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Human Whole Blood (human + whole_blood)
Selected AbstractsEx Vivo Biocompatibility of Avidin-Agarose: A New Device for Direct Adsorption of Biotinylated Antibodies from Human Whole BloodARTIFICIAL ORGANS, Issue 9 2000T. Bosch Abstract: Radioimmunotherapy using radiolabeled antitumor antibodies (RAA) is limited by the toxicity of unbound antibodies in the circulation. Removal of excessive antibodies by affinity-adsorption could therefore allow the administration of increased dosages of RAA while decreasing their adverse effects. Recently, avidin-agarose (AA) minicolumns were used in animal experiments for the removal of biotinylated antibodies from whole blood exploiting the high affinity binding of biotin to avidin (pK 1015 M,1). This study was performed to evaluate the ex vivo biocompatibility of AA minicolumns with human blood. Ten ml AA minicolumns were perfused online ex vivo in the single pass mode with fresh blood from 8 healthy donors at a flow rate of 6.25 ml/min. The anticoagulation consisted of 0.5 IU heparin plus 0.0,2.1 mg citrate per ml of blood. In Part 1 of the study (40 min perfusion, n = 4), the optimal anticoagulation was found to be 0.5 IU heparin plus about 1 mg citrate per ml of blood. In Part 2 of the study, four 80 min test-runs were performed. No signs of hemolysis were found, and the thrombogenicity of the AA gel was negligible. Cell counts and column inlet pressures remained constant; toward the end of the 80 min test-runs, some activation of blood cells (elastase, ,-thromboglobulin), the complement system (C3a, C5a) and the plasmatic coagulation (thrombin-antithrombin complex) was detectable. A moderate initial bradykinin release rapidly subsided to very low levels. In summary, AA minicolumns showed good biocompatibility upon contact with human whole blood and merit further investigation in a closed-loop system for a potential application of direct tumor antibody removal by hemoperfusion. [source] Triclosan inhibition of acute and chronic inflammatory gene pathwaysJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 5 2010Silvana P. Barros Barros SP, Wirojchanasak S, Barrow DA, Panagakos F, Devizio W, Offenbacher S. Triclosan inhibition of acute and chronic inflammatory gene pathways. J Clin Periodontol 2010; 37: 412,418. doi: 10.1111/j.1600-051X.2010.01548.x. Abstract Aim: We sought to determine whether triclosan (2,4,4,-trichloro-2,-hydroxydiphenylether), an extensively used anti-plaque agent with broad-spectrum anti-microbial activity, with reported anti-inflammatory effects via inhibition of prostaglandin E2 and interleukin 1 (IL-1),, could also more broadly suppress multiple inflammatory gene pathways responsible for the pathogenesis of gingivitis and periodontitis. Materials and Methods: As an exploratory study, the effects of triclosan on the inflammatory gene expression profile were assessed ex vivo using peripheral whole blood samples from eight periodontally healthy donors. Ten-millilitres whole blood aliquots were incubated 2 h with 0.3 ,g/ml Escherichia coli lipopolysaccharide (LPS) with or without 0.5 ,g/ml triclosan. Affymetrix microarray gene expression profiles from isolated leucocytes and pathway-specific quantitative polymerase chain reaction arrays were used to investigate changes in expression of target cytokines and cell signalling molecules. Results: Ex vivo human whole blood assays indicated that triclosan significantly down-regulated the LPS-stimulated expression of Toll-like receptor signalling molecules and other multiple inflammatory molecules including IL-1 and IL-6 and the dampening of signals that activate the T-helper type 1 acquired immune response via suppression of CD70 with concomitant up-regulation of growth factors related to bone morphogenetic protein (BMP)2 and BMP6 synthesis. Conclusions: Anti-inflammatory effects were found in this exploratory survey, including suppression of microbial-pathogen recognition pathway molecules and the suppression of acute and chronic mediators of inflammation. [source] Expression of p16INK4a in peripheral blood T-cells is a biomarker of human agingAGING CELL, Issue 4 2009Yan Liu Summary Expression of the p16INK4a tumor suppressor sharply increases with age in most mammalian tissues, and contributes to an age-induced functional decline of certain self-renewing compartments. These observations have suggested that p16INK4a expression could be a biomarker of mammalian aging. To translate this notion to human use, we determined p16INK4a expression in cellular fractions of human whole blood, and found highest expression in peripheral blood T-lymphocytes (PBTL). We then measured INK4/ARF transcript expression in PBTL from two independent cohorts of healthy humans (170 donors total), and analyzed their relationship with donor characteristics. Expression of p16INK4a, but not other