Intra-individual, randomised comparison of the MRI contrast agents gadobutrol versus gadoteridol in patients with primary and secondary brain tumours, evaluated in a blinded read.

OBJECTIVE: To prove that 1.0 M gadobutrol provides superior contrast enhancement and MRI image characteristics of primary and secondary brain tumours compared with 0.5 M gadoteridol, thereby providing superior diagnostic information. METHODS: Brain MRI was performed in two separate examinations in patients scheduled for neurosurgery. Independent injections of 1.0 M gadobutrol and 0.5 M gadoteridol at doses of 0.1 mmol Gd/kg body weight were administered per patient in randomised order. Evaluation was performed in an off-site blinded read. RESULTS: Fifty-one patients in the full analysis set (FAS) were eligible for efficacy analysis and 44 for the per-protocol analysis. For the primary efficacy variable "preference in contrast enhancement for one contrast agent or the other", the rate of "gadobutrol preferred" was estimated at 0.73 (95 % confidence interval 0.61; 0.83), showing significant superiority of gadobutrol over gadoteridol. Calculated lesion-to-brain contrast and the results of all qualitative secondary efficacy variables were also in favour of gadobutrol. Keeping a sufficient time delay after contrast application proved to be essential to get optimal image quality. CONCLUSION: Compared with 0.5 M gadoteridol, 1.0 M gadobutrol was proven to have significantly superior contrast enhancement characteristics in a routine MRI protocol of primary and secondary brain tumours.

High spatial resolution magnetic resonance imaging of experimental cerebral venous thrombosis with a blood pool contrast agent.

PURPOSE: The purpose of this study was to investigate the feasibility of clot visualization in small sinus and cortical veins with contrast enhanced MRA in a cerebral venous thrombosis animal model using a blood pool contrast agent, Gadofosveset, and high spatial resolution imaging. MATERIAL AND METHODS: For induction of cerebral venous thrombosis a recently developed combined interventional and microsurgical model was used. Cerebral sinus and cortical vein thrombosis was induced in six pigs. Two further pigs died during the procedure. Standard structural, time-of-flight- and phase contrast-angiograms were followed by fast time resolved high resolution 3D MRA (4D MRA) and subsequent high spatial resolution 3D MRA in the equilibrium phase with and without addition of parallel imaging. Visualization of the clots using the different sequences was subjectively compared and contrast-to-noise ratio (CNR) was assessed. RESULTS: In the remaining six animals the procedure and MR-imaging protocol including administration of Gadofosveset was successfully completed. The 3D high resolution MRA in the equilibrium phase without the addition of parallel imaging was superior to all the other applied MR measurement techniques in terms of visualization of the clots. Only applying this sequence bridging vein thromboses were also seen as a small filling defect with a high CNR of >18. CONCLUSION: Only the non-accelerated high spatial resolution 3D MRA in the equilibrium in conjunction with the blood pool agent Gadofosveset allows for high-contrast visualization of very small clots in the cerebral sinus and cortical veins. STATEMENT CLINICAL IMPACT: Detection of cortical vein thrombosis is of high clinical impact. Conventional MRI sequences often fail to visualize the clot. We could demonstrate that, in contrast to conventional sequences, with high spatial resolution 3D MRA in the equilibrium in conjunction with the blood pool agent Gadofosveset very small clots in the cerebral sinus and cortical veins could be successfully visualized. We think that with the presented approach cortical vein thrombosis might also be sufficiently visualized in patients.

New experimental model of sinus and cortical vein thrombosis in pigs for MR imaging studies.

