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As cobots become more popular in manufacturing, the close cooperation between the robot and the human operator becomes more complex. This paper proposes a method for optimizing the robot's work pace by interactive bacterial evolutionary algorithm. One of the main contributions is the interactivity of the optimization via the operator's feedback. Beside the operator's subjective evaluation, heart rate signals are also measured. The paper focuses mainly on the concept of this method tested with the help of some volunteers. Preliminary tests show that the algorithm is able to find comfortable configurations with less then 1% of them evaluated.

This paper proposes a novel supervised learning algorithm for spiking neural networks. The algorithm combines Hebbian learning and least mean squares method and it works well for small training datasets and short training cycles. The proposed method is applied in human-robot interaction for recognizing musical hand gestures based on the work of Zoltan Kodaly. The MNIST dataset is also used as a benchmark test to´ verify the proposed algorithm’s capability to outperform shallow ANN architectures. Experiments with the robot also provided promising results by recognizing the human hand signs correctly.

The number of gears in a transmission system has increased significantly in the last decades, in order to always keep the engine in the optimal operating point, whether the vehicle is accelerating, decelerating or traveling with constant velocity. Several transmission control algorithms have been developed for road vehicles equipped with Automated Manual Transmission (AMT) or Automatic Transmission (AT) in order to find out and serve the drivers will and choose gear according to the actual driving status. The aim of this work is to investigate different gear shifting strategies, and to compare them in various aspects in simulation environment and tests including a real vehicle. When selecting the appropriate gear in the transmission, a number of parameters can be taken into consideration, e.g. actual engine speed, actual engine torque, fuel consumption, acceleration pedal position and the speed of the position change (position derivative), etc. To consider all of these parameters four gear selection algorithms have been implemented and tested.

BACKGROUND: The in vitro quality of small-volume platelet (PLT) aliquots for pediatric transfusions was assessed to determine the best practice approach. STUDY DESIGN AND METHODS: Small volumes (50 mL) of single apheresis PLT components (APCs), collected on either CaridianBCT Trima or Haemonetics MCS+ instruments, were aliquoted on Days 2, 3, 4, and 5 postcollection into Fenwal PL1240 or 4R2014 bags or 60-mL polypropylene syringes. Samples were tested for in vitro quality at their recommended expiry times (4 hr for 4R2014 bags and syringes or Day 5 for PL1240 bags). Assays included pH, CD62P expression, and metabolic measures. RESULTS: CD62P expression increased throughout storage in all containers. Among the small-volume containers, pH, pCO2, lactate, and bicarbonate varied considerably. Regardless of the day of aliquoting, pCO2 was significantly higher and pO2 was significantly lower in gas-impermeable syringes than other containers. No bacterial growth was detected in any sample. CONCLUSION: The quality of APCs aliquoted into small-volume containers meets regulatory requirements and is generally equivalent to that of full-volume APCs at expiry.

BACKGROUND: A quality monitoring program (QMP) for platelet concentrates (PCs) was implemented at Canadian Blood Services (CBS) to improve standards and to better understand platelet (PLT) products by supplementing routine quality control (QC). STUDY DESIGN AND METHODS: Annual surveys of PCs from CBS production sites were conducted, with four completed to date (QMP Cycles 1-4) spanning two different PC production methods: PLT-rich plasma (PRP) and buffy coat (BC). Randomly selected PCs were sent to a central laboratory and tested 1 day after expiry. An expanded panel of tests including CD62P expression by flow cytometry, mean PLT volume, PLT count and morphology, extent of shape change, and PLT metabolic parameters, were applied. RESULTS: QMP data on the implementation of the BC production method across CBS indicated that BC PCs have less variable in vitro quality measures than PRP PCs. For the QC parameters pH and PLT count per unit, the range of mean values from each site for QMP 3 and 4 fell well within the range defined by regulatory standards, a first step in defining quality benchmarks for PCs. Of the extended panel of quality parameters, CD62P expression was the most sensitive indicator of change and identified an issue with the implementation of the BC PC production method at one site, which was subsequently remedied. CONCLUSION: A QMP was found to be useful to monitor production processes across sites and highlights best practice approaches while deepening understanding of the quality of PLT products at CBS.