INK4/ARF transcripts, appeared to exponentially increase with donor chronologic age. Importantly, p16INK4a expression did not independently correlate with gender or body-mass index, but was significantly associated with tobacco use and physical inactivity. In addition, p16INK4a expression was associated with plasma interleukin-6 concentration, a marker of human frailty. These data suggest that p16INK4a expression in PBTL is an easily measured, peripheral blood biomarker of molecular age. [source] LETTERS TO THE EDITOR: Inhibition of thrombin activatable fibrinolysis inhibitor augments fibrinolysis in human whole bloodJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2005N. L. M. CRUDEN No abstract is available for this article. [source] Superantigens from Staphylococcus aureus induce procoagulant activity and monocyte tissue factor expression in whole blood and mononuclear cells via IL-1,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2003E. Mattsson Summary.,Background:,Staphylococcus aureus is one of the most common bacteria in human sepsis, a condition in which the activation of blood coagulation plays a critical pathophysiological role. During severe sepsis and septic shock microthrombi and multiorgan dysfunction are observed as a result of bacterial interference with the host defense and coagulation systems. Objectives:,In the present study, staphylococcal superantigens were tested for their ability to induce procoagulant activity and tissue factor (TF) expression in human whole blood and in peripheral blood mononuclear cells. Methods and results:,Determination of clotting time showed that enterotoxin A, B and toxic shock syndrome toxin 1 from S. aureus induce procoagulant activity in whole blood and in mononuclear cells. The procoagulant activity was dependent on the expression of TF in monocytes since antibodies to TF inhibited the effect of the toxins and TF was detected on the surface of monocytes by flow cytometry. In the supernatants from staphylococcal toxin-stimulated mononuclear cells, interleukin (IL)-1, was detected by ELISA. Furthermore, the increased procoagulant activity and TF expression in monocytes induced by the staphylococcal toxins were inhibited in the presence of IL-1 receptor antagonist, a natural inhibitor of IL-1,. Conclusions:,The present study shows that superantigens from S. aureus activate the extrinsic coagulation pathway by inducing expression of TF in monocytes, and that the expression is mainly triggered by superantigen-induced IL-1, release. [source] Effect of isoflurane on monocyte adhesion molecule expression in human whole blood,ACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 5 2003L. W. De Rossi Background: Recruitment of monocytes to inflamed tissue is a crucial step in the acute inflammatory reaction. Adherence of monocytes to endothelial cells followed by transmigration depends on monocyte surface adhesion molecules, inflammatory cytokines and chemoattractant chemokines. In the present study, we determined the effect of isoflurane on monocyte adhesion receptor expression in vitro. Methods: Citrated whole blood was incubated for 60 min with either 0.5 or 1 MAC isoflurane. In unstimulated blood samples and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) monocyte cell-surface expression of the selectins PSGL-1 and L-selectin, and the ,2 -integrins CD11a and CD11b were evaluated by flow cytometry. Results: Isoflurane reduced significantly the expression of PSGL-1 on unstimulated monocytes, whereas the remaining selectins and ,2 -integrins were not affected. At both concentrations, the FMLP-induced removal of PSGL-1 from the monocyte surface was increased. Furthermore, at 1 MAC isoflurane the FMLP-induced increase in CD11a expression was significantly inhibited. The surface expression of L-selectin and CD11b was not affected following exposure to isoflurane. Conclusion: Isoflurane increases the removal of the selectin PSGL-1 from the monocyte surface. Since PSGL-1 is important during the initial step of monocyte adhesion to endothelial P-selectin, the decrease in monocyte surface PSGL-1 may have profound effects on monocyte,endothelial interactions. Furthermore, the effects of isoflurane on monocyte adhesion molecule expression are different from those reported for neutrophils. [source] More pronounced inhibition of cyclooxygenase 2, increase in blood pressure, and reduction of heart rate by treatment with diclofenac compared with celecoxib and rofecoxibARTHRITIS & RHEUMATISM, Issue 1 2006Burkhard Hinz Objective Recent findings suggest that permanent blockade of cyclooxygenase 2 (COX-2) is one factor contributing to the cardiovascular side effects of selective COX-2 inhibitors (coxibs) and nonsteroidal antiinflammatory drugs (NSAIDs). The present study compared the extent and time course of COX-2 inhibition and the effects on cardiovascular parameters (changes in