INTRODUCTION: Diagnosis of cerebral sinus vein thrombosis is still a challenge for imaging. MRI and MRA play a major role in sinus imaging. For further development of MR techniques, MR-compatible animal models are required. The aim of this study was to develop an animal model for sinus thrombosis and additional cortical vein thrombosis with a clot of human blood for MR imaging studies. METHODS: A combined surgical and interventional approach was carried out in 13 pigs. After minimal invasive surgical access to the anterior superior sagittal sinus and cortical vein, thrombosis with human blood was induced using an interventional catheter approach. MR imaging was performed prior to and after thrombus induction. RESULTS: Sinus thrombosis was induced in 12 of 13 animals. Three animals suffered acute subdural haemorrhage; one of these animals died during the intervention, and one died after thrombus induction. MR imaging of the thrombosed sinus could easily be performed without significant artefacts in 11 of 13 animals. CONCLUSION: This new model of sinus and cortical vein thrombosis with a clot of human blood allows artefact-free imaging studies on MR.

Advanced neuronavigation in skull base tumors and vascular lesions.

OBJECTIVE: The purpose of this study was to describe the usefulness of recent advances of neuronavigational technology in the management of skull base tumors and of vascular lesions, treated via a skull base approach. METHODS: In 16 patients (skull base meningioma n = 9, petrous apex epidermoid n = l, craniopharyngeoma n = 1, giant internal carotid artery aneurysm n = 1, basilar/vertebral artery aneurysm n = 2, brain stem cavernoma n = 2), "advanced" neuronavigation was used. In contrast to "conventional" neuronavigation, the information for the neurosurgeon was enhanced by the intraoperative screen display of 3-dimensional reconstructions of the lesion, vessels, nerves and fiber tracts at risk. The 3-dimensional reconstructions were obtained by preoperative manual or automated segmentation processes. In addition, different imaging modalities (computed tomography [CT] with magnetic resonance imaging [MRI], CT with CT angiography, T (l)- with diffusion-weighted MRI) were fused and shown on the screen. RESULTS: In the cases of tumors, "advanced" neuronavigation facilitated the approach (n = 4), contributed to tailor the approach (n = 2) and helped to identify hidden neurovascular structures (n = 9). In the cases of aneurysms, "advanced" neuronavigation allowed us to reduce the skull base approach to the needs of safe aneurysm clipping (n = 3). In both cases of brain stem cavernoma, "advanced" neuronavigation was deemed useful for definition of the best surgical approach in relation to the pyramidal tract and brain stem nuclei. CONCLUSION: The authors' experiences suggest that neuronavigation, which displays 3-dimensional reconstructions of lesion, vessels, nerves and fiber tracts during surgery and makes use of image fusion techniques, is an important tool in the neurosurgical management of skull base lesions.

[Diffusion Weighted Imaging Combined with Intraoperative 3D-Ultrasound and fMRI for the Resection of an Optic Radiation Cavernoma]. / Diffusionsgewichtete MRT kombiniert mit navigiertem 3D-Ultraschall und fMRT zur Entfernung eines Kavernoms der Sehstrahlung.

A 17-year-old patient with a symptomatic cavernoma of the optic radiation underwent surgery supported by functional magnetic resonance imaging (fMRI), diffusion weighted magnetic resonance imaging (DWI) and navigated 3D-ultrasound. The primary visual cortex was visualized with fMRI. The optic radiation was delineated by means of DWI. The diffusion weigthed images were used for 3-dimensional reconstruction of the optic radiation. During surgery, the information of the localisation of functional brain regions were used together with the 3D-ultrasound, enabling the surgeon to remove the cavernoma without morbidity. This is the first report of the combined use of fMRI, fiber tract imaging and 3D-ultrasound for the safe resection of an optic radiation lesion.

Modulation of the regioselectivity of a Bacillus alpha-galactosidase by directed evolution.

Alpha-galactosidase AgaB of Bacillus stearothermophilus was subjected to directed evolution in an effort to modify its regioselectivity. The wild-type enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used random mutagenesis and staggered extension process (StEP) to obtain mutant enzymes displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselectivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of their activity towards the 1,6 linkage both in hydrolysis and in synthesis, one (E901) has retained its 1,3 activity. However the transglycosylation level reached by this mutant is quite low versus that of the native enzyme. This work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.

Inhibition of leukocyte chemiluminescence by platelets: role of platelet-bound fibrinogen.

