Connexin 37 limits thrombus propensity by downregulating platelet reactivity.

Background- Formation of platelet plug initiates hemostasis after vascular injury and triggers thrombosis in ischemic disease. However, the mechanisms leading to the formation of a stable thrombus are poorly understood. Connexins comprise a family of proteins that form gap junctions enabling intercellular coordination of tissue activity, a process termed gap junctional intercellular communication. Methods and Results- In the present study, we show that megakaryocytes and platelets express connexin 37 (Cx37). Deletion of the Cx37 gene in mice shortens bleeding time and increases thrombus propensity. Aggregation is increased in murine Cx37(-/-) platelets or in murine Cx37(+/+) and human platelets treated with gap junction blockers. Intracellular microinjection of neurobiotin, a Cx37-permeant tracer, revealed gap junctional intercellular communication in platelet aggregates, which was impaired in Cx37(-/-) platelets and in human platelets exposed to gap junction blockers. Finally, healthy subjects homozygous for Cx37-1019C, a prognostic marker for atherosclerosis, display increased platelet responses compared with subjects carrying the Cx37-1019T allele. Expression of these polymorphic channels in communication-deficient cells revealed a decreased permeability of Cx37-1019C channels for neurobiotin. Conclusions- We propose that the establishment of gap junctional communication between Cx37-expressing platelets provides a mechanism to limit thrombus propensity. To our knowledge, these data provide the first evidence incriminating gap junctions in the pathogenesis of thrombosis.

Urokinase receptor (uPAR, CD87) is a platelet receptor important for kinetics and TNF-induced endothelial adhesion in mice.

BACKGROUND: Urokinase plasminogen activator receptor (uPAR, CD87) is a widely distributed 55-kD, glycoprotein I-anchored surface receptor. On binding of its ligand uPA, it is known to increase leukocyte adhesion and traffic. Using genetically deficient mice, we explored the role of uPAR in platelet kinetics and TNF-induced platelet consumption. METHODS AND RESULTS: Anti-uPAR antibody stained platelets from normal (+/+) but not from uPAR-/- mice, as seen by fluorescence-activated cell sorter analysis. 51Cr-labeled platelets from uPAR-/- donors survived longer than those from +/+ donors when injected into a +/+ recipient. Intratracheal TNF injection induced thrombocytopenia and a platelet pulmonary localization, pronounced in +/+ but absent in uPAR-/- mice. Aprotinin, a plasmin inhibitor, decreased TNF-induced thrombocytopenia. TNF injection markedly reduced the survival and increased the pulmonary localization of 51Cr-labeled platelets from +/+ but not from uPAR-/- donors, indicating that it is the platelet uPAR that is critical for their response to TNF. As seen by electron microscopy, TNF injection increased the number of platelets and polymorphonuclear neutrophils (PMNs) in the alveolar capillaries of +/+ mice, whereas in uPAR-/- mice, platelet trapping was insignificant and PMN trapping was slightly reduced. Platelets within alveolar capillaries of TNF-injected mice were activated, as judged from their shape, and this was evident in +/+ but not in uPAR-/- mice. CONCLUSIONS: These results demonstrate for the first time the critical role of platelet uPAR for kinetics as well as for activation and endothelium adhesion associated with inflammation.

Bi-ventricular axial micropump: impact on blood cell integrity.

