Plasma temperature during methylene blue/light treatment influences virus inactivation capacity and product quality.

BACKGROUND: Photodynamic treatment using methylene blue (MB) and visible light is in routine use for pathogen inactivation of human plasma in different countries. Ambient and product temperature conditions for human plasma during production may vary between production sites. The influence of different temperature conditions on virus inactivation capacity and plasma quality of the THERAFLEX MB-Plasma procedure was investigated in this study. METHODS: Plasma units equilibrated to 5 ± 2°C, room temperature (22 ± 2°C) or 30 ± 2°C were treated with MB/light and comparatively assessed for the inactivation capacity for three different viruses, concentrations of MB and its photoproducts, activity of various plasma coagulation factors and clotting time. RESULTS: Reduced solubility of the MB pill was observed at 5 ± 2°C. Photocatalytic degradation of MB increased with increasing temperature, and the greatest formation of photoproducts (mainly azure B) occurred at 30 ± 2°C. Inactivation of suid herpesvirus, bovine viral diarrhoea virus and vesicular stomatitis virus was significantly lower at 5 ± 2°C than at higher temperatures. MB/light treatment affected clotting times and the activity of almost all investigated plasma proteins. Factor VIII (-17·7 ± 8·3%, 22 ± 2°C) and fibrinogen (-14·4 ± 16·4%, 22 ± 2°C) showed the highest decreases in activity. Increasing plasma temperatures resulted in greater changes in clotting time and higher losses of plasma coagulation factor activity. CONCLUSIONS: Temperature conditions for THERAFLEX MB-Plasma treatment must be carefully controlled to assure uniform quality of pathogen-reduced plasma in routine production. Inactivation of cooled plasma is not recommended.

Evaluation of droplet digital PCR for quantification of residual leucocytes in red blood cell concentrates.

BACKGROUND AND OBJECTIVES: Enumeration of residual white blood cells in leucoreduced blood components is essential part of quality control. Digital PCR has substantially facilitated quantitative PCR and was thus evaluated for measurements of leucocytes. MATERIALS AND METHODS: Target for quantification of leucocytes by digital droplet PCR was the blood group gene RHCE. The SPEF1 gene was added as internal control for the entire assay starting with automated DNA extraction. The sensitivity of the method was determined by serial dilutions of standard samples. Quality control samples were analysed within 24 h, 7 days and 6 months after collection. Routine samples from leucodepleted red blood cell concentrates (n = 150) were evaluated in parallel by flow-cytometry (LeucoCount) and by digital PCR. RESULTS: Digital PCR reliably detected at least 0·4 leucocytes per assay. The mean difference between PCR and flow-cytometric results from 150 units was -0·01 (±1·0). DNA samples were stable for up to at least six months. PCR measurement of leucocytes in samples from plasma and platelet concentrates also provided valid results in a pilot study. CONCLUSION: Droplet digital PCR to enumerate leucocytes offers an alternative for quality control of leucoreduced blood products. Sensitivity, specificity and reproducibility are comparable to flow-cytometry. The option to collect samples over an extended period of time and the automatization introduce attractive features for routine quality control.

Effect of increased agitation speed on pathogen inactivation efficacy and in vitro quality in UVC-treated platelet concentrates.