Tethering of PMNL by platelets via CD62P has been shown to cause PMNL activation. Co-incubation of purified PMNL with platelets that were activated with thrombin and then fixed and washed, resulted in the formation of platelet-PMNL conjugates as well as in a generation of reactive oxygen species that were measured as luminol-enhanced chemiluminescence. When platelets were thrombin activated in the presence of RGDS to prevent binding of fibrinogen to membrane receptors, they had a reduced capacity to adhere to PMNL, but ROS generation was enhanced. In samples of citrated whole blood RGDS as well as the more specific platelet fibrinogen receptor antagonist GR144053F or a dissociation of the platelet glycoprotein IIb/IIIa complex markedly enhanced ROS generation that was induced by stirring the samples for 10 min at 1000 rpm, by 175%, 95% and 138%, respectively. Removal of platelets from the whole blood samples also resulted in an enhancement of stirring-induced ROS generation, which was inversely correlated to the platelet count. These data provide some evidence that platelets are capable of inhibiting ROS generation in PMNL by a mechanism that involves platelet-bound fibrinogen and probably depends on fibrinogen-mediated platelet-PMNL contact.

Identification by saturation mutagenesis of a single residue involved in the alpha-galactosidase AgaB regioselectivity.

The alpha-galactosidase AgaB of Bacillus stearothermophilus displays a major 1,6 and a minor 1,3 regioselectivity. The wild-type enzyme was subjected to directed evolution (random mutagenesis and in vitro recombination) using a double screening strategy based on the elimination of the 1,6 regioselectivity and the analysis by TLC of the transglycosylation products. One of the AgaB mutants (E500) exhibited a new 1,2 regioselectivity and a rather high level of transglycosylation. The corresponding gene contains 10 mutations compared to the agaB gene and we demonstrated by saturation mutagenesis that the G442R substitution strongly contributes to the emergence of this new regioselectivity. Moreover, other single point mutations at this position led to new mutants displaying other kinds of regioselectivity demonstrating the importance of this position in the subtle kinetic control of transglycosylation.

Comparative study of new alpha-galactosidases in transglycosylation reactions.

We have studied the potential of several newly cloned alpha-galactosidases to catalyze the regioselective synthesis of disaccharides using 4-nitrophenylgalactoside as a donor. The kinetics of the reactions were followed by in situ NMR spectroscopy. The following thermophilic enzymes have been tested: Aga A and an isoenzyme Aga B obtained from the strain KVE39 and Aga 285 from the strain IT285 of Bacillus stearothermophilus; Aga T is an alpha-galactosidase from Thermus brockianus (strain IT360). Two other non-thermophilic alpha-galactosidases have also been evaluated: Aga 1 (Streptococcus mutans, strain Ingbritt) and Raf A (Escherichia coli, strain D1021). For all of the enzymes studied, high regioselectivity was observed leading to two (1 --> 6)-disaccharides: 4-nitrophenyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside and methyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside, which were obtained in 54% (Aga B) and 20% (Aga T) yields, respectively.

Effects of cytochalasin H, a potent inhibitor of cytoskeletal reorganisation, on platelet function.

Platelets contain a well-developed and dynamic cytoskeleton composed mainly of actin and actin-associated proteins. Upon platelet activation there is rapid polymerisation of actin and a marked reorganisation of the platelet cytoskeleton. Cytochalasins are agents that interfere with the polymerisation of actin, and it has recently been discovered that cytochalasin H (CyH) is particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (ADP). Here we have used CyH to inhibit platelet cytoskeletal reorganisation and to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or whole blood obtained from human volunteers. PRP was treated with 10 microM CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorganisation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeletons and quantitated by densitometry. Platelet aggregation and aggregate stability in PRP were measured by monitoring changes in light absorbance; aggregation was measured in whole blood via platelet counting. Shape change, P-selectin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, alpha-actinin and actin-binding protein into the cytoskeleton that occurred following ADP activation, and incorporation of myosin was markedly reduced. Aggregation was only partially inhibited but, more dramatically, the rate of disaggregation following addition of certain agents that interfere with fibrinogen binding to glycoprotein IIb/IIIa on the surface of platelets was markedly increased. The ADP-induced shape change was also inhibited. CyH had no effect on calcium mobilisation. Curiously, expression of P-selectin was potentiated by CyH, suggesting a modulatory role of the cytoskeleton in platelet secretory activity. The results suggest that cytoskeletal reorganisation plays an important role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.