BACKGROUND AND OBJECTIVE: Off-pump coronary artery bypass grafting has stimulated the development of micro-pumps designed to prevent the hemodynamic instability induced by heart luxation for the exposure of target vessels of the posterior wall. Impella (Aachen, Germany) developed micro-pumps with a miniaturized propeller system for both sides of the heart. The aim of this study was to analyze the impact of both pumps working together on blood cell integrity. MATERIALS AND METHODS: Both right and left-sided micro-pumps were implanted in 5 calves (body weight, 72_4 Kg) during 3 h. Blood samples for hematology and hemolysis parameters were drawn hourly. RESULTS: Both pumps performed well with a flow of 3.6 L +/- 0.3 L during the 3 h of the experiment with stable hemodynamic conditions. Mixed venous oxygen saturation was 63.4 +/- 15.2% at baseline and 63.8 +/- 16.3% at the end of the experiment (P = ns). Red cell count, LDH and free plasma hemoglobin were 6.7 +/- 2.1 x 10(12)/L, 1807 +/- 437 IU/L, and 32 +/- 9 mg/L at baseline vs. 6.1 +/- 2.1 x 10(12)/L, 1871 +/- 410 IU/L, and 52 +/- 9 mg/L at the end of the experiment (P = ns for all comparisons). Platelet count exhibited a non-significant drop (872 +/- 126 vs. 715 +/- 22 x 10(9)/L). CONCLUSIONS: This double pump system based on the Archimed screw principle is hematologically well tolerated under conditions of prolonged cardiac assist.

Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing : a prospective randomized clinical trial on skin graft donor sites

La cicatrisation cutanée requiert de nombreux mécanismes et un nombre toujours croissant de molécules participant à ces réactions sont identifiées. La compréhension de cette cinétique et des différents facteurs impliqués dans ce processus permet d'accélérer la guérison des plaies. Certains facteurs de croissance (PDGF, FGF, EGF) ont déjà été utilisés localement sur des plaies mais leurs effets relatés sont inconsistants et contradictoires, probablement en raison de l'absence de cinétique ou de concentration physiologique. Les plaquettes jouent un rôle fondamental dans la cicatrisation cutanée, principalement par la formation du clou plaquettaire et le relargage de divers facteurs de croissance et de cytokines. De même, les kératinocytes ont un rôle similaire dans l'épithélialisation des plaies. Ainsi, l'apport de plaquettes et de kératinocytes sur une plaie pourrait accélérer sa cicatrisation. L'étude rapportée ici tente de vérifier si une solution de kératinocytes autologues en suspension dans un concentré plaquettaire ou un concentré plaquettaire seul pourrait stimuler la cicatrisation cutanée. Pour ce faire, nous avons comparé trois groupes de 15 patients bénéficiant, sur une prise de greffe de profondeur similaire, d'un traitement standard fait de pansement simple, d'un concentré plaquettaire seul ou de kératinocytes autologues suspendus dans un concentré plaquettaire. Sur ces plaies, la durée de cicatrisation ainsi que la douleur au cours de la guérison ont été étudiés. Nos résultats montrent une réduction significative de la durée de cicatrisation dans le groupe traité avec un concentré plaquettaire, passant de 13.9 +/- 0.5 à 7.2 +/- 0.2 jours. Cet effet est encore plus marqué dans le groupe traité avec des kératinocytes en suspension dans un concentré plaquettaire passant de 13.9 +/- 0.5 à 5.7 +/- 0.2 jours. De même, la douleur évaluée au cinquième jour montre une nette diminution dans les deux groupes traités avec des cellules. En conclusion, notre travail montre que l'application de plaquettes autologues ou de kératinocytes en suspension dans un concentré plaquettaire permet d'accélérer la cicatrisation cutanée et de diminuer les douleurs, sans aucun effet indésirable constaté. Notre étude montre également qu'un concentré plaquettaire peut être utilisé comme vecteur pour une suspension de kératinocytes. L'identification de la cinétique d'apparition et de la concentration de chacun des facteurs de croissance lors de la cicatrisation cutanée permettrait dans le futur d'analyser plus finement et de traiter physiologiquement des plaies chroniques.