blood pressure and heart rate) between various antirheumatic doses of diclofenac, celecoxib, and rofecoxib in healthy elderly volunteers. Methods A randomized, parallel-group study was conducted in volunteers receiving 75 mg diclofenac twice daily, 200 mg celecoxib twice daily, or 25 mg rofecoxib once daily for 8 days. Blood samples were obtained predose and at specified time points postdose, on days 1 and 8, for assay of drug plasma concentrations and COX-2 inhibition. Lipopolysaccharide-induced prostaglandin E2 synthesis was measured ex vivo as an index of COX-2 activity in human whole blood. Results COX-2 inhibition was significantly less pronounced after treatment with celecoxib and rofecoxib than with diclofenac. Maximal inhibitions after a single dose and at steady state, respectively, were as follows: 99% and 99% with diclofenac, 70% and 81% with celecoxib, and 56% and 72% with rofecoxib. At steady state, only diclofenac caused virtually complete COX-2 inhibition over the whole dose interval, and this corresponded to the highest increase in systolic blood pressure and greatest reduction in heart rate. Conclusion Diclofenac elicited the most pronounced COX-2 inhibition, blood pressure elevation, and suppression of heart rate. It is assumed that the extent and time course of intravascular COX-2 inhibition may determine the differential profile of cardiovascular side effects associated with NSAIDs and coxibs, but this has to be proven in future studies. [source] Ex Vivo Biocompatibility of Avidin-Agarose: A New Device for Direct Adsorption of Biotinylated Antibodies from Human Whole BloodARTIFICIAL ORGANS, Issue 9 2000T. Bosch Abstract: Radioimmunotherapy using radiolabeled antitumor antibodies (RAA) is limited by the toxicity of unbound antibodies in the circulation. Removal of excessive antibodies by affinity-adsorption could therefore allow the administration of increased dosages of RAA while decreasing their adverse effects. Recently, avidin-agarose (AA) minicolumns were used in animal experiments for the removal of biotinylated antibodies from whole blood exploiting the high affinity binding of biotin to avidin (pK 1015 M,1). This study was performed to evaluate the ex vivo biocompatibility of AA minicolumns with human blood. Ten ml AA minicolumns were perfused online ex vivo in the single pass mode with fresh blood from 8 healthy donors at a flow rate of 6.25 ml/min. The anticoagulation consisted of 0.5 IU heparin plus 0.0,2.1 mg citrate per ml of blood. In Part 1 of the study (40 min perfusion, n = 4), the optimal anticoagulation was found to be 0.5 IU heparin plus about 1 mg citrate per ml of blood. In Part 2 of the study, four 80 min test-runs were performed. No signs of hemolysis were found, and the thrombogenicity of the AA gel was negligible. Cell counts and column inlet pressures remained constant; toward the end of the 80 min test-runs, some activation of blood cells (elastase, ,-thromboglobulin), the complement system (C3a, C5a) and the plasmatic coagulation (thrombin-antithrombin complex) was detectable. A moderate initial bradykinin release rapidly subsided to very low levels. In summary, AA minicolumns showed good biocompatibility upon contact with human whole blood and merit further investigation in a closed-loop system for a potential application of direct tumor antibody removal by hemoperfusion. [source] An HPLC assay for the lipophilic camptothecin analog AR-67 carboxylate and lactone in human whole bloodBIOMEDICAL CHROMATOGRAPHY, Issue 10 2010Eleftheria Tsakalozou Abstract AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin, DB-67) is a camptothecin analog currently in early stage clinical trials. The lactone moiety of camptothecins hydrolyzes readily in blood to yield the pharmacologically inactive carboxylate form. However the lactone form of third-generation lipophilic congeners, such as AR-67, is more stable, possibly due to partitioning into red cell membranes. This prompted us to develop a reverse-phase HPLC method with fluorescence detection (excitation 380,nm/emission 560,nm), which could quantitate the concentration of AR-67 lactone and carboxylate in whole blood. Samples were prepared by red cell lysis, protein precipitation with methanol and centrifugation to remove denatured materials. Recovery was estimated to be >85%. Analytes were eluted isocratically with 0.15,m ammonium acetate buffer containing 10,mm TBAP (pH 6.5) and acetonitrile (65:35, v/v) on a Nova-Pak C18 column (4,µm; 3.9 × 150,mm). The assay was linear in the ranges 0.5,300 and 2.5,300,ng/mL for carboxylate and lactone, respectively. Accuracy and precision were acceptable. AR-67 forms were stable in whole blood and in methanolic supernatants. This assay has been successfully applied to measure AR-67 concentrations in whole blood of patients enrolled in a phase I study. Copyright © 2010 John Wiley & Sons, Ltd. [source] Development