Asthma and chemical bronchitis in vanadium plant workers.

BACKGROUND: Whether vanadium induces bronchial hyperresponsiveness and asthma in previously normal subjects is unresolved: the two reported series addressing this question both have shortcomings. OBJECTIVE: To determine the cause of cough and breathlessness in vanadium plant workers after variable periods of exposure. DESIGN: Case series of employees presenting with persistent symptoms over a 24-month study period. PATIENTS AND METHODS: Forty of an estimated 1,440 patients were investigated by 1) blood count and serum IgE, 2) intracutaneous allergen skin tests, 3) spirometry, and 4) bronchoprovocation by histamine inhalation or exercise challenge. Exposure was assessed by measurement of 1) ambient V2O5, NH3 and SO2 over 7 days during the 24-month study period, 2) urine vanadium concentration at time of first presentation. RESULTS: Twelve of 40 subjects had bronchial hyperreactivity (BHR), and these were compared to 12 age-matched companion subjects whose BHR was normal. In 10, BHR was diagnosed by histamine inhalation (PC20 0.25-1.82 mg/ml, nl > 8.0 mg/ml), and in six of these the abnormality was severe (PC20 < 0.5 mg/ml). A further two had BHR by exercise challenge (FEV1, 600 ml/30% and 770 ml/18% pre/post exercise). After removal from exposure, 9 of the 12 subjects returned for follow-up 5 to 23 months later. BHR was worse in one, still present although less severe in five, and was no longer found in one subject. Baseline spirometry measurements were normal in seven subjects and only mildly impaired in the remaining five of the 12 subjects with BHR. CONCLUSION: This study provides strong supporting evidence that inhaled V2O5 induces BHR and asthma in subjects previously free of lung disease; the abnormality may persist for up to 23 months following exposure; routine spirometry will not detect affected subjects.

Procoagulant activity in patients with isolated severe head trauma.

OBJECTIVE: To determine the degree of regional and systemic coagulation activation soon after isolated severe head injury. DESIGN: Prospective, controlled clinical study. SETTING: The emergency room and intensive care unit (ICU) of a trauma center in a university hospital serving a population of 5 million people. PATIENTS: Twenty-four trauma victims: 20 patients with isolated severe head injury (head trauma group, Glasgow Coma Score of < or =8) and four patients with isolated bone fracture (control group). INTERVENTIONS: Insertion of central venous, i.e. superior caval vein, jugular bulb, and arterial catheters for blood sampling. MEASUREMENTS AND MAIN RESULTS: Central venous (i.e., superior caval vein) global coagulation variables (i.e., prothrombin time, activated partial thromboplastin time, fibrinogen concentration, antithrombin III activity, and platelet count) and central venous and jugular bulb activation coagulation and fibrinolysis variables (i.e., prothrombin fragment F1+2, thrombin-antithrombin III complex, soluble fibrin, and D-dimer concentration) were measured soon after trauma (<6 hrs) and 3 hrs later. When compared with the control group patients, upon ICU admission, fibrinogen concentration (p < .005) and platelet count (p < .025) were significantly decreased in the head trauma group. Thrombin-antithrombin III complex (p < .025), prothrombin fragment F1+2 (p < .025), and D-dimer (p < .005) concentrations measured at the time of ICU admission were significantly higher in the head trauma group than in the control group. Only in the head trauma group were soluble fibrin concentrations increased above the normal range; thrombin-antithrombin III complex and the prothrombin fragment F1+2 were found to be significantly higher in cerebrovenous blood than in central venous blood (p < .025). There was no cerebrocentral venous difference in the concentrations of soluble fibrin or D-dimer in either group. CONCLUSIONS: Within 6 hrs after severe isolated head trauma, systemic procoagulant overflow from the traumatized cerebral microvasculature proceeds to the thrombin level and is then inhibited by antithrombin III. Regional and systemic hypercoagulability and increased D-dimer concentrations appear to be common among head trauma patients. Increased procoagulant and consecutive fibrinolytic turnover may, therefore, spark disseminated intravascular coagulation in this patient group.