Controls on ostracod valve geochemistry, Part 1: Variations of environmental parameters in ostracod (micro-)habitats

The variations of environmental conditions (T°, pH, δ13CDIC, [DIC], δ18O, Mg/Ca, and Sr/Ca) of ostracod habitats were examined to determine the controls of environmental parameters on the chemical and isotopic composition of ostracod valves. Results of a one-year monitoring of environmental parameters at five sites, with depths of between 2 and 70 m, in Lake Geneva indicate that in littoral to sub-littoral zones (2, 5, and 13 m), the chemical composition of bottom water varies seasonally in concert with changes in temperature and photosynthetic activity. An increase of temperature and photosynthetic activity leads to an increase in δ13C values of DIC and to precipitation of authigenic calcite, which results in a concomitant increase of Mg/Ca and Sr/Ca ratios of water. In deeper sites (33 and 70 m), the composition of bottom water remains constant throughout the year and isotopic values and trace element contents are similar to those of deep water within the lake. The chemical composition of interstitial pore water also does not reflect seasonal variations but is controlled by calcite dissolution, aerobic respiration, anaerobic respiration with reduction of sulphate and/or nitrate, and methanogenesis that may occur in the sediment pores. Relative influence of each of these factors on the pore water geochemistry depends on sediment thickness and texture, oxygen content in bottom as well as pore water. Variations of chemical compositions of the ostracod valves of this study vary according to the specific ecology of the ostracod species analysed, that is its life-cycle and its (micro-)habitat. Littoral species have compositions that are related to the seasonal variations of temperature, δ13C values of DIC, and of Mg/Ca and Sr/Ca ratios of water. In contrast, the compositions of profundal species are largely controlled by variations of pore fluids along sediment depth profiles according to the specific depth preference of the species. The control on the geochemistry of sub-littoral species is a combination of controls for the littoral and profundal species as well as the specific ecology of the species.

Community introduction of practice parameters for autistic spectrum disorders: Advancing early recognition.

OBJECTIVES: Within a strong interdisciplinary framework, improvement in the quality of care for children with autistic spectrum disorders through a 2 year implementation program of Practice Parameters, aimed principally at improving early detection and intervention. METHOD: We developed Practice Parameters (PPs) for Pervasive Developmental Disorders and circulated the PPs to all child and adolescent psychiatrists practicing in the region. RESULTS: PP development and parallel information strategies resulted in a significant decrease of 1.5 years in the mean-age-at-diagnosis. However, further analysis indicated that improvement was only transient. CONCLUSION: Despite the encouraging improvement in mean-age-at-diagnosis 2 years after PP implementation, other indicators showed a failure to maintain the improvements. A systematic screening program would be the most reliable method to reinforce the PPs.

Suitability, success and sinks: how do predictions of nesting distributions relate to fitness parameters in high arctic waders?

AimAlthough habitat suitability maps derived from species distribution models (SDMs) are often assumed to highlight locations that can sustain healthy populations over time, the relationship between suitability scores and fitness parameters has rarely been tested thoroughly. LocationZackenberg Valley, north-east Greenland. MethodsUsing 14years of data (1997-2010) representing three wader species (dunlin Calidris alpina, sanderling Calidris alba and ruddy turnstone Arenaria interpres), we tested the relationships between modelled suitability and fitness parameters at nesting locations. ResultsAmong the three species examined, only the ruddy turnstone exhibited significant relationships between suitability and nest success, but over time rather than space. During years with extensive snow cover in the landscape, the nesting sites of ruddy turnstone occurred in different habitats than were typically used across years. Moreover, in years with extensive snow cover, the ruddy turnstone initiated nests later and suffered from higher egg predation rates. Main conclusionOur results suggest that SDMs derived from species occurrences that include years of low reproductive success may over-estimate the potential suitable habitat in the landscape. Whenever possible, variation in reproductive success should be considered when building models to inform species' response to environmental change. species' response to environmental change.

Developmental effects of basic fibroblast growth factor and platelet-derived growth factor on glial cells in a three-dimensional cell culture system.