BACKGROUND AND OBJECTIVES: Pathogen inactivation technologies require continuous development for adjustment to different blood components and products. With Theraflex UV-Platelets, a system using shortwave ultraviolet C (UVC) light (254 nm), efficient mixing of platelet concentrates (PCs) during UVC treatment is essential to ensure homogeneous illumination of the blood components. In this study, we investigated the impact of increasing the agitation speed during UVC treatment on pathogen inactivation capacity and platelet quality. MATERIAL AND METHODS: The pathogen inactivation efficacy of UVC treatment was evaluated at two agitation speeds (110 vs. 180 rpm) using four different transfusion-relevant bacteria strains and three model viruses. Using a pool-and-split design, the in vitro quality of buffy coat-derived PCs stored in SSP+ additive solution for up to 7 days was assessed in UVC-treated PCs agitated at either 110 rpm (standard speed) or 180 rpm (increased speed) and in untreated controls. RESULTS: The higher agitation speed improved bacterial inactivation but did not influence viral inactivation. Metabolic activity (glucose consumption and lactate accumulation) in UVC-treated platelets was slightly higher than in untreated controls. Increases in parameters such as CD62P expression and annexin A5 binding indicated moderate activation of UVC-treated platelets. Quality variables for UVC-treated platelets agitated at standard vs. increased agitation speed were comparable. CONCLUSION: The mixing rate during illumination may be a process parameter for further development of UVC-based pathogen inactivation procedures for PLT concentrates.

Tolerance of platelet concentrates treated with UVC-light only for pathogen reduction--a phase I clinical trial.

BACKGROUND: The THERAFLEX UV-Platelets pathogen reduction system for platelet concentrates (PCs) operates with ultraviolet C light (UVC; 254 nm) only without addition of photosensitizers. This phase I study evaluated safety and tolerability of autologous UVC-irradiated PCs in healthy volunteers. METHODS: Eleven volunteers underwent two single (series 1 and 2) and one double apheresis (series 3). PCs were treated with UVC, stored for 48 h and retransfused in a dose-escalation scheme: 12·5, 25% and 50% of a PC (series 1); one complete PC (series 2); two PCs (series 3). Platelet counts, fibrinogen, activated partial thromboplastin time, prothrombin time, D-dimer, standard haematology, temperature, heart rate, blood pressure and clinical chemistry parameters were measured. One- and 24-h corrected count increments were determined in series 2 and 3. Platelet-specific antibodies were assessed before and at the end of the study. RESULTS: Neither adverse reactions related to transfusions nor antibodies against UVC-treated platelets were observed. Corrected count increments did not differ between series 2 and 3. CONCLUSIONS: Repeated transfusions of autologous UVC-treated PCs were well tolerated and did not induce antibody responses in all volunteers studied. EudraCT No. 2010-023404-26.

Long-term clinical and molecular remissions in patients with follicular lymphoma following high-dose therapy and autologous stem cell transplantation.

BACKGROUND: Long-term clinical and molecular remissions in patients with follicular lymphoma (FL) following high-dose therapy (HDT) and autologous stem cell transplantation (ASCT) have been evaluated in only a few studies. Results are especially limited for second-line HDT with BEAM (BCNU, etoposide, cytarabine and melphalan). PATIENTS AND METHODS: Sixty patients with FL received ASCT in our institution (18 first-line with total body irradiation and cyclophosphamide, 34 second-line with BEAM and 8 ≥ third-line with BEAM). In the case of long-term remission (>6 years; N = 17), peripheral blood was tested for minimal residual disease by t(14;18)- and IGH-PCR. RESULTS: Ten-year overall survival, progression-free survival and freedom from progression (FFP) after first-line ASCT were 79%, 57% and 64% after second-line ASCT 41%, 35% and 42%, respectively. Prognostic factors for FFP were treatment line and FLIPI (Follicular Lymphoma International Prognostic Index). Ten-year FFP for second-line ASCT and low-risk FLIPI was 57%, intermediate risk 37% and high risk 33%. No relapses occurred after 6 years following ASCT. Sixteen patients developed sustained long-term clinical and molecular remissions of up to 17.5 years. CONCLUSION: Sustained long-term clinical and molecular remissions can be achieved following ASCT, including HDT with BEAM in second line.

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. MATERIAL AND METHODS: Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. RESULTS: Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. CONCLUSION: The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

Blood donor screening with cobas s 201/cobas TaqScreen MPX under routine conditions at German Red Cross institutes.