Activation of leukocytes in whole blood samples by N-formyl-methionyl-leucyl-phenylalanine (FMLP) enhances platelet aggregability but not platelet P-selectin exposure and adhesion to leukocytes.

Adhesion of platelets to neutrophils and monocytes is believed to play an important role in intercellular communication. Evidence has been provided that such heterotypic cell-cell contacts via adhesion molecules may be directly involved in intercellular signal transduction as well as facilitate the action of soluble signal transmitters, e.g. cathepsin G, PAF or nitric oxide. With respect to platelet activation, stimulatory and inhibitory effects of leukocytes have been reported, and the results obtained seem to be influenced by the experimental conditions. We investigated the effect of leukocyte stimulation on platelet behaviour in samples of human citrated whole blood. Adding the chemotactic peptide FM LP, which stimulates neutrophils and monocytes but not lymphocytes and platelets, to stirred whole blood samples resulted in a significant enhancement ( P < 0.01) of spontaneous as well as ADP-induced platelet aggregation (25 vs 33% and 66 vs 69% , respectively). In contrast stirring-induced as well as ADP-induced increase of P-selectin exposure (33 and 107% , respectively) was not affected by FMLP. In unstirred whole blood samples, about 10 to 20% of neutrophils and monocytes had bound platelets to their surfaces, and the number of these heterotypic conjugates was enhanced about twofold during spontaneous platelet aggregation. Addition of FMLP significantly reduced the stirring-induced formation of platelet-neutrophil conjugates but not of platelet-monocyte conjugates. These results indicate that neutrophil and/or monocyte activation in whole blood may enhance platelet aggregation, but not secretion (CD62P exposure) and formation of heterotypic platelet-leukocyte conjugates.

GPIIb-IIIa antagonists cause rapid disaggregation of platelets pre-treated with cytochalasin D. Evidence that the stability of platelet aggregates depends on normal cytoskeletal assembly.

Platelet activation is accompanied by changes in the composition of the platelet cytoskeleton with rapid incorporation and displacement of certain proteins. Here we have inhibited cytoskeletal assembly by pretreating platelets with cytochalasin D (CyD) and investigated the effect on the stability of the aggregates that form. The experiments were performed in both citrated and hirudinized platelet-rich plasma (PRP) and aggregation was induced by adenosine diphosphate (ADP), collagen, the TXA2-mimetic U46619 and adrenaline. Platelets in the aggregates that formed, underwent rapid disaggregation on addition of EDTA or a GpIIb-IIIa antagonist such as MK-852 and GR144053F, all of which are agents that interfere with the ability of fibrinogen to interact with GpIIb-IIIa. This was the case irrespective of the aggregating agent used and occurred in both citrated and hirudinized PRP. In contrast, the rate of disaggregation brought about by some other agents, iloprost and ARL 66096, appeared to be unaffected by CyD. Information was also obtained on the effects of CyD on the cytoskeletal changes brought about by ADP and the effects on the cytoskeleton of subsequent addition of M K-852. The results show that CyD retards the incorporation of certain proteins (actin, myosin, alpha -actinin, actin binding protein and a 66 K protein) into the cytoskeleton and that subsequent addition of MK-852 results in rapid displacement of some of these with re-incorporation of a 31 K protein. The results suggest that the early changes in the cytoskeleton following platelet activation contribute to the stability of the aggregates that form, and that interference with these early changes results in aggregates that are easily disassembled by agents that interfere with GpIIb-IIIa-fibrinogen complex formation.

Role of GPIIb-IIIa in platelet-monocyte and platelet-neutrophil conjugate formation in whole blood.