In order to study peptide growth factor action in a three-dimensional cellular environment, aggregating cell cultures prepared from 15-day fetal rat telencephalon were grown in a chemically defined medium and treated during an early developmental stage with either bovine fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF homodimers AA and BB). A single dose (5-50 ng/ml) of either growth factor given to the cultures on day 3 greatly enhanced the developmental increase of the two glia-specific enzyme activities, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and glutamine synthetase (GS), whereas it had relatively little effect on total protein and DNA content. Distinct patterns of dose-dependency were found for CNP and GS stimulation. At low concentrations of bFGF (0.5-5 ng/ml) and at all PDGF concentrations applied, the oligodendroglial marker enzyme CNP was the most affected. A relatively small but significant mitogenic effect was observed after treatment with PDGF, particularly at higher concentrations or after repetitive stimulation. The two PDGF homodimers AA and BB were similar in their biological effects and potency. The present results show that under histotypic conditions both growth factors, bFGF and PDGF, promote the maturation rather than the proliferation of immature oligodendrocytes and astrocytes.

Coactivated platelet-derived growth factor receptor {alpha} and epidermal growth factor receptor are potential therapeutic targets in intimal sarcoma.

Intimal sarcoma (IS) is a rare, malignant, and aggressive tumor that shows a relentless course with a concomitant low survival rate and for which no effective treatment is available. In this study, 21 cases of large arterial blood vessel IS were analyzed by immunohistochemistry and fluorescence in situ hybridization and selectively by karyotyping, array comparative genomic hybridization, sequencing, phospho-kinase antibody arrays, and Western immunoblotting in search for novel diagnostic markers and potential molecular therapeutic targets. Ex vivo immunoassays were applied to test the sensitivity of IS primary tumor cells to the receptor tyrosine kinase (RTK) inhibitors imatinib and dasatinib. We showed that amplification of platelet-derived growth factor receptor α (PDGFRA) is a common finding in IS, which should be considered as a molecular hallmark of this entity. This amplification is consistently associated with PDGFRA activation. Furthermore, the tumors reveal persistent activation of the epidermal growth factor receptor (EGFR), concurrent to PDGFRA activation. Activated PDGFRA and EGFR frequently coexist with amplification and overexpression of the MDM2 oncogene. Ex vivo immunoassays on primary IS cells from one case showed the potency of dasatinib to inhibit PDGFRA and downstream signaling pathways. Our findings provide a rationale for investigating therapies that target PDGFRA, EGFR, or MDM2 in IS. Given the clonal heterogeneity of this tumor type and the potential cross-talk between the PDGFRA and EGFR signaling pathways, targeting multiple RTKs and aberrant downstream effectors might be required to improve the therapeutic outcome for patients with this disease.

Effects of smoking and physical exercise on platelet free cytosolic calcium in healthy normotensive volunteers.

Platelet free cytosolic calcium (PFCC) was measured in 21 healthy volunteers before and after cigarette smoking or physical exercise. The aim was to investigate whether acute blood pressure changes and increases in circulating levels of catecholamines and vasopressin modify PFCC. PFCC was determined using the Quin-2 method. Following cigarette smoking, significant increases in blood pressure, heart rate, plasma epinephrine (35 +/- 18 pg/ml before versus 51 +/- 31 pg/ml after smoking, P less than 0.05, mean +/- s.d.) and vasopressin levels (0.8 +/- 0.3 pg/ml before and 4.2 +/- 4.1 pg/ml after smoking, P less than 0.001) were observed. However, despite these acute hormonal and hemodynamic changes, PFCC remained stable at 156 +/- 55 nmol/l prior to the study and 157 +/- 29 nmol/l and 156 +/- 38 nmol/l at 20 and 80 min post-smoking, respectively. Acute physical exercise led to an increase in heart rate and systolic blood pressure but to a decrease in diastolic pressure. Moreover, a marked increase in plasma norepinephrine levels was observed after exercise (213 +/- 71 pg/ml before versus 747 +/- 501 pg/ml after exercise, P +/- 0.001). Again, PFCC was stable at 185 +/- 56 nmol/l at baseline versus 188 +/- 51 nmol/l at 20 min and 155 +/- 26 nmol/l at 80 min after exercise. These results therefore demonstrate that PFCC is not influenced acutely either by blood pressure increases, or by elevations in circulating catecholamine and vasopressin concentrations.

