIa) were 58.3 +/- 3.6% and 58.6 +/- 3.6% respectively, while the frequencies of HPA-1a (PLA1) and HPA-3a (Leka) negativity were 0/9 and 4/9 respectively. When both antigens were expressed they were detected at reduced levels. These results indicate that cytofluorometry is a simple technique for GT detection, but this method would nevertheless not appear to be sufficiently accurate to identify obligatory carriers in a general population.

Platelet membrane glycoproteins and platelet functions during storage in the presence of a proteinase inhibitor.

The effect of the proteinase inhibitor aprotinin on membrane glycoproteins and functions of platelets stored for 5 days in platelet-rich plasma was tested. Platelet membrane glycoprotein content was determined by flow cytometry or immunoblot techniques using different monoclonal antibodies. ADP- and ristocetin-induced platelet aggregation and adhesion to collagen were tested in parallel. Using the flow-cytometry technique i) a progressive decrease in the percentage of platelets reacting with the different monoclonal antibodies was observed during storage ii) a 30% reduction of the GPIb mean fluorescence intensity (MFI) was observed after 5 days storage while the MFI of the GP IIb-IIIa complex was not modified. Using the immunoblot technique, a decrease in the amount of both the GPIb alpha and the component of Mr 100,000 was observed, while a 50,000 Mr fragment appeared progressively. Platelet adhesion and aggregation were reduced after 24 hours of storage. Aprotinin prevented neither the GPIb alpha reduction nor the modifications of the functions of human platelets stored in their autologous plasma.

Association of autoimmune thrombocytopenic purpura (AITP), Graves' disease and ovarian carcinoma.

We report here the case of a patient suffering sequentially from autoimmune thrombocytopenic purpura (AITP), Grave's disease and an ovarian carcinoma. An autoimmune mechanism was indicated by the presence of platelet associated immunoglobulins and the detection of antithyroid microsomal antibodies. However, the exact mechanism associating these autoimmune manifestations with the ovarian tumour remains unexplained. Specific therapy for hyperthyroid led to a moderate increase of the platelet count. But complete remission of AITP now lasting for more than eight years was only obtained by ablation of the ovarian tumour.