BACKGROUND: Pathogen reduction technologies (PRTs) were developed to improve the safety of platelet concentrates (PCs) for transfusion purposes; however, several studies report a negative impact on the in vitro and in vivo platelet (PLT) quality. Therefore, analyses of the underlying molecular processes triggered by PRT treatments are necessary to understand their effects on PLT function. STUDY DESIGN AND METHODS: In two separate two-arm studies PCs prepared in plasma for storage either by the leukoreduced buffy coat (BC-PCs) or by the leukoreduced apheresis (AP-PCs) method were treated with or without riboflavin and ultraviolet (UV) light (Mirasol; 6.24 J/mL; 265-375 nm). Samples were drawn after treatment and after 1, 4, and 6 days of storage with subsequent analyses performed using in vitro measurements for PLT quality monitoring. Semiquantitative proteomic studies identified proteins that changed in band intensities in response to treatment or storage. Protein validation and subsequent biochemical studies were carried out by immunoblot analyses. RESULTS: The proteomic results identified changes mainly of proteins associated with the structure and regulation of the cytoskeleton. Focusing on the vasodilator-stimulated phosphoprotein (VASP) in AP-PCs revealed a storage-dependent, but treatment-independent, delocalization and a strong treatment-dependent phosphorylation at Ser-239 that was also present, but to a much lesser degree in BC-PCs. This modification correlated exponentially with PLT activation as determined by P-selectin expression. CONCLUSION: Treatment of PCs with Mirasol leads to the amplification of VASP Ser-239 phosphorylation, which is linked to actin dynamics and regulation of integrin αIIbβ3 activation. This change offers one explanation for Mirasol's impact on PLT in vitro quality measures. The Ser-239 phosphorylation level of VASP might be a useful protein marker for riboflavin and UV light–mediated PLT compromise.

BACKGROUND: Platelet additive solutions (PASs) are an alternative to plasma for the storage of platelet concentrates (PCs). However, little is known about the effect of PAS on the growth dynamics of contaminant bacteria. Conversely, there have been no studies on the influence of bacteria on platelet (PLT) quality indicators when suspended in PAS. STUDY DESIGN AND METHODS: Eight buffy coats were pooled, split, and processed into PCs suspended in either plasma or PAS (SSP+, MacoPharma). PCs were inoculated with 10 and 100 colony-forming units (CFUs)/bag of either Serratia liquefaciens or Staphylococcus epidermidis. Bacterial growth was measured over 5 days by colony counts and bacterial biofilm formation was assayed by scanning electron microscopy and crystal violet staining. Concurrently, PLT markers were measured by an assay panel and flow cytometry. RESULTS:S. liquefaciens exhibited an apparent slower doubling time in plasma-suspended PCs (plasma-PCs). Biofilm formation by S. liquefaciens and S. epidermidis was significantly greater in PCs stored in plasma than in PAS. Although S. liquefaciens altered several PLT quality markers by Days 3 to 4 postinoculation in both PAS- and plasma-PCs, S. epidermidis contamination did not produce measurable PLT changes. CONCLUSIONS:S. liquefaciens can be detected more quickly in PAS-suspended PCs (PAS-PCs) than in plasma-PCs by colony counting. Furthermore, reduced biofilm formation by S. liquefaciens and S. epidermidis during storage in PAS-PCs increases bacteria availability for sampling detection. Culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs, while changes of PLT quality can herald S. liquefaciens contamination when in excess of 108 CFUs/mL.

BACKGROUND: With buffy coat (BC) processing of whole blood (WB) donations, the preparation of plasma occurs within 24 hours rather than 8 hours of collection. The effect of this change on coagulation factor function in plasma and cryoprecipitate was evaluated during the validation of this production method and with routine production. STUDY DESIGN AND METHODS: Plasma frozen after an overnight hold of WB was prepared via BC or whole blood filtration (WBF) methods and quality control (QC) variables were measured. Additionally, plasma prepared with the BC method was compared to plasma produced using the platelet-rich plasma (PRP) method with an extended plasma factor analysis. Selected plasma factor levels were also measured in both cryoprecipitate and cryosupernatant plasma prepared using the WBF method from plasma frozen on the day of collection or after an overnight hold of WB. RESULTS: When comparing BC plasma to PRP plasma, coagulation factors (F)II, VII, VIII, IX, X, and XI had somewhat lower levels, and fibrinogen and antithrombin levels were elevated. As expected the most sensitive to the prolongation of production time was FVIII with 72 and 78% of the activity of PRP plasma and cryoprecipitate, respectively. However, both still met QC standards. Similarly, products made in routine production show acceptable levels of FVIII. CONCLUSION: Plasma and cryoprecipitate products, prepared using methods in which the plasma is frozen close to 24 hours after collection, meet current quality standards. The longer WB storage time has been implemented into general use in Canada.