BACKGROUND: In 1997 the German Red Cross (GRC) blood donor services introduced mini-pool nucleic acid testing (NAT) for human immunodeficiency virus (HIV)-1, hepatitis C virus (HCV) and hepatitis B virus (HBV) to increase blood safety. With the new cobas s 201/cobas TaqScreen MPX, a fully automated extraction method and a multiplex amplification system specifically adapted to the needs of blood donation services is available. METHODS: The cobas s 201 system was evaluated at the GRC BTS locations Hagen, Springe and Frankfurt. In phase A, the analytical sensitivity for the detection of HBV, HCV and HIV-1 was investigated and in phase B, at least 60,000 samples at each test site were screened in parallel with the MPX test on s 201 system and the existing routine mini-pool NAT system to compare the diagnostic specificity and the diagnostic sensitivity. RESULTS: Comparable analytical sensitivities in a range of 1.6-3.6 IU/ml, 4.9-10.9 IU/ml and 14.7-26.6 IU/ml for HBV, HCV HIV, respectively, for the MPX test on s 201 system (95% probability based on probit analysis) were determined at all test sites. The diagnostic sensitivity was 99.8% and the diagnostic specificity was 99.85%. CONCLUSIONS: The MPX test on s 201 system is a fully automated NAT system suitable for routine blood donor screening. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements of the Paul Ehrlich Institute for blood donor screening in mini-pools up to 96 donations per pool. A major benefit of the automated NAT system is the reduced personnel time and the extensive complete barcode-controlled process documentation.

Inhibition of high voltage-activated calcium currents by L-glutamate receptor-mediated calcium influx.

The modulation of high voltage-activated (HVA) Ca2+ currents by L-glutamate and its agonists was investigated in cultured rat hypothalamic neurons. L-Glutamate and agonists selective for NMDA or non-NMDA receptors reversibly inhibited HVA Ca2+ currents. The putative presynaptic glutamate receptor agonist L-2-amino-4-phosphonobutyric acid and the selective metabotropic agonist trans-ACPD were ineffective. Inhibition was dependent on the presence of extracellular Ca2+ and blocked by internal perfusion of the cells with BAPTA. The calmodulin antagonists trifluoperazine and calmidazolium completely prevented the inhibition. Increases in the intracellular Ca2+ concentration due to Ca2+ influx through non-NMDA receptor channels were visualized using fura-2. These results indicate that not only NMDA but also non-NMDA receptor channels in these neurons are permeable for Ca2+ and that Ca2+ influx through these channels activates a calmodulin-dependent mechanism, which leads to HVA Ca2+ current inhibition.

Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons.

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.

Two dye two wavelength excitation calcium imaging: results from bovine adrenal chromaffin cells.

We tested a mixture of Calcium-Green-1 (CG-1) and Brilliantsulfaflavine (BS) for dual excitation ratiometric measurements of the intracellular free calcium concentration ([Ca2+]i) in bovine adrenal chromaffin cells. Dyes were coloaded (without being molecularly linked to each other) in the whole-cell configuration of the patch clamp technique. We compared the loading time-courses of CG-1 and BS, investigated their intracellular distribution patterns and studied the time course of photobleaching. We determined the apparent dissociation constant of CG-1, both optically and by potentiometric titration. Our findings indicate that: (i) with excitation at 420/488 nm, calibrated fluorescence signals could be derived using a Grynkiewicz-type equation; (ii) BS is an ideal reference dye that displayed no interaction with CG-1 or cellular constituents; and (iii) that calibration requires diffusional equilibration between pipette and the accessible volume of the cell. Spatially resolved recordings of fluorescence excitation spectra revealed elevated fluorescence of CG-1 in the nucleus such that reported [Ca2+]i levels seemed 25% higher compared to cytosolic values. Comparing fluorescence emission from in vitro dye solutions with in vivo values, we could estimate the accessible volume fraction and amount of Ca(2+)-insensitive dye.