Assessment of liver tumor response by high-field (3T) MRI after radiofrequency ablation: Short- and mid-term evolution of diffusion parameters within the ablation zone.

PURPOSE: To compare the apparent diffusion coefficient (ADC) values of malignant liver lesions on diffusion-weighted MRI (DWI) before and after successful radiofrequency ablation (RF ablation). MATERIALS AND METHODS: Thirty-two patients with 43 malignant liver lesions (23/20: metastases/hepatocellular carcinomas (HCC)) underwent liver MRI (3.0T) before (<1month) and after RF ablation (at 1, 3 and 6months) using T2-, gadolinium-enhanced T1- and DWI-weighted MR sequences. Jointly, two radiologists prospectively measured ADCs for each lesion by means of two different regions of interest (ROIs), first including the whole lesion and secondly the area with the visibly most restricted diffusion (MRDA) on ADC map. Changes of ADCs were evaluated with ANOVA and Dunnett tests. RESULTS: Thirty-one patients were successfully treated, while one patient was excluded due to focal recurrence. In metastases (n=22), the ADC in the whole lesion and in MRDA showed an up-and-down evolution. In HCC (n=20), the evolution of ADC was more complex, but with significantly higher values (p=0.013) at 1 and 6months after RF ablation. CONCLUSION: The ADC values of malignant liver lesions successfully treated by RF ablation show a predictable evolution and may help radiologists to monitor tumor response after treatment.

Effect of weight reduction in moderately overweight patients on recorded ambulatory blood pressure and free cytosolic platelet calcium.

Although platelet cytosolic calcium has been shown to decrease during pharmacological treatment of hypertension, there is no evidence that cytosolic calcium also falls during a nonpharmacological reduction in blood pressure. To provide such evidence, we examined prospectively the relation between platelet cytosolic calcium and ambulatory blood pressure during weight reduction in moderately overweight (body mass index [BMI] greater than 25), mildly hypertensive individuals. The experimental group (responders: BMI reduction greater than 5%) consisted of 19 patients who lost 8.5 +/- 2.9 kg (mean +/- SD, p less than 0.05) during a 10-week hypocaloric diet, whereas the control group (nonresponders: BMI reduction less than 5%) consisted of 12 patients who showed no relevant change in body weight (-2.0 +/- 1.3 kg) during the same period of time. The moderate weight loss of the responders decreased blood pressure by 14/5 mm Hg (p less than 0.05), as measured by ambulatory monitoring, which renders a placebo effect unlikely. This nonpharmacological reduction in blood pressure was accompanied by a proportional 11% decrease (p less than 0.05) in platelet cytosolic calcium and also by significant (p less than 0.05) decreases in plasma catecholamines and serum cholesterol. These findings establish the concept of a nonpharmacological reduction in free cytosolic platelet calcium in humans and add further evidence suggesting a link between intracellular calcium homeostasis and blood pressure regulation.

Development of a cost-effective method for platelet-rich plasma (PRP) preparation for topical wound healing.

Platelet-rich plasma (PRP) is a volume of plasma fraction of autologous blood having platelet concentrations above baseline whole-blood values due to processing and concentration. PRP is used in various surgical fields to enhance soft-tissue and bone healing by delivering supra-physiological concentrations of autologous platelets at the site of tissue damage. These preparations may provide a good cellular source of various growth factors and cytokines, and modulate tissue response to injury. Common clinically available materials for blood preparations combined with a two-step centrifugation protocol at 280g each, to ensure cellular component integrity, provided platelet preparations which were concentrated 2-3 fold over total blood values. Costs were shown to be lower than those of other methods which require specific equipment and high-cost disposables, while safety and traceability can be increased. PRP can be used for the treatment of wounds of all types including burns and also of split-thickness skin graft donor sites, which are frequently used in burn management. The procedure can be standardized and is easy to adapt in clinical settings with minimal infrastructure, thus enabling large numbers of patients to benefit from a form of cellular therapy.