Background In Europe, buffy-coat processing allows for the use of platelet additive solutions (PAS). These solutions, however, have long been questioned for their lack of glucose, a potentially essential nutrient for platelet storage. Using a novel, practical, two-part system for incorporation of glucose into an additive solution (PAS-G), this study compares platelet storage in plasma to storage in PAS-G. Study Design and Methods A paired study design of platelet concentrates (PC) were prepared from leucoreduced pools of eight buffy coats (BCP) split into two equal pools, with suspension in autologous plasma, or PAS-G. On days 2, 5, 7 and 9 of storage, samples were tested using standard in vitro platelet parameters. Data were analysed by paired Student's t-tests. Results During storage, PCs in PAS-G maintain a quality profile that is strikingly similar to PCs stored in plasma in terms of platelet activation (CD62) morphology score, swirl, glucose metabolism and pH. However, PCs in PAS-G perform lower (P

BACKGROUND: Buffy coat (BC) production of platelets (PLTs) has been successfully used in Europe for more than two decades. Currently, Canadian Blood Services is implementing the BC method. This article summarizes results of the validation testing performed to qualify the process of PLT production from whole blood and compares the quality of PLTs produced in routine production by either the PLT-rich plasma method (PRPPCs) or the BC method (BC-PCs). STUDY DESIGN AND METHODS: Validation data included variables used for routine quality control (QC; pH, PLT count, volume, sterility, residual white blood cell count) as well as nonroutine testing of PLTs for PLT activation, metabolic changes during storage, and PLT responsiveness to hypotonic shock and the extent of shape change induced by adenosine 5′-diphosphate. BC-PCs were tested on Days 1 and 6. QC of production runs included the same routine tests performed on Day 6. RESULTS: PLTs produced by the BC method during validation and pilot implementation met all Canadian Standards Association standards with respect to yield, volume, pH, and leukoreduction. Additional validation testing indicated a moderate level of PLT storage lesion development. In comparison to PRP-PCs, in vitro variables of BC-PCs, either pH in this study, or other markers compared to the literature were better, suggesting that BC-PCs have less evidence of productionrelated damage and improved PLT quality during storage. CONCLUSIONS: PLT concentrates produced from whole blood by the BC method after an overnight hold have laboratory variables suggestive of a higher quality than those concentrates produced by the PRP method. C anadian Blood Services undertook to change its method of component production from whole blood from the North American standard platelet-rich plasma (PRP) method to the buffy coat (BC) method of platelet (PLT) production. Developed by investigators in the Netherlands and Sweden in the mid-1970s, 1 the BC production method reverses the sequence of centrifugation steps compared to PRP. A hard spin is used initially to separate whole blood into three components: plasma, red blood cells (RBCs), and a BC layer. Using semiautomated extraction, the most common configuration uses a so-called top-and-bottom collection set in which plasma and RBCs are transferred to storage containers and the BC is left in the donation bag. This BC contains PLTs, white blood cells (WBCs), plasma, and some RBCs. ABO-matched BCs are pooled together with 1 plasma unit from one of the donors or 1 unit of PLT additive solution using a sterile docking device. The pooled BC is then given a soft spin, and the PRP is extracted with or without leukofiltration to produce a pooled PLT concentrate. Although both methods produce a PLT product, the products may have somewhat different in vitro characteristics. There is noticeable quality improvement in laboratory markers of BC-produced PLTs

Although the disaccharide trehalose has been shown to protect cells during freezing and desiccation when present intracellularly, a major obstacle to using intracellular sugars for biopreservation applications is the impermeability of plasma membranes. We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of stabilizing sugars into mammalian cells. Previous work has shown that the mechanism of red blood cell (RBC)–liposome interaction includes both liposome fusion with the RBC membranes, as well as tight adsorption of the vesicles onto the RBC surface. However, the fusion efficiency of liposomes was low, with only micromolar concentrations of trehalose delivered to the RBC cytosol. The purpose of this study is to enhance the efficacy of liposomes’ delivery of trehalose into RBCs with minimal detrimental effects on RBC membrane quality, by manipulating liposome physical properties and liposome–RBC incubation conditions. Charged and uncharged un...