Signaling by E-selectin and ICAM-1 induces endothelial tissue factor production via autocrine secretion of platelet-activating factor and tumor necrosis factor alpha.

Based on previous studies showed adhesion molecule-dependent induction of tissue factor upon endothelium-lymphocyte interactions, we investigated whether E-selectin and ICAM-1 are linked to signaling pathways leading to tissue factor gene expression. Cellular interaction was mimicked by antibody cross-linking of E-selectin and ICAM-1 on the surface of human umbilical vein endothelial cells (HUVECs), resulting in induction of tissue factor mRNA and protein expression. Tissue factor production could be independently abolished by antibodies against TNF-alpha and by WEB 2086, a platelet-activating factor (PAF) receptor antagonist. Because WEB 2086 prevented the production and/or secretion of TNF-alpha by HUVECs, these results provide evidence for E-selectin- and ICAM-1-linked signal pathways leading to tissue factor synthesis in endothelial cells via an autocrine feedback loop involving PAF and TNF-alpha secretion.

Inhibition of thrombus formation by low-dose acetylsalicylic acid, dipyridamole, and their combination in a model of platelet-vessel wall interaction.

Effects of low-dose acetylsalicylic acid (ASA, 50 mg/day), dipyridamole (sustained-release preparation 400 mg/day), and their combination were investigated in a model of human platelet-vessel wall interaction. In a randomized, double-blind clinical pharmacology trial in 96 healthy subjects, the inhibition of mural platelet thrombus was measured ex vivo using blood samples collected both before and 2 hours after a 3.5-day treatment with ASA, dipyridamole, ASA combined with dipyridamole, or placebo. Both the size and the number of platelet thrombi adherent to a thrombogenic matrix after a 15-minute flow experiment were identified by automated fluorescence microscopy. ASA treatment alone reduced the mean size of all thrombi by about 45%, and dipyridamole alone achieved an approximate 17% reduction in the mean size of all thrombi. The combination of both agents had an additive effect. Formation of the subpopulation of very large thrombi was reduced by ASA and dipyridamole to a similar extent, with their combination producing an effect at least twice as strong as that witnessed in a single treatment. These results suggest that ASA and dipyridamole affect platelet thrombus growth by different mechanisms of action. These findings provide the pharmacologic rationale for the combination of ASA (suppressing the synthesis of prothrombotic thromboxane A2) and dipyridamole (by feedback inhibition of platelet activation via local accumulation of adenosine) as a highly effective and safe combination for secondary prevention of stroke. They are consistent with the clinical findings of the Second European Stroke Prevention Study (ESPS-2). In this large trial, the addition of dipyridamole (400 mg/day in a sustained-release preparation) to aspirin (50 mg/day) doubled the efficacy of aspirin in the secondary prevention of stroke without increasing the risk for bleeding.

Guanidine derivatives as combined thromboxane A2 receptor antagonists and synthase inhibitors.

A new series of omega-disubstituted alkenoic acid derivatives derived from samixogrel 5 were designed and synthesized as combined thromboxane A2 receptor antagonists/thromboxane A2 synthase inhibitors with improved solubility and reduced protein binding compared to 5. Hexenoic acid derivatives with a 3-pyridyl group and 3-(2-cyano-3-alkyl-guanidino)phenyl substituent were found to be optimal with regard to this dual mode of action. The most potent compound, E-6-(3-(2-cyano-3-tert-butyl-guanidino)phenyl)-6-(3-pyridyl)hex-5-eno ic acid, "terbogrel" 32 inhibits the thromboxane A2 synthase in human gel-filtered platelets with an IC50 value of 4.0 +/- 0.5 nM (n = 4). Radioligand binding studies with 3H-SQ 29,548 revealed that 32 blocks the thromboxane A2/endoperoxide receptor on washed human platelets with an IC50 of 11 +/- 6 nM (n = 2) and with an IC50 of 38 +/- 1 nM (n = 15) in platelet-rich plasma. Terbogrel inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an IC50 of 310 +/- 18 nM (n = 8) and 52 +/- 20 nM (n = 6), respectively. This was shown to translate into a potent antithrombotic effect in vivo as demonstrated in studies using a model of arterial thrombosis in rabbits (ED50 = 0.19 +/- 0.07 mg/kg; n = 20). Thus, terbogrel is the first compound with a guanidino moiety demonstrating both a potent TXA2 synthase inhibition and a potent TXA2 receptor antagonism and has been selected for further clinical development.