Platelets in stirred whole blood can be induced to form aggregates and also to form heterotypic platelet-monocyte (P/M) and platelet-neutrophil (P/N) conjugates. Here we have investigated the effects of three GPIIb-IIIa antagonists (GR144053F, MK-852 and Reopro, a CD62P-blocking antibody, GA6, and EDTA on the conjugate formation that occurs on stirring whole blood and in response to adding ADP and PAF. We have confirmed the identities of the conjugates by light microscopy after cell sorting. Platelet aggregation was measured by platelet counting. Monocytes, neutrophils, P/M and P/N were detected and quantitated using immunofluorescence and flow cytometry. Stirring whole blood resulted in both platelet aggregation and formation of P/M but not P/N. Adding ADP or PAF to whole blood caused rapid platelet aggregation and generation of both P/M and P/N. All of the GPIIb-IIIa antagonists studied had similar effects: inhibition of stirring-induced platelet aggregation and P/M formation, and inhibition of ADP-induced platelet aggregation and P/N formation. In contrast, they accelerated ADP induced-P/M conjugate formation and PAF-induced formation of both P/M and P/N. Both EDTA and GA6 completely inhibited P/M and P/N, which is commensurate with CD62P being involved in platelet-leucocyte conjugate formation. The results of these investigations suggest that GPIIb-IIIa has a dual role in determining the interaction between platelets and leukocytes.

Measurement of platelet activation and adhesion to leukocytes during haemodialysis.

In this study we used fluorescent-labelled antibodies to measure the extent of platelet adhesion to polymorphonuclear leukocytes (PMNLs), monocytes and lymphocytes. The activation of platelets, PMNLs and monocytes were also measured during the course of haemodialysis treatment using flow cytometric techniques established in our laboratory. Twenty patients were treated with either a cellulose membrane (TFU) or a polycarbonate filter (Pro 500). Blood samples were taken from the output line of the dialyser 2, 15, 30 and 180 min after commencing dialysis and just before starting treatment. Compared with the pre-dialysis sample, there was a marked increase in platelet-PMNL conjugate formation at 2 min, followed by a decrease in conjugates at 15 and 30 min, and a slight increase at 180 min. During extracorporeal circulation PMNLs become activated as measured by a CD11b upregulation at 15, 30 and 180 min, but not at 2 min. Platelet binding to monocytes was increased above 15 min after starting dialysis, and monocyte activation was slightly increased above basal levels during the same period. The activation state of circulating free platelets, as measured by surface P-selectin exposition, initially decreased slightly, but then returned to basal levels over the 3-h period. Changes in cell counts were also detected: there was a massive decrease in circulating PMNLs and monocytes, and a small decrease in circulating platelets, at 15 and 30 min. These reverted to basal values by the end of the 3-h period. There was no change in the number of circulating lymphocytes or erythrocytes. These results show that flow cytometric studies on whole blood samples may provide important information on the behaviour of circulating blood cells, which could supplement conventional clinical measurements, to give a better insight into changes that occur in the circulation during haemodialysis.

[Post-traumatic cerebrospinal rhinorrhea]. / Posttraumatische Rhinoliquorrhoe.

PURPOSE: Since frontobasal fractures after severe head trauma may cause serious late-term complications such as meningitis, their recognition and operative treatment are essential. Therefore, we analysed the importance of rhinoliquorrhoea as an early symptom of such fractures by comparison with neuroradiological methods. MATERIAL AND METHODS: In all patients undergoing operative revision of frontobasal fractures during a 7-year period, the clinical symptoms, results of neuroradiological examinations, concomitant injuries, as well as operative results, were studied retrospectively. RESULTS: 45 patients out of 688 with severe head injury showed frontobasal fractures (6.5%). The most common cause of these injuries were traffic accidents (55%) and precipitated falls or plunges (35%). Posttraumatic rhinoliquorrhoea was seen in 41 of 45 patients (91%), and 30 showed external periorbital injuries (66%). In 8 patients (18%) the frontobasal fractures could not be visualised by facultatively performed neuroradiological methods (coronary CT-scan, CT-cisternography, subarachnoid space scintigraphy). 15 patients (33%) were secondarily transferred to our institution, two of them more than two years after the causative injury. All patients were operated on successfully within 14 days after admission. CONCLUSION: Traffic accidents and precipitated falls or plunges are the main causes of head injuries with frontobasal fractures and about 2/3 of these patients show external periorbital injuries. Rhinoliquorrhoea as an early symptom allows diagnosis of frontobasal fractures in 90% of all cases rather than neuroradiological methods which failed to demonstrate frontobasal fractures in about 20% of our patients. Therefore, recognition of rhinoliquorrhoea in patients with severe head injury is essential.