A major obstacle in using intracellular sugars for the cryopreservation of mammalian cells is the inability of cells to synthesize or actively accumulate these sugars. We are investigating the use ...

RGD (arginine-glycine-aspartic acid) is a known peptide sequence that binds platelet integrin GPIIbIIIa and disrupts platelet-fibrinogen binding and platelet cross-linking during thrombosis. RGD peptides are unsuitable for clinical applications due to their high 50% inhibitory concentration (IC50) and low in vivo residence times. We addressed these issues by conjugating RGD peptides to biocompatible macromolecular carriers: hyperbranched polyglycerols (HPG) via divinyl sulfone. The GPIIbIIIa binding activity of RGD was maintained after conjugation and the effectiveness of the HPG-RGD conjugate was dependent upon molecular weight and the number of RGD peptides attached to each HPG molecule. These polyvalent inhibitors of platelet aggregation decreased the IC50 of RGD in an inverse linear manner based on the number of RGD peptides per HPG. Since HPG-RGD conjugates do not cause platelet activation by degranulation and certain substitution ratios do not increase fibrinogen binding to resting platelets, HPG-RGD may serve as a model for a novel class of antithrombotics.

Background and Objectives We developed a viscous platelet additive solution (PAS) based on MacoPharma's SSP+ but containing hydroxyethyl starch to address the poor osmotic balance and low yield associated with conventional PAS for the storage of buffy-coat platelet concentrates (PC). Materials and Methods Pools of four buffy-coats were made into leucoreduced PCs (n = 5) suspended either in plasma or viscous PAS. After determination of platelet recoveries, the PCs were stored under standard conditions. On days 1, 2, 3, 5, 7 and 9, PCs were tested for mean platelet volume, platelet concentration, soluble protein concentration, CD62 expression, platelet morphology, partial pressure of oxygen and partial pressure of carbon dioxide, glucose and lactate concentration, pH, extent of shape change, and hypotonic shock response (HSR). Results Platelets were prepared with greater ease using the viscous PAS and had improved platelet yield. PCs stored in either plasma or viscous PAS displayed similar storage characteristics to day 9. On days 7 and 9 of storage, platelets stored in viscous PAS displayed significantly lower (P

BACKGROUND Universal prestorage leukoreduction in Canada created the perception that stored red cells (RBCs) are more hemolyzed than their unfiltered predecessors. A pool-split design tested the effects of leukoreduction on hemolysis of stored RBCs. STUDY DESIGN AND METHODS Two ABO-matched units were pooled, divided, and then processed into leukoreduced (LR) and nonleukoreduced (NLR) units with the Pall LT-WB or RC-PL systems and sampled during standard processing and storage for testing of sterility, counts, hemolysis, and osmotic fragility. RESULTS Room temperature (RT) filtration of 10 pairs of LT-WB-LR and -NLR units showed significantly different percentage of hemolysis (0.39%) and osmotic fragility (0.643%) at 42 days. Cold-stored and -filtered units (2 days at 4 degrees C before processing) were less hemolyzed, but showed a similar proportional decrease of hemolysis in LR units (0.13% vs. 0.25% at 42 days). RBCs from RC-PL systems showed the lowest hemolysis although there was a filtration effect (0.05% vs. 0.12%, 42 days). Osmotic fragility paralleled hemolysis. Segment samples gave inaccurate results. Two-day prefiltration cold storage reduced hemolysis from 0.36 to 0.07 percent (42 days, p < 0.001). RT-LR hemolysis became significantly higher by Day 10 and 4 degrees C LR by Day 12. NLR units showed hemolysis by Day 7. LR units filtered cold were less hemolyzed (p < 0.05) than RT-LR but osmotic fragility was unchanged. CONCLUSIONS LR-RBCs prepared by any of three methods (LT-WB, RT or cold; RC-PL), filtered at 4 degrees C, were less hemolyzed during storage than nonfiltered concentrates: 4 degrees C leukoreduction is beneficial for RBCs and does not cause hemolysis or enhance fragility.