and validation of a sensitive assay for the quantification of imatinib using LC/LC-MS/MS in human whole blood and cell cultureBIOMEDICAL CHROMATOGRAPHY, Issue 12 2009Jelena Klawitter Abstract We developed and validated a semi-automated LC/LC-MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back-flushed onto the analytical column. Ion transitions [M + H]+ of imatinib (m/z = 494.3 , 394.3) and its internal standard trazodone (372.5 , 176.3) were monitored. The range of reliable response was 0.03,75 ng/mL. The inter-day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze,thaw cycles. This semi-automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. Copyright © 2009 John Wiley & Sons, Ltd. [source] Simultaneous determination of ten amphetamine designer drugs in human whole blood by capillary electrophoresis with diode array detectionBIOMEDICAL CHROMATOGRAPHY, Issue 10 2005Maria Nieddu Abstract In recent years, a number of new designer drugs have entered the illicit drug market. The methylenedioxyderivatives of amphetamine represent the largest group of designer drugs. This paper describes a method for screening for and simultaneously quantifying 10 2,5-methylenedioxy-derivatives of amphetamine and phenethylamine in human whole blood, using capillary electrophoresis (CE) with diode array detection (DAD). Using an aqueous pH 2.5 phosphate buffer, CE analysis gave peaks with good symmetry and reproducible migration times. Under these experimental conditions, the 10 amphetamines were resolved in 15 min and without interference from biological matrices (blood). Their identification by migration time was confirmed by their UV spectra recorded with a DAD (190,350 nm). The main advantages of the present method lie in its simplicity, clean and reliable extraction from human whole blood and simultaneous detection and quantification by CE-DAD. The applicability of the method was demonstrated by analysis of in vivo rat blood samples. The method was validated according to international guidelines. Copyright © 2005 John Wiley & Sons, Ltd. [source] Cloning and pharmacological characterization of the dog P2X7 receptorBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2009S Roman Background and purpose:, Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues. Experimental approach:, A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1, release in dog and human whole blood. Key results:, The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2,-&3,-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower. Conclusions and implications:, Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic. [source] Functional basis for complement evasion by staphylococcal superantigen-like 7CELLULAR MICROBIOLOGY, Issue 10 2010Jovanka Bestebroer Summary The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen-like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA-Fc,RI binding and serum killing of Escherichia coli. As C5a generation, in contrast to C5b-9-mediated lysis, is crucial for immune defence against staphylococci, we investigated the impact of SSL7 on staphylococcal-induced C5a-mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA-binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a-induced phagocytosis of S. aureus and oxidative burst in an in vitro whole-blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a-driven influx of neutrophils in mouse peritoneum. [source] 2-Acylaminopyridin-4-ylimidazoles as p38 MAP Kinase Inhibitors: Design, Synthesis, and Biological and Metabolic EvaluationsCHEMMEDCHEM, Issue 11 2009Katharina Ziegler Dr. Abstract Targeting cytokines has become an important focus in the treatment of many inflammatory disorders. p38 MAP kinase (MAPK) is the key enzyme in regulating the biosynthesis and release of pro-inflammatory cytokines such as IL-1, and TNF,. Inhibition of p38 MAPK results in decreased expression of these cytokines. Tri- and tetrasubstituted pyridinylimidazoles are potent inhibitors of p38 MAPK. Substitution on the pyridinyl moiety allows the design of inhibitors that show increased selectivity and activity by targeting the enzyme's hydrophobic region,II. The objective of this study was to synthesize novel 1,2,4,5-tetrasubstituted imidazole derivates and to characterize them not only for their ability to inhibit p38 MAPK and modulate cytokine release in human whole blood, but also to evaluate their metabolic stability. Biological data and metabolic studies demonstrate that the introduction of a 2-acylamino function at C2 of the pyridine results in highly efficient and metabolically stable inhibitors relative to C2-alkylamino derivatives. A series of novel candidates was investigated for metabolic stability in human liver microsomes and in human whole blood. Additionally, metabolic S-oxidation was investigated, and possible metabolites were synthesized. [source] | ||||||||||||||||