Formation of platelet-leukocyte conjugates in whole blood.

The purpose of this investigation was to obtain information on platelet-leukocyte conjugate formation in whole blood and on factors that affect it. We also measured platelet and leukocyte activation by quantitating the expression of CD62P and CD11b. In both cases a flow cytometric approach was used. The results show that platelet-monocyte and platelet-polymorphonuclear leukocyte (PMNL) conjugate formation is enhanced by simply stirring blood, with optimum conjugate formation occurring after 10 min. In the case of monocytes,conjugate formation was enhanced by adenosine diphosphate (ADP). Both monocyte and PMNL conjugate formation was enhanced by phorbol myristate acetate (PMA), but L-formyl methionyl lysyl proline (FMLP) was either without effect (monocytes) or inhibitory (PMNL). EDTA also inhibited conjugate formation (implying involvement of divalent cations), as did dextran sulphate (implying involvement of P-selectin = CD62P). Interestingly GR144053F, which acts at GpIIb-IIIa on platelets to interfere with fibrinogen binding, and also glycyl prolyl arginyl proline (GPRP), a peptide that interferes with the interaction between CD11c on leukocytes and fibrinogen, did not inhibit platelet-monocyte conjugate formation, but did inhibit the platelet-PMNL interaction; this indicates that GpIIb-IIIa on platelets and CD11c on leukocytes and fibrinogen are involved in mediating the interaction between platelets and PMNL but not platelets and monocytes. Surprisingly arginyl-glycyl aspartyl serine (RGDS) inhibited the formation of both types of conjugate but this may be because it also inhibited both platelet and leukocyte activation as measured by CD62P and CD11b exposure and/or interferes with the binding of adhesion molecules other than fibrinogen. The results show that a flow cytometric procedure can be effective in obtaining rapid information on platelet-leukocyte conjugate formation in whole blood and on factors that are involved in its regulation. It is suggested that the technique may be applicable to the study of platelet-leukocyte conjugate formation in whole blood in disease, and also to study the effects of drugs interfering with conjugate formation.

Contact-induced modulation of neutrophil elastase secretion and phagocytic activity by platelets.

It has been reported that platelets stimulate generation of reactive oxygen species in neutrophils and monocytes by a mechanism that requires mutual cell-cell contact and the presence of P-selectin on the platelet surface. In the present study we investigated the effect of platelet-neutrophil contacts on neutrophil elastase secretion and phagocytic activity. Non-activated or thrombin-activated platelets were fixed with formaldehyde, washed and incubated with neutrophils in the absence or presence of various neutrophil agonists. Elastase secretion was determined by measuring the enzyme activity in cell-free supernatants using a chromogenic substrate. Platelet-neutrophil adhesion and ingestion of zymosan particles by neutrophils were quantitated by light microscopy. Platelets significantly reduced elastase secretion from neutrophils but had no effect on the elastase activity in the supernatant of neutrophil lysates. When neutrophils were stimulated with the ionophore A23187 or the chemotactic peptide FMLP, thrombin-activated platelets were more potent to inhibit elastase secretion when compared with non-activated platelets. Neutrophils that were not able to bind platelets to their surface had a significantly lower phagocytic activity when compared with neutrophil with adherent platelets or neutrophils that were incubated in the absence of platelets. The results indicate that platelet-neutrophil contacts may also lead to an inhibition of neutrophil functions and that such inhibition could be due to a transient contact rather than due to a firm platelet-neutrophil adhesion.