BACKGROUND: There are few reports about throm-bopoietic responses in whole blood (WB) and platelet-pheresis donors. This study compares the thrombo-poietic responses of such donors and their platelet values. STUDY DESIGN AND METHODS: The effect of WB donation or selective platelet loss (plateletpheresis) was evaluated prospectively. WB and platelet donor samples before donation and for 7 days thereafter were assessed for platelet count, mean platelet volume, and platelet reticulocytes. RESULTS: Reticulated platelets appeared in the circulation of plateletpheresis donors by 24 hours. The proportion of reticulated platelets was highest on Day 2, and above-normal levels of reticulated platelets persisted until Day 7. The mean platelet volume was high on Days 2 and 3, which corresponded with the appearance of reticulated platelets. After plateletpheresis, platelet counts were higher than could be accounted for by new platelets, which suggested the release of sequestered platelets. WB donors manifested no changes in platelet counts but had a peak of circulating platelet reticulocytes 2 days after the donation. CONCLUSION: The thrombopoietic peak in WB and plateletpheresis donors occurs 2 days after donation, and the response level is related to the amount of platelets lost. The impact of platelet loss on the number of circulating platelets is modulated by the release of platelets from the spleen.

BACKGROUND: The introduction of prestorage white cell (WBC) reduction in random-donor platelet concentrates in Canada has increased the occurrence of particulate material in PCs. The effects of filtration on platelet activation state and the activation of plasma enzyme systems were assessed. STUDY DESIGN AND METHODS: Particulate material was examined by light microscopy, electron microscopy, protein electrophoresis, and biochemical analysis. Thirty PCs (10 unfiltered, 20 filtered) were examined during processing and 5-day storage for pH, platelet count and mean volume, morphology, activation marker expression, and hypotonic shock response. Complement activation, thrombin generation, and fibrinolysis were assessed by using specific enzyme immunoassays or chromogenic assays. RESULTS: By all analyses, the particulate material appeared to be platelet aggregates. Platelets exposed to WBC-reduction filters expressed a significantly higher level of activation markers CD62 and CD63, altered morphology, and increased platelet microparticles throughout the storage period than did unfiltered platelets. Complement activation at the C3 level was significantly increased in filtered units with little evidence of coagulation or fibrinolytic system activation. CONCLUSION: Exposure of platelets to filters during prestorage WBC reduction increased platelet activation and mildly increased complement activation over the levels during the storage period. These alterations can contribute to the formation of irreversible platelet aggregates during processing.

To assess alterations in coagulation proteins in stored platelet concentrates, we used a series of platelet parameters and measures of coagulation activation to compare samples collected before unit donation, during the processing of platelet concentrates (PC) from CPDA-1 blood, and in storage up to 5 days as well as stored platelet-poor plasma (PPP). Storage-dependent increases in activated partial thromboplastin time and prothrombin time were seen in both PC and PPP. However, FVII, FXI, FXII, kallikrein activity and prothrombin F1.2 levels remained unchanged in stored PC. Surprisingly, in stored PPP, significant alterations in FXII, FVII, kallikrein and prothrombin F1.2 levels were seen. Platelet morphology and surface marker studies demonstrated platelet activation during storage. These data suggest that the presence of platelets in the CLX storage container partially suppresses coagulation activation at significant cost to platelet viability.

Here, we report on a patient with pseudoexfoliation syndrome who developed acute angle-closure glaucoma with a marked myopic shift due to anterior dislocation of the posterior chamber intraocular lens almost 16 months after an uneventful phacoemulsification. Examination with a Scheimpflug camera was extremely useful in confirming the diagnosis. This is the fist case of late-onset angle-closure glaucoma with a significant myopic shift due to anterior dislocation of the posterior chamber intraocular lens, which resulted in a permanent alteration of the postoperative target refraction. Copyright © 2011 S. Karger AG, Basel.