6,6-Disubstituted Hex-5-enoic acid derivatives as combined thromboxane A2 receptor antagonists and synthetase inhibitors.

A series of omega-disubstituted alkenoic acid derivatives were designed and synthesized as antithrombotic inhibitors of thromboxane A2 synthetase and thromboxane A2 receptor antagonists. Hexenoic acid derivatives with a 3-pyridyl group and a 4-(2-benzenesulfonamidoethyl)phenyl substituent were found to be optimal with regard to the dual mode of action. The most potent compound, (E)-6-(4-(2-(((4-chlorophenyl)sulfonyl)amino)ethyl)phenyl)-6-(3-pyridyl) hex-5-enoic acid (36), inhibits thromboxane A2 synthetase in gel-filtered human platelets with an IC50 value of 4.5 +/- 0.5 nM (n = 4), whereas an inhibitory effect on cyclooxygenase is seen only at a much higher concentration (IC50: 240 microM). Radioligand-binding studies with [3H]SQ 29,548 in washed human platelets revealed that 36 blocks the prostaglandin H2/thromboxane A2 receptor with an IC50 of 19 +/- 5 nM (n = 5) and is therefore 85-fold more potent than another combined thromboxane A2 synthetase inhibitor/receptor antagonist, Ridogrel (4). Compound 36 inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an EC50 of 1 microM (Ridogrel: 16 microM) and 100 nM, respectively, and was selected for further development.

Thrombin stimulates inositol phosphate accumulation and prostacyclin synthesis in human endothelial cells from umbilical vein but not from omentum.

We have compared the effects of thrombin on the accumulation of inositol phosphates and the synthesis of prostacyclin in cultured human endothelial cells from umbilical vein and the microvasculature of omentum. Active human thrombin induced a dose-dependent accumulation of inositol phosphates and a concomitant synthesis of prostacyclin in endothelial cells from human umbilical vein. However, thrombin at all concentrations tested was unable to stimulate inositol phosphate accumulation and prostacyclin synthesis in microvascular endothelial cells from human omentum. Bradykinin was able to stimulate these effects in both types of cell. These results demonstrate that although inositol phosphate turnover is an initial event associated with prostacyclin synthesis in endothelial cells, there are differences in the way microvascular endothelial cells respond to thrombin.

The effects of heparin and annexin V on fibrin accretion after injury in the jugular veins of rabbits.

We compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely de-endothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p < 0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins.(ABSTRACT TRUNCATED AT 250 WORDS)

Different effects of aspirin, dipyridamole and UD-CG 115 on platelet activation in a model of vascular injury: studies with extracellular matrix covered with endothelial cells.

Cultured endothelial cells produce an extracellular matrix (ECM) which activates platelets, similarly to deendothelialized vascular segments. Platelet-rich plasma (PRP) was incubated with endothelial cells cultures seeded in various densities on ECM. The interaction of the platelets with this artificial intima was evaluated by phase microscopy and by thromboxane A2 (TXA2) and prostacyclin (PGI2) measurement. Large platelet aggregates were formed on exposed ECM. Platelets aggregation but not adhesion on the ECM was markedly inhibited by the presence of endothelial cells. Pretreatment of the endothelial cells with 0.1 mM aspirin reduced their PGI2 synthesis and was associated with platelet aggregation on the ECM. 10 microM dipyridamole markedly inhibited platelet activation by ECM when the drug was added to citrated whole blood before PRP preparation. UD-CG 115 which elevates cyclic AMP in cardiac muscle, inhibited platelet aggregation and TXA2 production induced by ECM, in the presence as well as in the absence of endothelial cells, without any effect on endothelial PGI2 production.