The rural life will have a new aspect within the European Union. The paper deals with the general term of ‘network’ which can refer to any interconnected group or system. It shows the lessons, experiences and the main obstacle of the networking activity, implemented is the former programming period, i.e. 2000-2006, and so for the current programming period, i.e. 2007-2013. It introduces the steps and structures to the Hungarian National Rural Network (HNRN), as an example. Officially the Article 68 of the Regulation 1698/2005/EC contains provisions as to the establishment of the National Rural Network, which aims at (a) identifying and analysing the best practises on rural development, providing information about them and organizing exchanges of experiences and know-how, and (b) preparing training programmes for local action groups in the process of formation and giving technical assistance for inter-territorial and trans-national cooperation between LAGs. Networking activity is looked upon as a permanent, improvable tool that can assist in developing the rural quality and economy. The paper introduces an evaluation on the willingness for cooperation on international field, which analysis was launched June 2009 among the Hungarian Local Action Groups (LAGs) by the HNRN. It shows that the successful adaptation to persistent rural acts will depend on many elements, as a result mainly on good practices and experiences. It is visible, that Hungary is also on the way of learning, and it has to draw the conclusion from time to time, the process of network building is drawn from the rural stakeholders and the wider rural economy point of view.

Preserving cell viability and function is an essential component in the translation and delivery of existing and emerging cell-based therapeutics from the research lab to the patient bedside. This workshop provided a summary of the advances and challenges that currently face the preservation sciences, together with a glimpse at the future applications and instrumentation that will enhance our ability to process, preserve, and store red blood cells (RBCs), platelets, and stem cells. It is clear from the presentations made during the workshop and the discussions that ensued after that, for us to overcome the challenges that face blood biopreservation, it will require a concerted effort from clinicians, scientists, and engineers from a variety of disciplines. Through this interdisciplinary research effort, significant progress will be made to improve the safety, quality, and potency of the blood products that are used in reparative medicine. As the need for effective preservation technologies will be the motivation for more concerted efforts in the biopreservation sciences, there are encouraging prospects for the future applications of biopreserved blood cells.

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3M trehalose or liposome solution, then cooled with slow (0.95+/-0.02 degrees C/min), medium (73+/-3 degrees C/min) and fast (265+/-12 degrees C/min) cooling rates and storage in liquid nitrogen, followed by a 37 degrees C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66%+/-5% vs 29%+/-4%) compared to control RBCs (c-RBC, p=0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p=0.039 and p=0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p=0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p=0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.

Conventional antithrombotic drug discovery requires testing of large numbers of drug candidates. We used computer-aided macromolecular interaction assessment (MIAX) to select antithrombotic molecules that mimic and therefore block platelet GPIb’s binding to von Willebrand factor (vWf), an early step in thrombus formation. We screened a random array of 15-mer D-amino acid peptides for binding vWf. Structures of 4 candidate peptides were inferred by comparison to sequences in protein databases, conversion from the L to D conformations and molecular dynamics (MD) determinations of those most energetically stable. By MIAX, we deduced the amino acids and intermolecular hydrogen bonds contributing to the GPIb-vWf interaction interface. We docked the peptides onto vWf in silico to localize their binding sites and consequent potential for preventing GPIb-vWf binding. In vitro inhibition of ristocetin-initiated platelet agglutination confirmed peptide function and suitability for antithrombotic development, thereby validating this novel approach to drug discovery.

BACKGROUND: Buffy-coat processing allows for the use of platelet additive solutions (PASs). PASs reduce plasma-associated transfusion reactions and conserve plasma for transfusion or fractionation. Platelet (PLT) storage in plasma was compared to storage in three commercially available PASs compared to assess their influence on in vitro laboratory variables. STUDY DESIGN AND METHODS: Platelet concentrates (PCs) were prepared from leukoreduced pools of four buffy coats (BCPs) suspended in autologous plasma or one of PASs (Composol, Fresenius-Kabi; T-Sol, Baxter Corp.; or SSP+, MacoPharma). On Days 1, 2, 3, 5, and 7 of storage, samples were tested for PLT concentration, mean PLT volume (MPV), CD62P, morphology, pO2, pCO2, glucose, lactate and total protein concentration, pH, extent of shape change (ESC), and hypotonic shock response (HSR). Data were analyzed by analysis of variance (ANOVA) with repeated measures and t tests. RESULTS: PLT recoveries from BCPs were higher (p

Following treatment of Lewis lung carcinomas (LLC) by Photofrin-mediated photodynamic therapy (PDT), tumor tissues and sera of host mice were collected for the analysis of complement activity. Elevated tumor C3 levels were detected between 1 and 24 h after PDT, while serum C3 levels increased significantly at 24 h post therapy. Increased alternative complement pathway activity in the serum was evident between 1 and 3 days post PDT. Blocking C3a- or C5a-receptors in the host mice decreased the efficacy of PDT in producing LLC tumor cures, supporting the importance of complement action in PDT-mediated tumor destruction.