Lymphocyte adhesion to human endothelial cells induces tissue factor expression via a juxtacrine pathway.

To study the effect of lymphocyte adhesion on the procoagulant activity of endothelial cells, we have stimulated HUVECs with interferon-gamma to upregulate adhesion molecules. Subsequent addition of lymphocytes induced the expression of tissue factor (TF) by HUVECs. Both CD4+ and CD8+ T-cells promoted this TF synthesis via distinct adhesion molecules (CD4+ T-cells: E-selectin and ICAM-1; CD8+ T-cells: MHC-I molecules). In addition, tumor necrosis factor-alpha and -beta (TNF alpha, TNF beta) and platelet-activating factor (PAF) were involved in lymphocyte-mediated TF expression on HUVECs. We demonstrate that PAF plays a pivotal role in this process. Adhesion of lymphocytes to endothelial cell surface molecules induced the release of PAF. PAF, in turn, caused the production of TNF alpha and TNF beta, both of which are potent stimulators of TF expression.

Ultrastructural, biochemical, and cell-culture studies of a presumed extraskeletal Ewing's sarcoma with special reference to differential diagnosis from neuroblastoma.

The history of a 6-year-old girl with a tumor originating from thoracic spine and finally becoming resistant to surgery, radio-, and chemotherapy is reported. Tumor-biopsy material was studied by light and electron microscopy, in cell culture, by acetylcholinesterase ultracytochemistry, and by quantitative catecholamine analysis and this led to the rejection of the initial diagnosis of a neuroblastoma. Light microscopy revealed a uniform population of undifferentiated cells incompletely lobulated by broad fibrovascular septa. Using the electron microscope, cells were characterized by large intracellular pools of glycogen, little cytoplasm with an abundance of free ribosomes and a paucity of organelles. A few cells displayed desmosome-like attachment sites. Staining for specific and unspecific acetylcholinesterase was negative with light and electron microscopy, as were the results of catecholamine histofluorescence using the glyoxylic acid method. The latter result was confirmed by the negative outcome of quantitative analyses of dopamine, noradrenaline, and adrenaline with high pressure liquid chromatography nd electrochemical detection in tissue samples. Tumor cells could easily be maintained in culture for up to 4 weeks. None of a variety of treatments that are known to favor expression of neuronal characteristics in neuroblastoma cells (serum withdrawal, nerve growth factor, dbcAMP, dexamethasone) induced morphological differentiation in cultured tumor cells. On the basis of the clinical history, morphology, and of our experiments with tumor cells, the diagnosis of a so-called extraskeletal Ewing's sarcoma is most likely. Our results strengthen the view that a cell biology approach may be valuable in neuroblastoma differential diagnosis.

Pharmacodynamic models to evaluate antithrombotics in clinical pharmacology.

The need to prevent thromboembolic events effectively and safely has stimulated an intense search for novel antithrombotics. Parameters derived from in vitro tests with patients' blood are essential for therapeutic monitoring of anticoagulants. Clinical pharmacologic evaluation of novel antithrombotic therapies based on such parameters can easily fail, however, by neglecting pivotal pathophysiologic determinants of thrombus formation. When vascular injury occurs, blood cells, plasma proteins, and the vessel wall intimately cooperate for an adequate local repair. Much remains to be learned about the local and transient interaction of these components. In most tests of platelet function and coagulation proteins, blood samples from treated individuals are stimulated in vitro to assess inhibitory effects. Limitations of such a test strategy for dose-finding studies with antithrombotics may be overcome by measuring activation markers specifically generated on the surface of blood cells or in plasma at the site of thrombosis in patients.