To clarify the interactions of liposomes with blood cells, this study examined the behaviour of liposomes of a range of compositions in the presence of purified human blood cells in buffer or plasma; or in whole blood, or in mice in vivo. Liposomes, labeled with the hydrophilic fluorochrome, carboxy fluorescein (CF), or with membrane-sequestering R18 or FITC-labeled phospholipids, were mixed with blood cells and the appearance of the fluorochromes in the blood cell population was monitored by flow cytometry. Irrespective of composition, with or without poly(ethylene glycol), all types of liposomes were found to interact rapidly and dose-dependently with red cells, leukocytes and platelets, both in vitro and in vivo. This took place equally in the presence and the absence of plasma proteins and functional enzyme cascades, suggesting that the prime facie interaction is opsonization-independent and is consistent with liposome-blood cell fusion.

Immune thrombocytopenic purpura's diagnosis (ITP) is based on low platelet count and exclusion of clinical conditions rather than a specific diagnostic test. We used the reticulated platelet (RP) assay to study ITP and thrombocytopenia associated with HIV infection (HIV-ITP). Data from 96 ITP and 23 HIV-ITP patients showed low platelet counts (PC) with both high or low %RP suggesting that individuals have different degrees of thrombopoiesis. About 20% of ITP and 46% of HIV-ITP patients had %RP in the 'low' or 'normal' ranges. Grouped by platelet count

We investigated the effect on in vitro platelet function of hirutonin, a modified hirutonin with an RGD-like motif, a pseudo-RGDS peptide and a linear RGDS peptide. Inhibition of expression of surface fibrinogen on ADP-activated platelets with 40 microM of the peptide was as follows: hirutonin 10+/-3%, modified chimeric peptide 26+/-5%, pseudo-RGDS 66+/-11% and linear RGDS 93+/-13%. Both hirutonin and the chimeric peptide significantly inhibited ADP-induced platelet activation as detected by CD62 expression. Unlike the RGDS and pseudo-RGDS controls, neither the chimeric peptide nor the parent hirutonin inhibited ADP-induced platelet aggregation even at 140 microM. The chimeric hirutonin peptide reduced ATP release from ADP-stimulated platelets by 40+/-4%. This inhibition was stronger than that caused by hirutonin (23+/-13%), but less than the RGDS (90+/-2%) and pseudo RGDS-peptides (59+/-11%). Primary platelet haemostasis was slightly but not significantly affected by the peptide at 40 and 80 microM. However, shear-induced platelet adhesion to vWF and especially subsequent aggregate formation was interrupted after the addition of the chimeric peptide. Similar results were obtained with hirutonin. This inhibition was not as marked as with the RGDS- and pseudo-RGDS peptides. Both the parent hirutonin and the chimeric peptide caused prolongation of the clinical coagulation assays aPTT and TT. In conclusion, the chimeric hirutonin peptide with introduction of the RGD motif retained its anticoagulant effect but had little formal disintegrin activity. Instead, it appeared to have novel anti-platelet effects that may be of therapeutic use.

With the advent of liposomes as drug carriers, there arises a need for efficient targeted delivery in vivo. Proteins coupled to liposomes usually yield heterogeneous products that are ill-defined both chemically and in terms of spatial orientation. We now report on the disulfide linkage to the surface of intact liposomes of a peptide representing one-half of a ligand-receptor pair. An RGD-motif-containing peptide was coupled to the phospholipid PDP-DOPE of the liposomes by a thiol-disulfide exchange. The resulting lipopeptides were amenable to definition by TLC, HPLC, and MS and found to be in a functional orientation allowing biochemical interaction with their receptor, the integrin glycoprotein IIb-IIIa.